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Apoptosis, Cell Death and

Cell Proliferation
3rd edition
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Apoptosis, Cell Death, and

Cell Proliferation
3rd edition
Table of Contents
Table of Contents
A Cell Death Apoptosis and Necrosis Page
1. Introduction .......................................................................................................................................................... 2
1.1 Terminology of cell death ................................................................................................................................... 2
1.2 Differences between necrosis and apoptosis ............................................................................................ 3
1.3 Apoptotic Pathways.............................................................................................................................................. 5
2. Apoptosis Assay Methods............................................................................................................................ 7
Method/Product selection guide .................................................................................................................... 8
2.1 Methods for studying apoptosis in cell populations............................................................................. 10
2.1.1 Assays that measure DNA fragmentation ....................................................................................... 10
Apoptotic DNA Ladder Kit ............................................................................................................... 13
Cell Death Detection ELISAPLUS .................................................................................................... 15
2.1.2 Assays that measure apoptosis-induced proteases (caspases) ............................................. 18
M30 CytoDEATH ................................................................................................................................... 19
M30 CytoDEATH, Fluorescein ......................................................................................................... 19
Caspase 3 Activity Assay .................................................................................................................. 22
Homogeneous Caspases Assay ..................................................................................................... 25
Anti-PARP ............................................................................................................................................... 27
2.1.3 Summary of methods for studying apoptosis in cell populations ........................................... 30
2.2 Methods for studying apoptosis in individual cells............................................................................... 32
2.2.1 The TUNEL enzymatic labeling assay ............................................................................................... 33
In Situ Cell Death Detection Kit, Fluorescein ............................................................................. 36
In Situ Cell Death Detection Kit, TMR red .................................................................................. 36
In Situ Cell Death Detection Kit, AP .............................................................................................. 39
In Situ Cell Death Detection Kit, POD .......................................................................................... 39
2.2.2 Assays that measure membrane alterations .................................................................................. 43
Annexin-V-FLUOS ............................................................................................................................... 44
Annexin-V-FLUOS Staining Kit ....................................................................................................... 44
Annexin-V-Alexa 568 .......................................................................................................................... 44
Annexin-V-Biotin .................................................................................................................................. 47
2.2.3 Assays that use DNA stains .................................................................................................................. 49
DAPI, Propidium iodide ...................................................................................................................... 50
2.2.4 Summary of methods for studying apoptosis in individual cells ............................................. 52
2.3. Detection of apoptosis-related proteins.................................................................................................... 54
Anti-Fas (CD95/Apo-1) ...................................................................................................................... 55
p53 pan ELISA ...................................................................................................................................... 56
3. Cytotoxicity Assay Methods..................................................................................................................... 58
3.1 Relationship between cytotoxicity, apoptosis and necrosis............................................................... 58
3.2 Methods for studying cytotoxicity................................................................................................................ 59
3.2.1 Assays that measure plasma membrane leakage ........................................................................ 59
Cytotoxicity Detection Kit (LDH) .................................................................................................... 61
Cellular DNA Fragmentation ELISA .............................................................................................. 64
3.2.2 Assays that measure metabolic activity ............................................................................................ 68
Cell Proliferation Kit I (MTT) ............................................................................................................ 85
Cell Proliferation Kit II (XTT) ............................................................................................................ 86
Cell Proliferation Reagent WST-1 ................................................................................................... 87
3.2.3 Summary of methods for studying cytotoxicity .............................................................................. 70
II Apoptosis, Cell Death, and Cell Proliferation Manual

Table of Contents
B Cell Proliferation and Viability Page
1. Introduction ........................................................................................................................................................ 74
1.1 Terminology of cell proliferation and viability........................................................................................... 74
1.2 Cell Cycle .............................................................................................................................................................. 75
2. Cell proliferation/viability assay methods......................................................................................... 78
Method/Product selection guide ................................................................................................................. 80
2.1 Methods for studying cell proliferation and viability in cell populations...................................... 82
2.1.1 Assays that measure metabolic activity ........................................................................................... 82
Cell Proliferation Kit I (MTT) ............................................................................................................ 85
Cell Proliferation Kit II (XTT) ............................................................................................................ 86
Cell Proliferation Reagent WST-1 .................................................................................................... 87
2.1.2 Assays that measure DNA synthesis ................................................................................................ 89
5-Bromo-2-deoxy-uridine Labeling and Detection Kit III .................................................. 91
Cell Proliferation ELISA, BrdU (colorimetric) ............................................................................ 94
Cell Proliferation ELISA, BrdU (chemiluminescence) ............................................................ 94
2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations 98
2.1.4 Single reagents for the measurement of DNA synthesis .......................................................... 98
2.2 Methods for studying cell proliferation and viability in individual cells ...................................... 100
2.2.1 Assays that measure DNA synthesis................................................................................................ 100
5-Bromo-2-deoxy-uridine Labeling and Detection Kit I .................................................... 101
5-Bromo-2-deoxy-uridine Labeling and Detection Kit II .................................................. 101
In Situ Cell Proliferation Kit, FLUOS ............................................................................................ 102
Anti-BrdU, formalin grade .............................................................................................................. 105
Anti-BrdU-Fluorescein ..................................................................................................................... 105
Anti-BrdU-Peroxidase, Fab fragment ......................................................................................... 105
2.2.2 Summary of methods for studying cell proliferation and viability in individual cells .... 108
Introduction III
Table of Contents
C Appendix Page
1. Technical Tips ................................................................................................................................................. 112

1.1 Selected frequently asked questions (FAQs) about cell death assays ....................................... 112
1.2 Technical tips on the TUNEL method ..................................................................................................... 113
1.2.1 TUNEL: Improvement and evaluation of the method for in situ apoptotic
cell identification ..................................................................................................................................... 113
1.2.2 TUNEL protocol for tissues which tend to give false positives ............................................. 114
1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts .................................... 115
1.3 Technical tips on the use of Annexin-V-Biotin for light microscope detection ....................... 117
1.4 Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples ..................... 118
1.5 Technical tips on the Cell Proliferation ELISA Kits ............................................................................. 118
2. Special applications of cell death and cell proliferation methods .................................. 119
2.1 TUNEL assays .................................................................................................................................................. 119
2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay
and surface labeling as detected by flow cytometry ................................................................. 119
2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell
cycle analysis ............................................................................................................................................ 119
2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL 120
2.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial
cell marker ................................................................................................................................................. 120
2.2 Metabolic assays ............................................................................................................................................ 121
2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts . 121
2.3 Annexin assays ................................................................................................................................................ 121
2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype ... 121
2.4 BrdU assays ...................................................................................................................................................... 122
2.4.1 Detection of bromodeoxyuridine in paraffin-embedded tissue sections using
microwave antigen retrieval is dependent on the mode of tissue fixation ........................ 122
3. References ...................................................................................................................................................... 123
3.1 Apoptosis-related parameters Abbreviations and References .................................................. 123
3.2 Examples for applications of Roche Applied Science products ................................................... 128
3.3 General references ......................................................................................................................................... 154
4. General abbreviations .............................................................................................................................. 158
5. Ordering Guide ............................................................................................................................................. 161
6. Index ................................................................................................................................................................... 164
IV Apoptosis, Cell Death, and Cell Proliferation Manual

Overview of this Guide
Overview of this Guide

How this guide can help you study cell death and cell proliferation?
When and why do cells die? Does the concentration of environmental pollutants exert
cytotoxic or cytostatic effects on cells? What factors influence the rate and timing of cell
proliferation? Researchers in basic, industrial, and medical research are asking these ques-
tions and looking for answers. Understanding the normal regulation of cell death and cell
proliferation will be critical e.g., for the development of new and more successful thera-
pies for preventing and treating cancer and for the screening of new anti-cancer com-
pounds.
Many assays exist to measure cell death and cell proliferation. However, if you have only
recently become interested in cell death or cell proliferation, you may find the diversity of
such assays bewildering. You may not be able to determine what each assay measures nor
decide which assays are best for your purposes. This guide is designed to help you make
such decisions. It presents a brief overview of cell death and cell proliferation, along with
the major assays currently available to measure each. In addition, it clearly lists the
advantages and the disadvantages of these assays.
For those who want to eliminate radioactivity from their laboratories, this review also
describes a number of non-radioactive assays that can serve as alternatives to radioactive
assays. Wherever possible, the review will compare the sensitivity of the radioactive and
non-radioactive assays.
Introduction V
CELL DEATH by Andrew H. Wyllie
Over the past five or six years there has been a near-exponential increase in publications
on apoptosis. Around 30 new molecules have been discovered whose known functions
are exclusively to do with the initiation or regulation of apoptosis. A further 20 molecules
at least, although already associated with important roles in signalling or DNA replica-
tion, transcription or repair, have been recognised as affecting the regulation of apopto-
sis. This article is dedicated to young scientists thinking of entering this exploding area of
biology, and to those more mature ones who happened to be looking elsewhere when the
blast reached them, and consequently are in need of a rapid introduction to the present
state of affairs.
The term apoptosis first appeared in the biomedical literature in 1972, to delineate a
structurally-distinctive mode of cell death responsible for cell loss within living tissues1.
The cardinal morphological features are cell shrinkage, accompanied by transient but
violent bubbling and blebbing from the surface, and culminating in separation of the cell
into a cluster of membrane-bounded bodies. Organellar structure is usually preserved
intact, but the nucleus undergoes a characteristic condensation of chromatin, initiated at
sublamellar foci and often extending to generate toroidal or cap-like, densely heterochro-
matic regions. Changes in several cell surface molecules also ensure that, in tissues, apop-
totic cells are immediately recognised and phagocytosed by their neighbours. The result is
that many cells can be deleted from tissues in a relatively short time with little to show for
it in conventional microscopic sections.
This remarkable process is responsible for cell death in development, normal tissue turn-
over, atrophy induced by endocrine and other stimuli, negative selection in the immune
system, and a substantial proportion of T-cell killing. It also accounts for many cell deaths
following exposure to cytotoxic compounds, hypoxia or viral infection. It is a major fac-
tor in the cell kinetics of tumors, both growing and regressing. Many cancer therapeutic
agents exert their effects through initiation of apoptosis, and even the process of carcino-
genesis itself seems sometimes to depend upon a selective, critical failure of apoptosis
that permits the survival of cells after mutagenic DNA damage. Apoptosis probably con-
tributes to many chronic degenerative processes, including Alzheimers disease, Parkin-
sons disease and heart failure. So how does it work?
Molecular genetic studies on the hard-wired developmental cell death programme of the
nematode Caenorhabditis elegans led to discovery of a set of proteins, widely represented
by homologues in other species, and responsible for turning on or off the final commit-
ment to death2. In the nematode these proteins include the products of the ced3 and ced4
genes (which initiate cell suicide), ced9 (which prevents it) and a series of some seven
genes involved in recognition and phagocytosis of the doomed cell.
CED3 is the prototype of a family of around a dozen mammalian proteases, called

caspases because of the obligatory cysteine in their active site and their predilection for
cutting adjacent to aspartate residues. Mammalian caspases appear to constitute an auto-
catalytic cascade, some members (notably caspase 8 or FLICE) being apical and more
susceptible to modification by endogenous regulatory proteins, whilst others (notably
caspase 3 also called CPP32, Yama and apopain) enact the final, irreversible commit-
ment to death. Study of caspase substrates is providing interesting insights into the ways
in which cells dismantle their structure and function. Such substrates include not sur-
prisingly cytoskeletal proteins such as actin and fodrin and the nuclear lamins, but also
an array of regulatory and chaperone-like proteins whose function is altered by cleavage
in subtle and suggestive ways3. A recent example is the nuclease chaperone ICAD, whose
cleavage permits nuclear entry by a distinctive apoptosis nuclease responsible for chro-
matin cleavage to oligonucleosome fragments4.
VI Apoptosis, Cell Death, and Cell Proliferation Manual

Caspases appear to be present in most if not all cells in inactive pro-enzyme form, await-
ing activation by cleavage. One of the killing mechanisms of cytotoxic T cells is a protease,
granzyme B, that is delivered to the target cell by the T cell granules and triggers these
latent pro-enzymes. There are endogenous triggers also, and the first to be discovered
the C. elegans CED4 protein and its mammalian homologue is particularly intriguing
because of its mitochondrial origin5. Thus CED4 could be the signal that initiates apop-
tosis under conditions of shut-down of cellular energy metabolism, or when there is a
critical level of cell injury affecting mitochondrial respiration. In this way CED4 may act
as the link between agents long known to be associated with mitochondrial injury, such
as calcium and reactive oxygen species, and the initiation of apoptosis.
A second mitochondrial protein of enormous significance in apoptosis is BCL2, a mam-

malian homologue of the nematode CED9 protein. BCL2 has the tertiary structure of a
bacterial pore-forming protein, and inserts into the outer membrane of mitochondria. It
abrogates apoptosis, probably through binding CED4 and another protein BAX, with
which it forms heterodimers and which, like CED4, is also a killer protein6. Both BCL2
and BAX have several structurally and functionally similar homologues and some of this
family at least also tap into other cell membranes such as the outer nuclear membrane
and the endoplasmic reticulum.
So are there other sources of death transducers, activating the caspase cascade because of
injury to or signals arising in other parts of the cell than mitochondria? There are already
examples that show that the answer is yes. Thus, the oncosuppressor protein p53 is acti-
vated following some types of DNA damage and can trigger apoptosis. One way but
only one of several whereby this happens is through transcriptional activation of BAX7.
The second messenger ceramide, a product of membrane-linked acid sphingomyelinase
activation, may act as a signal for plasma membrane damage8. And a powerful caspase-
activating system is mediated by cytokine receptors of the tumor necrosis factor family,
notably fas/apo-1/CD95, TNF receptor I, and others. These receptors, on receiving a
death stimulus from binding their ligand, initiate a series of protein-protein interactions,
building a complex (the death initiating signalling complex or DISC) which eventually
recruits and activates caspase 89.
These mechanisms for coupling cell injury to apoptosis have mostly depended on activa-
tion of pre-formed proteins. Apoptosis can also be initiated (and forestalled) by tran-
scriptional mechanisms, although rather little is known about most of them. An
outstanding example is the Drosophila gene reaper, transcriptionally activated around
two hours prior to developmental and injury-induced deaths in this organism. Droso-
phila apoptosis can occur without reaper transactivation, but requires very substantially
enhanced stimuli, suggesting that reaper adjusts a threshold for apoptosis initiation10.
Another gene whose transcription can initiate death is the familiar immediate early gene
c-myc11. Transcriptional activation of c-myc initiates entry into DNA synthesis and is
required for sustained re-entry in repeated cell cycles, but c-myc activation in the absence
of concurrent cytokine support triggers apoptosis. This can also be interpreted as a
threshold regulatory effect: c-myc expression increases the cellular requirement for
survival factors such as IGF-1.
Impressive confirmation of the significance of these pathways to apoptosis is available

from study of transforming viruses. These are hardened survivors in the labyrinth of cell
regulation, and have found keys to allow escape from cell death in a variety of ways. Thus
the transforming papovavirus SV40, adenovirus type 12, Human Papilloma Virus type 16
and Epstein-Barr Virus all have proteins that inactivate apoptosis through inactivation of
p53 or binding of BAX12. Even lytic viruses possess mechanisms to postpone death, such
as the cowpox crmA serpin protein and the baculovirus p35 protein, which are caspase
inhibitors.
Introduction VII
So far so good: there are transcriptional and non-transcriptional pathways for activation
of apoptosis, and they play through common effector events mediated by caspases and
regulated by members of the BCL2 family. Underlying this simple scheme, however, is an
extraordinary complexity. Thus, inactivation of fas signalling appears to neuter the abil-
ity of both c-myc and p53 to initiate apoptosis13,14. Maybe fas signalling is yet another
example of threshold regulation. New proteins have been discovered that are recruited
to the DISC but appear to inhibit rather than activate death15, some of them of viral ori-
gin. Many of the proteins mentioned above have alternative splice variants that have
opposite effects. And we still have little idea of the relevance of intracellular location or of
cell lineage to the activity of most of the apoptosis proteins. Susceptibility to apoptosis
can be influenced by many other gene products, including oncoproteins such as RAS and
ABL16, but in some cases a single oncoprotein may either increase or decrease susceptibil-
ity depending on the context. Perhaps it is not surprising that a cellular function as
important and irreversible as death should be subject to a huge range of coarse and fine
controls. The reagents and protocols in this book should help unravel these.
Andrew H. Wyllie FRS,

Professor of Experimental Pathology,
Sir Alastair Currie CRC Laboratories, University Medical School,
Edinburgh, Scotland
References
1. Kerr, J. R. F., Wyllie, A. H., Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging
implications in tissue kinetics. Br. J. Cancer 26, 239257.
2. Hengartner, M. O., Horvitz, H. R. (1994) The ins and outs of programmed cell death during C. elegans develop-
ment. Phil. Trans. R. Soc. Lond. B 345, 243248.
3. Thornberry, N.A. (1997) The caspase family of cysteine proteases. Brit. Med. Bull. 53, 478490.
4. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S. (1998) A caspase-activated DNAse
that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 4350.
5. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., Wang, X. (1997) Apaf-1, a human protein homologous to C. elegans
CED-4, participates in cytochrome c-dependent activation of caspase 3. Cell 90, 405413.
6. Oltvai, Z. N., Milliman, C. L., Korsmeyer, S. J. (1993) Bcl-2 heterodimerises in vivo with a conserved homo-
logue BAX, that accelerates programmed cell death. Cell 74, 609619.
7. Miyashita, T., Reed, J. C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax
gene. Cell 80, 293299.
8. Jarvis, D. W., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., Grant, S. (1994) Induction of apoptotic
DNA degradation and cell death by activation of the sphingomyelin pathway. Proc. Natl. Acad. Sci. USA 91,
7377.
9. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., ORourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D.,
Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., Dixit, V. M. (1996) FLICE, a novel FADD-homo-
logous ICE/CED-3-like protease, is recruited to the CD 95 (FAS/APO-1) death-inducing signalling complex.
Cell 85, 817827.
10. White, K., Tahaoglu, E., Steller, H. (1996) Cell killing by the Drosophila gene reaper. Science 271, 805807.
11. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Land, H., Brooks, M., Littlewood, T., Waters, C., Hancock, D. (1992)
Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119128.
12. Young, L. S., Dawson, C. W., Eliopoulos, A. G. (1997) Viruses and apoptosis. Brit. Med. Bull. 53, 509521.
13. Hueber, A. O., Zornig, M., Lyon, D., Suda, T., Nagata, S., Evan, G. I. (1997) Requirement for the CD95 receptor-
ligand pathway in c-myc-induced apoptosis. Science 278, 13051309.
14. Krammer, P. H. (1997) The tumor strikes back: new data on expression of the CD-95 (APO-1/Fas) receptor/
ligand system may cause paradigm changes in our view on drug treatment and tumor immunology. Cell Death
and Differentiation 4, 362364.
15. Irmler, M., Thorne, M., Hanne, M., Schneider, P., Hofmann, B., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K.,
Mattmann, C., Rimoldi, D., French, L. E., Tschopp, J. (1997) Inhibition of death receptor signals by cellular FLIP.
Nature 388, 190195.
16. Evan, G. (1997) A question of DNA repair. Nature 387, 450.
VIII Apoptosis, Cell Death, and Cell Proliferation Manual

A
Cell Death-
Apoptosis and Necrosis
Introduction
Terminology of cell death
1 Introduction
1.1 Terminology of cell death
Cell death can occur by either of two distinct1,2 mechanisms, necrosis or apoptosis. In
addition, certain chemical compounds and cells are said to be cytotoxic to the cell, that is,
to cause its death.
Someone new to the field might ask, whats the difference between these terms? To clear
A
up any possible confusion, we start with some basic definitions.
Necrosis and apoptosis

The two mechanisms of cell death may briefly be defined:
Necrosis (accidental cell death) is the pathological process which occurs when cells are
exposed to a serious physical or chemical insult.
Apoptosis (normal or programmed cell death) is the physiological process by which

unwanted or useless cells are eliminated during development and other normal biological
processes.
Cytotoxicity
Cytotoxicity is the cell-killing property of a chemical compound (such as a food, cos-
metic, or pharmaceutical) or a mediator cell (cytotoxic T cell). In contrast to necrosis and
apoptosis, the term cytotoxicity does not indicate a specific cellular death mechanism.
For example, cell-mediated cytotoxicity (that is, cell death mediated by either cytotoxic
T lymphocytes [CTL] or natural killer [NK] cells) combines some aspects of both necro-
sis and apoptosis3,4.
Figure 1: Illustration of the morphological features of necrosis and apoptosis.
2 Apoptosis, Cell Death, and Cell Proliferation Manual

Introduction
Differences between necrosis and apoptosis
1.2 Differences between necrosis and apoptosis

There are many observable morphological (Figure 1, Table 1) and biochemical differ-
ences (Table 1) between necrosis and apoptosis2.
Necrosis occurs when cells are exposed to extreme variance from physiological conditions
(e.g., hypothermia, hypoxia) which may result in damage to the plasma membrane.
Under physiological conditions direct damage to the plasma membrane is evoked by
agents like complement and lytic viruses.
A
Necrosis begins with an impairment of the cells ability to maintain homeostasis, leading
to an influx of water and extracellular ions. Intracellular organelles, most notably the
mitochondria, and the entire cell swell and rupture (cell lysis). Due to the ultimate break-
down of the plasma membrane, the cytoplasmic contents including lysosomal enzymes
are released into the extracellular fluid. Therefore, in vivo, necrotic cell death is often
associated with extensive tissue damage resulting in an intense inflammatory response5.
Apoptosis, in contrast, is a mode of cell death that occurs under normal physiological
conditions and the cell is an active participant in its own demise (cellular suicide). It is
most often found during normal cell turnover and tissue homeostasis, embryogenesis,
induction and maintenance of immune tolerance, development of the nervous system
and endocrine-dependent tissue atrophy.
Cells undergoing apoptosis show characteristic morphological and biochemical features6.

These features include chromatin aggregation, nuclear and cytoplasmic condensation,
partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies)
which contain ribosomes, morphologically intact mitochondria and nuclear material. In
vivo, these apoptotic bodies are rapidly recognized and phagocytized by either macroph-
ages or adjacent epithelial cells7. Due to this efficient mechanism for the removal of apo-
ptotic cells in vivo no inflammatory response is elicited. In vitro, the apoptotic bodies as
well as the remaining cell fragments ultimately swell and finally lyse. This terminal phase
of in vitro cell death has been termed secondary necrosis (Figure 1).
Cell Death Apoptosis and Necrosis 3

Introduction
Differences between necrosis and apoptosis
Necrosis Apoptosis
Morphological features
Loss of membrane integrity Membrane blebbing, but no loss of integrity
Aggregation of chromatin at the nuclear membrane
Begins with swelling of cytoplasm and mitochondria Begins with shrinking of cytoplasm and condensation of
nucleus
Ends with total cell lysis Ends with fragmentation of cell into smaller bodies
No vesicle formation, complete lysis Formation of membrane bound vesicles (apoptotic bodies)
Disintegration (swelling) of organelles Mitochondria become leaky due to pore formation involving
A
proteins of the bcl-2 family.
Biochemical features
Loss of regulation of ion homeostasis Tightly regulated process involving activation and
enzymatic steps
No energy requirement (passive process, also occurs Energy (ATP)-dependent (active process, does not occur at
at 4C) 4C)
Random digestion of DNA (smear of DNA after agarose gel Non-random mono- and oligonucleosomal length frag-
electrophoresis) mentation of DNA (Ladder pattern after agarose gel
electrophoresis)
Postlytic DNA fragmentation (= late event of death) Prelytic DNA fragmentation
Release of various factors (cytochrome C, AIF) into
cytoplasm by mitochondria
Activation of caspase cascade
Alterations in membrane asymmetry (i.e., translocation of
phosphatidylserine from the cytoplasmic to the extracellular
side of the membrane)
Physiological significance
Affects groups of contiguous cells Affects individual cells
Evoked by non-physiological disturbances (complement Induced by physiological stimuli (lack of growth
attack, lytic viruses, hypothermia, hypoxia, ischemica, factors, changes in hormonal environment)
metabolic poisons)
Phagocytosis by macrophages Phagocytosis by adjacent cells or macrophages
Significant inflammatory response No inflammatory response
Table 1: Differential features and significance of necrosis and apoptosis.

Introduction
Apoptotic Pathways
1.3 Apoptotic Pathways

Scientists now recognize that most, if not all, physiological cell death occurs by apoptosis,
and that alteration of apoptosis may result in a variety of malignant disorders. Conse-
quently, in the last few years, interest in apoptosis has increased greatly. Great progress
has been made in the understanding of the basic mechanisms of apoptosis and the gene
products involved (Figure 2 below, Table 20, see Appendix, page 123).
Figure 2: Apoptotic pathways. This apoptotic pathways chart represents a compendium of information on
different cell lines, from various sources. As the dynamic field of apoptosis changes, the information shown here
will likely change. Table 20 in the Appendix, page 123 contains a list of sources that can be consulted for more
information about the items on this chart. Have a look on our website www.roche-applied-science/apoptosis
to find this chart under Scientific Information-Apoptotic Pathways and click on the name of a molecule to get
information about its function.
Key elements of the apoptotic pathway include:

Death receptors
Apoptosis has been found to be induced via the stimulation of several different cell sur-
face receptors in association with caspase activation. For example, the CD95 (APO-1, Fas)
receptor ligand system is a critical mediator of several physiological and pathophysiologi-
cal processes, including homeostasis of the peripheral lymphoid compartment and CTL-
mediated target cell killing. Upon cross-linking by ligand or agonist antibody, the Fas
receptor initiates a signal transduction cascade which leads to caspase-dependent pro-
grammed cell death.
Membrane alterations
In the early stages of apoptosis, changes occur at the cell surface and plasma membrane.
One of these plasma membrane alterations is the translocation of phosphatidylserine
(PS) from the inner side of the plasma membrane to the outer layer, by which PS
becomes exposed at the external surface of the cell.

Introduction
Apoptotic Pathways
Protease cascade
Signals leading to the activation of a family of intracellular cysteine proteases, the
caspases, (Cysteinyl-aspartate-specific proteinases) play a pivotal role in the initiation
and execution of apoptosis induced by various stimuli. Different members of caspases in
mammalian cells have been identified. Among the best-characterized caspases is
caspase-1 or ICE (Interleukin-1-Converting Enzyme), which was originally identified as
a cysteine protease responsible for the processing of interleukin 1.
Mitochondrial changes
A
Mitochondrial physiology is disrupted in cells undergoing either apoptosis or necrosis.
During apoptosis mitochondrial permeability is altered and apoptosis specific protease
activators are released from mitochondria. Specifically, the discontinuity of the outer
mitochondrial membrane results in the redistribution of cytochrome C to the cytosol fol-
lowed by subsequent depolarization of the inner mitochondrial membrane. Cytochrome
C (Apaf-2) release further promotes caspase activation by binding to Apaf-1 and there-
fore activating Apaf-3 (caspase 9). AIF (apoptosis inducing factor), released in the cyto-
plasm, has proteolytic activity and is by itself sufficient to induce apoptosis.
DNA fragmentation
The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an
irreversible event that commits the cell to die and occurs before changes in plasma mem-
brane permeability (prelytic DNA fragmentation). In many systems, this DNA fragmen-
tation has been shown to result from activation of an endogenous Ca2+ and Mg2+-
dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites located
between nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA
fragments.
Note: For more information about the elements of the pathways as well as synonyms and
abbreviations, please see Table 20 in the Appendix, page 123.

Apoptosis Assay Methods
2 Apoptosis Assay Methods

Originally, to study both forms of cell death, necrosis and apoptosis, cytotoxicity assays
were used. These assays were principally of two types:
Radioactive and non-radioactive assays that measure increases in plasma membrane

permeability, since dying cells become leaky.
Colorimetric assays that measure reduction in the metabolic activity of mitochondria;
A
mitochondria in dead cells cannot metabolize dyes, while mitochondria in live cells
can.
Note: For a detailed discussion of both types of cytotoxicity assay, see Section A 3, beginning
on page 58 of this guide.
However, as more information on apoptosis became available, researchers realized that

both types of cytotoxicity assays vastly underestimated the extent and timing of apop-
tosis. For instance, early phases of apoptosis do not affect membrane permeability, nor do
they alter mitochondrial activity. Although the cytotoxicity assays might be suitable for
detecting the later stages of apoptosis, other assays were needed to detect the early events
of apoptosis.
In concert with increased understanding of the physiological events that occur during
apoptosis, a number of assay methods have been developed for its detection. For instance,
these assays can measure one of the following apoptotic parameters:
Fragmentation of DNA in populations of cells or in individual cells, in which apop-

totic DNA breaks into different length pieces.
Alterations in membrane asymmetry. Phosphatidylserine translocates from the cyto-

plasmic to the extracellular side of the cell membrane.
Activation of apoptotic caspases. This family of proteases sets off a cascade of events
that disable a multitude of cell functions.
Release of cytochrome C and AIF into cytoplasm by mitochondria.
For practical reasons, we have divided this chapter into two broad categories: assays that
measure apoptosis in cell populations (Section A 2.1 of this guide) and assays that mea-
sure apoptosis in individual cells (Section A 2.2 of this guide).
For discussions of particular assays, turn to the pages indicated in the product selection
guide (Figure 3).

Start
Are you studying
A
Apoptosis Cytotoxicity*
Are you studying
cell populations single cells
Which techniques
do you perform?
Do you analyze data by
FACS microscopy
ELISA
DNA fragmentation, quantitative
Cell Death Detection 11 774 425 001
ELISAPLUS 11 920 685 001
Cell Death Detection ELISA 11 544 675 001
Fluorescence microplate Reader

Caspase activity, quantitative
Caspase 3 Activity Assay 12 012 952 001
Homogeneous Caspases 03 005 372 001 DNA fragmentation
Assay, fluorimetric 12 236 869 001 In Situ Cell Death Detection Kit, 12 156 792 001
TMR red
InSitu Cell Death Detection Kit, 11 684 795 001
Fluorescein
Western blotting
ICE protease activity Membrane modification
Anti-PARP 11 835 238 001 Annexin-V-Alexa 568 03 703 126 001
Annexin-V-FLUOS 11 828 681 001
Annexin-V-FLUOS Staining Kit 11 858 777 001
Gel electrophoresis 11 988 549 001
DNA fragmentation, qualitative
Apoptotic DNA Ladder Kit 11 835 246 001
Proteolytic activity
M30 CytoDEATH, Fluorescein 12 156 857 001
8 Figure 3: Product Selection Guide Apoptosis, Cell Death, and Cell Proliferation Manual
Cellular DNA Fragmentation ELISA 11 585 045 001
Cell Proliferation Reagent WST-1 11 644 807 001
Cell Proliferation Kit II (XTT) 11 465 015 001
Cell Proliferation Kit I (MTT) 11 465 007 001
yes
Do your cells proliferate in vitro?
no
Cytotoxicity Detection Kit (LDH) 11 644 793 001
cytotoxitic
A
substances
Are you studying
cell-mediated
cytotoxicity Cellular DNA Fragmentation ELISA 11 585 045 001
yes
Do your cells proliferate in vitro?
no
Cytotoxicity Detection Kit (LDH) 11 644 793 001L
Are you studying
cell cultures, tissue sections

blood cells,
etc.
DNA fragmentation
In Situ Cell Death Detection Kit, 12 156 792 001
Do you use TMR red
fluorescence light InSitu Cell Death Detection Kit, 11 684 795 001
microscopy microscopy Fluorescein
In Situ Cell Death Detection Kit, POD 11 684 817 001
In Situ Cell Death Detection Kit, AP 11 684 809 001
DNA fragmentation Proteolytic activity

In Situ Cell Death Detection Kit, POD 11 684 817 001 M30 CytoDEATH 12 140 322 001
In Situ Cell Death Detection Kit, AP 11 684 809 001 12 140 349 001
M30 CytoDEATH, Fluorescein 12 156 857 001
Membrane modification
Annexin-V-Biotin 10 828 690 001
Proteolytic activity
M30 CytoDEATH 12 140 322 001
12 140 349 001
Methods for studying apoptosis in cell populations
2.1 Methods for studying apoptosis in cell populations

A number of methods have now been developed to study apoptosis in cell populations.
We focus on two key apoptotic events in the cell:
Apoptosis and cell mediated cytotoxicity are characterized by cleavage of the genomic
DNA into discrete fragments prior to membrane disintegration. Because DNA cleav-
age is a hallmark for apoptosis, assays which measure prelytic DNA fragmentation are
especially attractive for the determination of apoptotic cell death. The DNA fragments
may be assayed in either of two ways:
A
As ladders (with the 180 bp multiples as rungs of the ladder) derived from pop-
ulations of cells, e.g., with the Apoptotic DNA Ladder Kit (described on page 13 of
this guide).
By quantification of histone complexed DNA fragments with an ELISA (described
on page 15 of this guide).
Further, researchers discovered that proteases were involved in the early stages of apo-
ptosis. The appearance of these caspases sets off a cascade of events that disable a mul-
titude of cell functions. Caspase activation can be analyzed in different ways:
By an in vitro enzyme assay. Activity of a specific caspase, for instance caspase 3, can
be determined in cellular lysates by capturing of the caspase and measuring pro-
teolytic cleavage of a suitable substrate (described on page 22 of this guide).
By detection of cleavage of an in vivo caspase substrate. For instance caspase 3 is
activated during early stages (as shown in Figure 2). Its substrate PARP (Poly-ADP-
Ribose-Polymerase) and the cleaved fragments can be detected with the anti PARP
antibody (described on page 27 of this guide).
If youre just starting out in the field, however, it may be difficult to decide how best to
assay apoptosis in your system. Thus, in the following sections, we will describe details of
each of these apoptosis assays.
2.1.1 Assays that measure DNA fragmentation
The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an

irreversible event that commits the cell to die. In many systems, this DNA fragmentation
has been shown to result from activation of an endogenous Ca2+ and Mg2+-dependent
nuclear endonuclease. This enzyme selectively cleaves DNA at sites located between
nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA frag-
ments (Figure 4). These DNA fragments reveal, upon agarose gel electrophoresis, a dis-
tinctive ladder pattern consisting of multiples of an approximately 180 bp subunit8.
Radioactive as well as non-radioactive methods to detect and quantify DNA fragmenta-

tion in cell populations have been developed. In general, these methods are based on the
detection and/or quantification of either low molecular weight (LMW) DNA which is
increased in apoptotic cells or high molecular weight (HMW) DNA which is reduced in
apoptotic cells (Figure 5). The underlying principle of these methods is that DNA, which
has undergone extensive double-stranded fragmentation (LMW DNA) may easily be sep-
arated from very large, chromosomal length DNA (HMW DNA), e.g., by centrifugation
and filtration.

A
Figure 4: The biochemistry of DNA fragmentation and the appearance of the DNA ladder.
For the quantification of DNA fragmentation, most methods involve a step in which the
DNA of the cells has to be labeled: Prior to the addition of the cell death-inducing agent
or of the effector cells, the (target) cells are incubated either with the [3H]-thymidine
([3H]-dT) isotope or the nucleotide analog 5-bromo-2-deoxyuridine (BrdU). During
DNA synthesis (DNA replication) these modified nucleotides are incorporated into the
genomic DNA. Subsequently, those labeled cells are incubated with cell death-inducing
agents or effector cells and the labeled DNA is either fragmented or retained in the cell
nucleus. Finally each type of DNA (HMW and LMW) is quantitated. Because the labeling
of the cellular DNA has to be done prior to the induction of cell death, this labeling is also
called prelabeling.
The prelabeling of one cell population (e.g., the target cells) allows the behavior of the
labeled cells to be traced specifically when different cell populations are mixed.
Note: Because cell-mediated cytotoxicity (CMT) proceeds, at least in part, by apoptotic

mechanisms, the DNA fragmentation assay may also be used as a CMT assay.
In a study of cell-mediated cytotoxicity the target cell population is labeled before the
effector cells (e.g., CTL) are added. Subsequently, due to pore formation in the target cell
plasma membrane, the fragmented LMW DNA is released from the cytoplasm of the tar-
get cell into the culture supernatant (Table 2). The cytotoxic potential of the effector cells
is measured by quantification of the label released from the damaged target cells.
Because this metabolic prelabeling of the genomic DNA requires DNA synthesis, only
cells proliferating in vitro (e.g., cell lines) may be labeled in this way; cells which do not
proliferate in vitro (e.g., primary cell cultures, tumor cells ex vivo) do not replicate their
DNA and therefore, do not incorporate labeled nucleotides (see also Section A 3.2.1.
Cellular DNA Fragmentation ELISA page 64).

To detect fragmented DNA in cells which do not replicate in vitro, the DNA has to be iso-
lated and analyzed by agarose gel electrophoresis (DNA ladder assay, Figure 6, see also
Figure 4). Roche Applied Science offers a kit, the Apoptotic DNA Ladder Kit, that simpli-
fies this assay.
A
Figure 5: Compartmentalization of HMW and LMW DNA in normal and apoptotic cells.
( = decreasing, = increasing)
Apoptosis Cell mediated Note: In the early phases of apopto-

cytotoxicity sis, no DNA is released into the
supernatant (prelytic DNA frag-
Compartment HMW LMW HMW LMW
mentation). However, in vitro, the
DNA DNA DNA DNA
apoptotic cells will lyse (secondary
Nucleus + + + + necrosis). Therefore, LMW DNA
Cytoplasm + + is found in the supernatant late in
apoptosis.
Supernatant +
Table 2: Distribution of HMW and LMW DNA in cells under-

going apoptosis and target cells during cell mediated cytotoxicity.
An alternative method which circumvents the isolation and electrophoretic analysis of

DNA is the immunological detection of LMW DNA (histone-complexed DNA frag-
ments) by an immunoassay (Cell Death Detection ELISAPLUS, see page 15).
This nonradioactive immunoassay, offered by Roche Applied Science quantifies that hall-
mark of apoptosis. The Cell Death Detection ELISAPLUS has been designed for relative
quantification of DNA fragmentation in cells which do not proliferate in vitro (since the
kit requires no prelabeling of the cells). This kit measures the enrichment of histone-
complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of apop-
totic cells.
Each of the methods to detect and measure apoptosis has its advantages and limitations.
Because the cellular mechanisms that result in apoptosis are complex, most published
methods cannot by themselves detect apoptosis unambiguously.
To ensure that the mode of cell death in the individual cell system or experiment is apop-
totic, one also has to consider other criteria like the cellular morphology. Morphologic
criteria for apoptotic cell death include, for example, chromatin condensation with
aggregation along the nuclear envelope and plasma membrane blebbing followed by sep-
aration into small, apoptotic bodies. When internucleosomal DNA fragmentation is
accompanied by these morphological features it provides an additional useful criterion to
define cell death as apoptotic.

Apoptotic DNA Ladder Kit

Cat. No. 11 835 246 001 20 tests
Type DNA purification kit

Useful for Preparation of apoptotic DNA fragments for display on electrophoretic gels
Samples Whole blood or cells in culture
Method Cell lysis, followed by binding of cellular DNA on glass fiber, removal of
impurities, and DNA recovery
Time DNA preparation: < 20 min (after induction of apoptosis)
Significance of kit: This kit offers the easiest way to isolate apoptotic DNA fragments for
DNA ladder analysis. The purification method outlined in the kit is much faster than
other DNA purification methods (e.g., phenol/chloroform extraction, DNA precipita-
tion). Purified DNA may be mixed directly with gel loading buffer and analyzed on an
agarose gel.
A
Test principle: Apoptotic DNA binds quickly to glass fiber fleece in the presence of a
chaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities are
washed off the fleece, the DNA is released from the fleece with a low salt buffer. The pro-
cedure (see Flow Chart 1) involves:
Incubating an aliquot of apoptotic cells with an equal volume of binding/lysis buffer.

After the incubation, the lysed sample is poured into a filter tube containing glass
fiber fleece.
Using centrifugation to separate the DNA in the lysate (which binds to the glass fiber
fleece) from unbound lysate components (which flow through the fleece into a
collection tube).
Washing the bound DNA twice.
Eluting the purified DNA from the filter tube and collecting it by centrifugation.
Sample Preparation
Treat sample with apoptosis-inducing agent (124 h)
Incubate treated sample with binding/lysis buffer (10 min, RT)
Mix isopropanol with sample and pipette mixture into filter tube
Centrifuge tube assembly (8000 rpm) and discard the flow-through (1 min, RT)
Add wash buffer to the filter tube, then centrifuge as before (1 min, RT)
Repeat the wash step, then add a final high speed spin (13,000 rpm) (1 min, then 10 sec, RT)
Insert the filter tube into a 1.5 ml centrifuge tube, and add warm elution buffer to the filter tube
Collect the eluted DNA by centrifugation (1 min, RT)

DNA Ladder Assay

Mix the eluted DNA sample with gel loading buffer
Apply sample to a 1% agarose gel which contains ethidium bromide
Run the gel in TBE (Tris-borate EDTA) buffer at 75 V (1.5 h, RT)
Place the gel on a UV light box to visualize the DNA ladder pattern
Flow Chart 1: Assay procedure, Apoptotic DNA Ladder Kit.
A Sample size: 200 300 l whole blood or cell suspension (for instance, 2 x 106 cells). The
kit allows simultaneous processing of multiple samples.
Yield
Sample Sample volume Yield of purified
DNA
Whole blood (human) 200 l 36 g
Cultured cells (K562) 2 x 106 cells 10 g
Specificity: Only nucleic acid will bind to the glass fiber filters under the conditions out-
lined in the kit. Salts, proteins, and other cellular components do not bind.
Kit contents
1. Nucleic acid binding/lysis buffer, ready-to-use
2. Washing buffer (ethanol to be added before use)
3. Elution buffer, ready-to-use
4. Glass fiber filter tubes, 700 l capacity
5. Polypropylene collection tubes, 2 ml (for washes)
6. Positive control, apoptotic U937 cells, lyophilized
Typical results: see Figure 6
Other applications: For more examples of how the Apoptotic DNA Ladder Kit can be
used in the lab, see Appendix, pages 128129.
Figure 6: DNA ladder assayed with the Apoptotic DNA Ladder Kit
M = Size marker
= Control cells without camptothecin
+ = Cells treated with camptothecin
C = Positive control from the kit

Cell Death Detection ELISAPLUS

Cat. No. 11 774 425 001 96 tests
Cat. No. 11 920 685 001 10 x 96 tests
Type One-step sandwich ELISA, colorimetric

Useful for Relative quantification of apoptosis without cell labeling;
differentiating apoptosis from necrosis
Samples Cell lysates, cell culture supernatants, serum, or plasma
Method
Time
Cell lysis, followed by immunochemical determination of histone-com-
plexed DNA fragments in a microplate well (Note: For detection of necrosis,
histone-complexed DNA fragments are detected directly in the culture superna-
tant, without cell lysis)
Approx. 3 h (after induction of apoptosis)
Significance of kit: Use this kit for relative quantification of histone-complexed DNA
A
fragments (mono- and oligonucleosomes) out of the cytoplasm of cells after the induc-
tion of apoptosis or when released from necrotic cells. Since the assay does not require
prelabeling of cells, it can detect internucleosomal degradation of genomic DNA during
apoptosis even in cells that do not proliferate in vitro (for example, freshly isolated tumor
cells). The antibodies used in the assay are not species-specific, so the kit may be used to
assay cells from a wide variety of species (see Other applications in this article).
Test principle: The assay uses an one-step sandwich immunoassay to detect nucleosomes.
The procedure (Figure 7 and Flow Chart 2) involves:
Incubating cells in a microplate well (for instance, 104 human cells in 200 l culture)
with an agent that induces cell death (for example, campothecin (CAM)). After the
incubation, the cells are pelleted by centrifugation and the supernatant is (containing
DNA from necrotic cells that leaked through the membrane during incubation) dis-
carded.
Resuspending and incubating cells in lysis buffer. After lysis, intact nuclei are pelleted
by centrifugation.
Transferring an aliquot of the supernatant to a streptavidin-coated well of a

microplate.
Binding nucleosomes in the supernatant with two monoclonal antibodies, anti-

histone (biotin-labeled) and anti-DNA (peroxidase-conjugated). Antibody-nucleo-
some complexes are bound to the microplate by the streptavidin.
Washing the immobilized antibody-histone complexes three times to remove cell

components that are not immunoreactive.
Incubating sample with peroxidase substrate (ABTS).
Determining the amount of colored product (and thus, of immobilized antibody-

histone complexes) spectrophotometrically.


After incubating cells with an apoptosis-inducing agent,
pellet the cells by centrifugation.
Discard the supernatant, which may contain necrotic DNA
that leaked through the membrane during the incubation.
Incubate cells with lysis buffer.
A
Pellet the intact nuclei by centrifugation. Take an aliquot of
the supernatant (cell lysate) and determine the amount of
apoptotic nucleosomes present.
Figure 7: How the Cell Death Detection ELISAPLUS works.

Panel A: Sample preparation
Panel B: ELISA
Treat cells with apoptosis-inducing agent in the well of a microplate (124 h, 37C)
Centrifuge microplate (200 x g ) and remove supernatant (10 min, RT)
Incubate treated cells with lysis buffer (30 min, RT)
Repeat microplate centrifugation (200 x g ) (10 min, RT)
Transfer aliquot of supernatant (lysate) to streptavidin-coated microplate
Incubate supernatant with immunoreagent (containing anti-histone and anti-DNA) (2 h, RT)
Wash microplate wells three times with incubation buffer at RT
Add substrate solution to wells and incubate (approx. 15 min, RT)
Measure absorbance at 405 nm
Flow Chart 2: Assay procedure, Cell Death Detection ELISAPLUS.
Sensitivity: In a model system, nucleosomes were detectable in as few as 600 campothe-

cin-induced U937 cells (Figure 8). However, the lower limit for detecting dying/dead cells
in a particular sample varies with the kinetics of the apoptotic process, the cytotoxic
agent used, and the number of affected cells in the total cell population.

Figure 8: Sensitivity of Cell Death Detection

ELISAPLUS.
Different cell concentrations of U937 cells were incu-
bated with camptothecin (CAM) (2 g/ml) or without
CAM for 4 h at 37C. 20 l of cell culture supernatant
A
and cell lysates were analyzed in the ELISA. Substrate
reaction time: 10 min. Lysate with CAM, Lysate
without CAM, Supernatant with CAM, Supernatant
without CAM.
Result: The ELISA can clearly detect apoptosis-related
nucleosomes in as few as 600 cells.
Specificity: The ELISA is specific for nucleosomes containing single- or double-stranded

DNA (Figure 9). It is not species specific.
Figure 9: Dose-response experiment analyzed by

the Cell Death Detection ELISAPLUS.
U937 cells (104 cells/well, in 200 l) were incubated with
different concentrations of camptothecin (CAM) for 4 h
at 37C. Before and after lysis, cells were centrifuged and
a 20 l aliquot of the supernatant was analyzed with the
Cell Death Detection ELISAPUS. Results were plotted as
dose vs. response. Substrate reaction time: 5 min.
Lysate, Supernatant, Enrichment factor of the
lysate.
Result: Amounts of cytoplasmic oligonucleosomes (an
indicator of apoptosis) increase as CAM concentration
increases. Cell culture supernatants removed from the
cells after treatment (but before lysis) gave no signal,
indicating that there are no necrotic cells during the
treatment.
Can be used to assay:

Adherent cells
Cells in suspension culture
Cell culture supernatant
Lysates of cells obtained ex vivo
Serum, or plasma
Kit contents
1. Anti-histone antibody (clone H11-4), biotin-labeled
2. Anti-DNA antibody (clone M-CA-33), peroxidase-conjugated
3. DNA-histone complex (positive control)
4. Incubation buffer, ready-to-use
5. Lysis buffer, ready-to-use
6. Substrate buffer, ready-to-use
7. ABTS substrate tablets
8. Microplate modules (12 x 8 wells)
9. Adhesive plate cover
Typical results: see Figure 8 and 9
Other applications: For more examples of how the Cell Death Detection ELISAPLUS and
the Cell Death Detection ELISA can be used in the lab, see Appendix, pages 129131.

2.1.2 Assays that measure apoptosis-induced proteases (caspases)
Several caspases (see Table 20, in the Appendix, page 123) are thought to mediate very
early stages of apoptosis10. For instance, one of these, caspase 3 (CPP32) is required for
the induction of apoptosis by certain effectors [especially tumor necrosis factor and the
cytotoxic T cell ligand effector, CD95 (also called Fas)] Enari et al. (1996) , Nature 380,
723726.
These proteases cleave numerous substrates at the carboxy site of an aspartate residue. All
are synthesized as pro-enzymes; activation involves cleavage at aspartate residues that
A
could themselves be sites for the caspase family. As caspases are probably the most impor-
tant effector molecules for triggering the biochemical events which lead to apoptotic cell
death, assays for determination of caspase activation can detect apoptosis earlier than
many other commonly used methods.
The most elucidatory assay for these caspases involves western blot detection of pro-
teolytic cleavage products found in apoptotic cells. An antibody, Anti-PARP, sold by
Roche Applied Science, can be used in such an assay. The antibody can detect intact and
cleaved forms of Poly-ADP-Ribose Polymerase, a target for some caspases.
For specific and quantitative measurement of caspase activity Western blotting is not
suitable. To quantify caspase activation enzyme activity assays based on detection of
cleaved caspase substrates have been developed recently. However most of the caspase
substrates are not exclusively cleaved by a specific caspase but only preferentially, while
other members of the caspases family act on these substrates to a lower extent. Roche
Applied Science offers a caspase 3 activity assay with highest specificity by the use of an
immunosorbent enzyme assay principle.

M30 CytoDEATH*
Cat. No. 12 140 322 001 50 tests
Cat. No. 12 140 349 001 250 tests
M30 CytoDEATH, Fluorescein

Cat. No. 12 156 857 001 250 tests
A
Type Monoclonal antibody, clone M30, IgG2b, mouse
Useful for Detection of apoptosis in epithelial cells and tissues (formalin grade)
Samples Adherent cells, tissue samples (routinely fixed and paraffin-embedded tissue
sections, cryostat sections)
Method Detect apoptosis by applying the M30-antibody to fixed samples, then using
secondary detection systems. Suitable for immunohistochemistry, immuno-
cytochemistry, and flow cytometry
Time 2 h for immunofluorescence on cells, 3.5 h for staining of tissues (excluding
dewaxing)
Background: During Apoptosis, vital intracellular proteins are cleaved. The proteases
that mediate this process are called caspases (Cysteinyl-aspartic acid proteases). Caspases
are expressed as zymogenes, which are activated by different apoptosis inducers. Once
activated, a single caspase activates a cascade of caspases.
Until recently cytokeratins, in particular cytokeratin 18, were not known to be affected by
early events of apoptosis. Recently, it has been shown that the M30 antibody recognizes a
specific caspase cleavage site within cytokeratin 18 that is not detectable in native CK18 of
normal cells. Consequently, the M30 CytoDEATH antibody is a unique tool for the easy
and reliable determination of very early apoptotic events in single cells and tissue sec-
tions.
Significance of reagent: Use the M30 CytoDEATH antibody for the determination of
early apoptotic events in cells and tissue sections by detection of a specific epitope of
cytokeratin 18 that is presented after cleavage by caspases.
Test principle of M30 CytoDEATH
for formalin-embedded tissue involves:
Dewax formalin-fixed, paraffin- Add Streptavidin-POD.

embedded tissue sections.
Retrieve antigen by heating in Add substrate solution (DAB or AEC).

citric acid buffer.
Add M30 antibody. Counterstain with Harries hematoxilin.
Add Anti-Mouse-Biotin. Analyze under a light microscope.
* The M30 CytoDEATH antibody is made under a license agreement form BEKI AB/BEKI Diagnostics AB, Sweden.
for immunofluorescence and flow cytometry on cells

for M30 CytoDEATH and M30 CytoDEATH, Fluorescein:
Fix cells. Add Anti-Mouse-Ig-Fluorescein (not nec-

essary for M30 CytoDEATH, Fluorescein).
Add M30 CytoDEATH antibody Analyze under a fluorescence microscope

or M30 CytoDEATH, Fluorescein or in FACS.

Staining procedure for fluorescence microscopy and flow cytometry (FACS)
M30 CytoDEATH M30 CytoDEATH, Fluorescein

Wash cells with PBS
Fix cells in ice-cold pure methanol (20C) for 30 min
Wash cells twice with PBS/BSA
Incubate with M30 CytoDEATH working solution Incubate with M30 CytoDEATH, Fluorescein working
A
(for 30 min, at RT) solution (for 30 min, at RT)
Wash cells twice with PBS
Incubate with Anti-Mouse-Ig-Fluorescein (for 30 min,

at RT)
Wash cells twice with PBS
Analyze by fluorescence microscope or flow cytometry (dilute cells in PBS, and store the samples in the dark
until analysis).
Staining procedure for immunohistochemistry
Incubate paraffin-embedded sections (over night) at 37C
Dewax formalin-fixed, paraffin-embedded tissue sections:

2x xylol, 2x ethanol (96%), 1x ethanol (70%), 1x methanol/H2O2 (3%)
(10 min, RT). Rinse for 10 min in demineralized water
Antigen retrieval:
Prepare 500 ml of 10 mM citric acid buffer. Incubate in a microwave oven at 750 W until boiling. Place slides into
the heated citric acid solution. Incubate once more at 750 W. When solution is boiling, turn setting of microwave
oven to keep warm (about 100 W). Incubate for 15 min. Cool the slides down (for 5 min, at RT)
Rinse three times in PBS; incubate 2 min in a separate jar of PBS
Block with PBS + 1% BSA (for 10 min, at RT)
Remove blocking solution. Add M30 CytoDEATH working solution (1 h, RT)
Wash slides three times in PBS
Cover with Anti-Mouse-Biotin (for 30 min, at RT)
Cover with Streptavidin-POD (for 30 min, at RT)
Incubate slides in a freshly prepared substrate solution (DAB or AEC at RT) until a clearly visible color develops
Counterstain with hematoxilin, and mount the section
Analyze by a light microscope
Flow Chart 3: Assay procedure, M30 CytoDEATH, immunohistochemistry and cytometry.

Specificity: The M30 CytoDEATH antibody binds to a caspase-cleaved, formalin-resis-

tant epitope of the cytokeratin 18 (CK 18) cytoskeletal protein.
The immunoreactivity of the M30 CytoDEATH antibody confined to the cytoplasma of

apoptotic cells.
Antibody supplied as: Mouse monoclonal antibody (clone M30), lyophilized, stabi-
lized. Formalin grade.
Typical results: see Figures 1012.
Other applications: For more examples of how the M30 CytoDEATH and M30
CytoDEATH, Fluorescein can be used in the lab, see Appendix, pages 131132.
A
Figure 10: Detection of apoptosis in HeLa cells, treated with TNF and Actinomycin D, using
M30 CytoDEATH. Secondary detection with Anti-Mouse-Fluorescein and propidium iodide.
Figure 11: Detection of apoptosis in human colon using M30 CytoDEATH (blue filter). Secondary
detection with Anti-Mouse-Biotin, Streptavidin-POD and AEC as substrate, counterstained with hematoxilin.
Figure 12: FACS analysis of apoptosis in HeLa cells, using M30 CytoDEATH, Fluorescein.
White: untreated control cells. Red: Cells treated with TNF and Actinomycin D.

Caspase 3 Activity Assay

Cat. No. 12 012 952 001 96 tests
Type Immunosorbent enzyme assay, fluorometric

Useful for Specific, quantitative in vitro determination of caspase 3 activity
Samples Cell lysates, recombinant caspase 3 (CPP32)
Method Cell lysis, followed by capturing of caspase 3 by a specific antibody and
fluorometric determination of proteolytic cleavage of the substrate
A Time Approx. 5 h (after induction of apoptosis)
Significance of kit: This kit allows specific, quantitative detection of caspase 3 activity in
cellular lysates after induction of apoptosis. Caspase 3 activation play a key role in initia-
tion of cellular events during the early apoptotic process. The immunosorbent enzyme
assay principle of this kit guarantees high specificity without cross-reactions with other
known caspases. The fluorochrome generated by proteolytic cleavage of the caspase sub-
strate is proportional to the concentration of activated caspase 3 in the lysates.
Test principle: The assay uses a fluorometric immunosorbent enzyme assay (FIENA)
principle. The procedure (Figure 13 and Flow Chart 4) involves:
Inducing apoptosis in cells by desired method (for instance 2 x 10 6 cells). After the
induction, the cells are washed and pelleted by centrifugation.
Preparing samples by resuspending and incubating cells in lysis buffer. After lysis
and following centrifugation, samples can be removed for direct analysis or storage.
Coating microplate with anti-caspase 3 solution and blocking of unspecific

binding.
Transferring a sample to the anti-caspase 3-coated well of a microplate and

capturing of caspase 3.
Washing the immobilized antibody-caspase 3 complexes three times to remove cell

Incubating sample with caspase substrate (Ac-DEVD-AFC) that is proteolytically

cleaved into free fluorescent AFC.
Measuring generated AFC fluorometrically.
Figure 13: How the Caspase 3 Activity Assay works.

Sample preparation
Treat sample (2 x 106 cells) with apoptosis-inducing agent. Include a negative control without induction (124 h)
Wash treated and control cells with ice-cold PBS and centrifuge (300 x g ) (5 min, RT)
Incubate cell pellet in lysis buffer (1 min, on ice)
Centrifuge at maximum speed in a tabletop centrifuge (1 min, RT)
A
Remove 100 l sample for direct analysis or storage for 1 week at 20C
Coating microplate
Incubate microplate with anti-caspase 3 coating solution (either at 37C for 1h or at 4C over night)
Block unspecific binding by incubation with blocking buffer (30 min, RT)
Remove blocking solution and wash 3 times with incubation buffer (3 x 1 min, RT)
Assaying protease activity

Add sample (100 l lysate, positive control) into microplate well (1 h, 37C)
Remove sample and wash 3 times with incubation buffer (3 x 1 min, RT)
Add freshly prepared substrate solution (13 h, 37C)
Measure fluorometrically (excitation filter 400 (370425) nm and emission filter 505 (490530) nm)
Optional: for calibration, set up a calibration curve with different dilutions of AFC as standard
Flow Chart 4: Assay procedure, Caspase 3 Activity Assay.
Sensitivity: In a model system, caspase 3 activity was clearly detectable in lysates of 106
cells with 5 % apoptotic cells (Figure 14). However, the lower limit for determination of
caspase 3 activity in cellular lysates of dying cells in a particular sample varies with the
kinetics of the apoptotic process, the apoptotic agent used, and the number of affected
cells within the total cell population.
Specificity: This fluorometric immunosorbent enzyme assay is highly specific for caspase
3 by the use of an anti-caspase 3-specific monoclonal capture antibody in combination
with a specific caspase substrate. Enzyme activity of natural and recombinant human
caspase 3 is detected by this assay. Cross-reactions with other caspases are not known.

Lysates of adherent cells, of cells in suspension culture, of cells obtained ex vivo or
recombinant caspase 3.

Kit contents
1. Coating buffer, 10x
2. Anti-caspase-3, 20x
3. Blocking buffer, ready-to-use
4. Incubation buffer, 5x
5. DTT, 100x
6. Substrate solution Ac-DEVD-AFC, 20x
7. AFC
8. Positive control, apoptotic U937 cell lysate
9. Microplate modules (12 x 8-wells)
A
10. Adhesive plate cover
Typical results: see Figures 1415
Other applications: For more examples of how the Caspase 3 Activity Assay can be used
in the lab, see Appendix, pages 132133.
The caspase 3 activity assay has been used to detect caspase 3 activation in U937 cells
exposed to different concentrations of the apoptosis inducing agent camptothecin
(CAM) (Figure 14, dose response curve). In this model system, the induction of apopto-
tis in only 5% of U937 cells is sufficient for detection of caspase 3 activation. Caspase 3
activity/fluorochrome development is proportional to the percentage of apoptotic cells.
Figures 14 and 15 demonstrate that Caspase 3 activity and Annexin-V binding correlate
very closely in both dose-response and kinetic studies.
Figure 14: Dose-response experiment analyzed by

the caspase 3 Activity Assay. U937 cells were exposed to
different concentrations of camptothecin (CAM) for 4 h at
37C. Lysates were analyzed for caspase 3 activity and
standardized values are plotted versus concentration. Addi-
tionally, an aliquot of the same cells was analyzed for
Annexin-V binding.
Figure 15: Kinetic study of caspase 3 activation by

camptothecin exposure in U937 cells. U937 cells were
exposed to 4 g/ml camptothecin for different time intervals
at 37C. Lysates were analyzed for caspase 3 activity and
fluorescence (minus fluorescence of blank) is plotted ver-
sus time. Additionally, an aliquot of the same cells was ana-
lyzed for Annexin-V binding in parallel.

Homogeneous Caspases Assay

Cat. No. 03 005 372 001 100 assays (96-well plates)
400 assays (384-well plates)
Cat. No. 12 236 869 001 1000 assays (96-well plates)
4000 assays (384-well plates)
Type One step assay, fluorimetric

Useful for Specific, quantitative in vitro determination of caspases in microplates
A
Samples Cell cultures, recombinant caspases
Method Cell lysis, followed by detection of caspases activity
(fluorimeric determintion of proteolytic cleavage of the substrate)
Time Approx. 2 h (after induction of apoptosis)
Significance of kit: The Homogeneous Caspases Assay is a fluorimetric assay for the
quantitative in vitro determination of caspases activity in microplates, which makes it
especially useful for high throughput screening. Apoptotic cells are incubated with
DEVD-Rhodamine 110 for 124 h. Upon cleavage of the substrate by activated caspases,
fluorescence of the released Rhodamine 110 is measured.
Test principle: The kit can be used for the quantification of activated caspases of human
as well as animal origin, or screening for caspase inhibitors. It is one step assay, including
the cell lysis step.
Figure 16: How the Homogeneous Caspases Assay work.
Dispense double concentrated apoptosis inducing agent into microplate. Include negative control (diluent only).
Volume should be 50 l (96 well plate) or 12.5 l (384 well plate).
Onto prediluted apoptosis inducing agents, seed cells (4 x 104 per well, volume 50 l on 96 well plate or 104 cells
per well, volume 12.5 l on 384 well plate) and incubate for desired interval for induction of apoptosis.
Add 100 l (96-wells) or 25 l (384-wells) substrate working solution, freshly prepared. Cover the microplate with
a lid and incubate more than 1 h at 37 C.
Measure with an excitation filter 470500 nm and emission filter 500560 nm

(maxima ex = 499 nm and em = 521 nm)
Flow Chart 5: Assay procedure, Homogeneous Caspase Assay.

Specificity: Specifically detects caspases 2, 3 and 7, caspases 6, 8, 9 and 10 to a lesser

extent
Can be used to assay: Cell cultures, recombinant caspases
Kit contents
1. Substrate stock solution, 10x
2. Positive control, 10x
3. Rhodamine 110, standard
4. Incubation buffer
A Typical results: see Figures 1718
Other applications: For more examples of how the Homogeneous Caspases Assay can be
used in the lab, see Appendix, page 133.
Figure 17: Kinetics of caspase activation in U937 cells by camptothecin (384-well plate). U937 cells
were exposed to 4 g/ml camptothecin for different time intervals at 37 C, analyzed for caspase activity with the
Homogeneous Caspases Assay and fluorescence plotted versus time.
Figure 18: Dose-response curve of U937 cells exposed to different concentrations of camptothecin
(CAM) (384-well plate). U937 cells were exposed to camtothecin for 4 h at 37 C, analyzed for caspase activity
with the Homogeneous Caspases Assay and standardized values plotted versus concentration.

Anti-PARP
Cat. No. 11 835 238 001 100 l (50 blots)
Type Polyclonal antiserum, from rabbit

Useful for Detection on Western blots of PARP cleaved by caspases during early stages
of apoptosis
Samples Crude cell extracts
Western blot of apoptotic cell extracts, followed by indirect immunodetec-
A
Method
tion of PARP cleavage fragment
Time Approx. 5.5 h (immunodetection only)
Significance of reagent: Anti-PARP recognizes Poly-ADP-Ribose-Polymerase (PARP), a

113 kD protein that binds specifically at DNA strand breaks. PARP is also a substrate for
certain caspases (for example, caspase 3 and 7) activated during early stages of apoptosis.
These proteases cleave PARP to fragments of approximately 89 kD and 24 kD. Detection
of the 89 kD PARP fragment with Anti-PARP thus serves as an early marker of apoptosis.
Test principle: The Anti-PARP antibody may be used to detect the 89 kD PARP fragment
(and intact PARP) from apoptotic cell extracts on a Western blot. The procedure (Flow
Chart 6) involves:
Preparing crude extracts of apoptotic cells (for instance, by sonication and incubation
of 105107 cells in the presence of urea, 2-mercaptoethanol, and SDS).
Separating proteins in the crude cell extracts on an SDS-polyacrylamide gel.
Transferring the separated proteins to a membrane by electroblotting.
Detecting PARP fragments (and intact PARP) on the membrane with the Anti-PARP
antibody.
Visualizing the antibody-protein complexes with an enzyme-conjugated anti-rabbit

IgG secondary antibody and a chromogenic or chemiluminescent enzyme substrate
(see Table 3).

Prepare crude extracts from apoptotic cells in extraction buffer (approx. 15 min, RT; then 15 min, 65C)
Separate proteins in crude extracts by SDS-PAGE
Electroblot proteins to nitrocellulose or PVDF membrane (approx. 1 h, RT)
Incubate membrane with blocking buffer (1 h, RT)
Incubate membrane with Anti-PARP (diluted 1:2000) (2 h, RT)
A Wash membrane twice with blocking buffer (25 min, RT)
Visualize antibody-antigen complexes with secondary antibody and chromogenic/chemiluminescent detection

(approx. 2 h, RT)
Flow Chart 6: Assay of caspase activity with Anti-PARP.
Antibody supplied as: Polyclonal antiserum from rabbit, stabilized.
Sensitivity: PARP cleavage fragments from 3 x 105 apoptotic cells could be detected on a
Western blot (Figure 19).
Specificity: On Western blots, Anti-PARP recognizes intact PARP from primates or

rodents, as well as the large PARP fragment generated by caspases. Anti-PARP will immu-
noprecipitate intact PARP from primates or rodents.

Crude cell extracts
Typical results: see Figure 19.
The appearance of a large (89 kD) cleavage fragment is indicative of caspase proteolytic
activity.
Other applications: For more examples of how the Anti-PARP can be used in the lab, see
Appendix, pages 133134.
Figure 19: Detection of cleaved PARP in cell extracts of apoptotic CEM T cells. CEM T cells were incu-
bated with one of three apoptosis-inducing drugs. Cell extracts from 3 x 105 treated or untreated cells were frac-
tionated on an 10% polyacrylamide gel in the presence of SDS. After electrophoresis, proteins on the gel were
transferred to a PVDF membrane by electroblotting and the blot was blocked with 5% powdered milk. The
blocked membrane was incubated with a 1:3000 dilution of Anti-PARP. Subsequent incubations with a peroxi-
dase-conjugated anti-rabbit secondary antibody and a peroxidase substrate revealed the presence of PARP
cleavage products on the blot. Note that the antibody recognizes both uncleaved PARP (113 kD) and the larger
cleavage fragment (89 kD).
Lane 1: Untreated control cells
Lane 2: Cells treated with 100 ng/ml doxorubicin for 24 h
Lane 3: Cells treated with 1 mg/ml methotrexate for 24 h
Lane 4: Cells treated with 1 mg/ml cytarabin for 24 h.
(Data is courtesy of Dr. Ingrid Herr, German Cancer Research Institute, department of molecular oncology,
Heidelberg, Germany)

Product Cat. No. Pack Size

BM Chromogenic Western Blotting Kit 11 647 644 001 for 2000 cm2 membrane
(Mouse/Rabbit)
BM Chemiluminescence Western Blotting Kit 11 520 709 001 for 2000 cm2 membrane
(Mouse/Rabbit)
BM Chemiluminescence Blotting Substrate 11 500 708 001 for 1000 cm2 membrane
(POD) 11 500 694 001 for 4000 cm2 membrane
CSPD (chemiluminescent AP substrate), 11 755 633 001 2 x 50 ml
ready-to-use
CDP Star (chemiluminescent AP substrate) 11 685 627 001 1 ml
A
ready-to-use 11 759 051 001 2 x 1 ml
BM Blue POD Substrate, precipitating 11 442 066 001 100 ml
BM Purple AP Substrate, precipitating 11 442 074 001 100 ml
Table 3: Related products for visualization of Anti-PARP.

2404 A poptosis K ap_A Tab_04_A K 1.fm Seite 1 M ontag,23.A ugust2004 11:51 11
Apoptosis Assay Methods Apoptosis Assay Methods

Methods for studying apoptosis in cell populations Methods for studying apoptosis in cell populations
2.1.3 Summary of methods for studying apoptosis in cell populations
Method/Roche Applied Science Parameter Label Assay principle Advantages Limitations For
product analyzed product
informa-
tion, see
DNA Fragmentation Assay, DNA [3H]-TdR or DNA fragments are released from the cytoplasm of Quantitative measurement over a large range (several Radioactive isotope
radioactive11, 12 fragmentation [125I]- UdR, apoptotic cells after lysis with non-ionic detergent. orders of magnitude) Requires prelabeling and extensive washing of the target
(LMW and prelabel The LMW DNA is separated from nuclear HMW DNA Sensitive (103104 cells/test required) cells
A A
HMW-DNA) by centrifugation. Suitable for analysis of cell-mediated (cytotoxicity) effects Limited to target cells proliferating in vitro
The radioactivity in the supernatant and in the pellet is Increased background due to free [3H]-TdR in the
determined by LSC. cytoplasm
DNA Fragmentation Assay, DNA BrdU, prelabel DNA fragments are released from the cytoplasm of Sensitive (103104 cells/test required) Prelabeling of the target cells required page 64
non-radioactive13 fragmentation apoptotic cells after lysis with a non-ionic detergent. Labeled cells do not have to be washed Can only assay target cells proliferating in vitro of this guide
(LMW DNA) The LMW DNA is separated from nuclear HMW DNA Optimal for microtiter plate format Narrow range of quantitative measurement (only one
Cellular DNA Fragmentation by centrifugation. Non-radioactive order of magnitude)
ELISA The supernatant is analyzed by ELISA. Suitable for analysis of cell-mediated (cytotoxicity) effects
JAM Test14 DNA [3H]-TdR, Cells are harvested by vacuum aspiration onto glass Sensitive (103104 cells/test required) Radioactive isotope
fragmentation prelabel fiber filters. While LMW-DNA is washed through the Only 1 washing step required for the labeled cells Prelabeling of the target cells required
(HMW DNA) filters, the HMW DNA is retained on these filters. Low spontaneous release: cytotoxic events causing low Limited to target cells proliferating in vitro
The radioactivity retained on the filters is measured by cell lysis over prolonged period of time (824 h) can be In apoptotic cells, DNA is only partially lost: viable and
LSC. studied damaged cells are separated by only a narrow range of
Optimal for microtiter plate format assay values
Alkaline Elution Analysis15 DNA [3H]-TdR, Cells are loaded onto polycarbonate filters. Differential elution allows the detection of strand breaks in Radioactive isotope
fragmentation prelabel The filters are incubated with three different buffer DNA, DNA-interstrand crosslinks and DNA-protein Prelabeling and washing of the target cells required
(LMW and solutions containing SDS, pH 10, SDS + Proteinase K, crosslinks Limited to target cells proliferating in vitro
HMW-DNA) pH 7, or SDS, pH 12.3. Insensitive (106 cells/test required)
The radioactivity in each fraction (LMW DNA) as well as Labor-intensive and time-consuming: only a few tests
the radioactivity retained on the filter (HMW DNA) is may be performed simultaneously
quantified by LSC.
DNA Ladder Assay16 DNA none Cellular DNA is isolated by extraction and quickly Hallmark of apoptosis: demonstration of the mono- and No quantitative measurement page 13
(LMW and HMW DNA by size) fragmentation purified. oligonucleosomal DNA fragments (180 bp multimers) Insensitive: More than 106 cells/test required of this guide
Purified total DNA (LMW and HMW DNA) is analyzed No prelabeling of the cells required: not limited to cells Labor-intensive and time-consuming:
Apoptotic DNA Ladder Kit by agarose gel electrophoresis and visualized by which proliferate in vitro only a few tests may be performed simultaneously
staining with ethidium bromide. Non-radioactive
Nucleosome Quantification DNA none Histone complexed DNA-fragments (mono- and Sensitive (102104 cells/test required) Samples have to be analyzed immediately because page 15
ELISA13 fragmentation oligonucleosomes, LMW DNA) are released from the No prelabeling of the cells required: not restricted to cells storage reduces ELISA signal of this guide
(LMW DNA in cytoplasm of apoptotic cells after lysis. which proliferate in vitro Not recommended for tissue homogenates. Increased
Cell Death Detection ELISAPLUS association with The LMW DNA is separated from nuclear HMW DNA Non-radioactive background could occur due to activation of nucleases
histones) by centrifugation. Detection of DNA and histones in one immunoassay dem- during sample preparation.
The supernatant is analyzed by ELISA. onstrates mono- and oligonucleosomal DNA fragments
DNA Ladder Assay, radioactive 17 DNA -[32P]-ATP, Cellular DNA is isolated by extraction and quickly Definitive marker of apoptosis: demonstration of the Labor-intensive and time-consuming: only a few tests
fragmentation postlabel purified. mono- and oligonucleosomal DNA fragments (180 bp may be performed simultaneously
(LMW and HMW Purified total DNA (LMW and HMW DNA) is labeled at multimers) Radioactive assay (32P)
by size) the 5end with -[32P]-ATP by T4 Polynucleotide Kinase. No prelabeling of the cells required: not limited to cells End-labeling of purified DNA required
[32P]-labeled DNA is separated by agarose gel which proliferate in vitro
electrophoresis and quantitated in the dried gel by a Highly sensitive (1000 x more sensitive than ethidium bro-
blot analyzer. mide): allows earlier detection of DNA fragmentation after
induction of apoptosis
Protease Activity Assay Activation of none Apoptotic process including activation of the caspase Quantitative assay, cleavage of substrate is proportional to High cell numbers needed page 22
caspases cascade is induced in cells by desired method. concentration of activated caspase 3 in samples Fluorescence reader, equipped with special fluorescence of this guide
Caspase 3 Acitivity Assay (Caspase 3) Cells are lysed and cell extracts are prepared. Detection of very early stages of apoptosis filters needed
Activated caspase 3 is captured out of cellular lysates Highly specific for caspase 3, no cross reactions with other
by an Anti-caspase 3 antibody members of the caspase family
Quantification of fluorochromes cleaved from a caspase
specific substrate.
Protease Activity Assay Discrete none Cells are treated with an apoptosis-inducing agent, Flexible, can be used with many different types of cells Insensitive (requires 105106 cells/test) page 27
cleavage of DNA which leads to induction of caspase 3 and the cleavage No prelabeling of cells required: not limited to cells which Labor-intensive and time-consuming: of this guide
Anti-PARP repair enzyme of Poly-ADP-Ribose-Polymerase (PARP). proliferate in vitro only a few tests may be performed simultaneously
(PARP) Cell extracts are prepared with SDS, fractionated by Non-radioactive
SDS-PAGE, and transferred to a PVDF membrane by Marker for very early stage of apoptosis
western blotting.
Blot is probed with an antibody to PARP, then with a
peroxidase-labeled secondary antibody.
Cleavage products of PARP (about 85 kD) on the mem-
brane are revealed after an incubation with a peroxi-
dase substrate.
Table 4: Methods for studying apoptosis in cell populations.

30 Apoptosis, Cell Death, and Cell Proliferation Manual Cell Death Apoptosis and Necrosis 31
Methods for studying apoptosis in individual cells
2.2 Methods for studying apoptosis in individual cells

A number of methods have now been developed to study apoptosis in individual cells. In
the following sections, we will describe details of several of these apoptosis assays.
We focus on two key apoptotic events in the cell:

DNA fragmentation used to study death in cell populations may also be used to study
death in individual cells. As described in Section A 2.1.1, DNA cleavage is a hallmark
for apoptosis, and assays which measure prelytic DNA fragmentation are especially
attractive for the determination of apoptotic cell death.
A
The methods used to assess DNA strand breaks are based on labeling/staining the cel-
lular DNA. The labeled/stained DNA is subsequently analyzed by flow cytometry, fluo-
rescence microscopy or light microscopy (Figure 20). In general, two different labeling
methods may be used to identify DNA in apoptotic cells:
Enzymatic labeling: Cellular DNA is labeled with modified nucleotides (e.g.,
biotin-dUTP, DIG-dUTP, fluorescein-dUTP) using exogenous enzymes (e.g., termi-
nal transferase, DNA polymerase). This labeling detects extensive DNA strand
breaks. Enzymatic labeling is discussed in detail below (Section A 2.2.1 of this
guide).
Staining with fluorochromes: Cellular DNA is stained with fluorescent DNA-bind-
ing dyes (DNA-fluorochromes) capable of intercalating into DNA. Upon binding to
DNA these dyes become highly fluorescent. Apoptotic cells are binding less dye mol-
ecules, since they characteristically lose DNA during the staining process (described
in Section A 2.2.3 of this guide).
In addition, individual cell death may be studied by assays that measure alterations in
plasma membranes (alterations in the asymmetry or permeability of individual cell
membranes, which occur as the membrane shrinks and becomes increasingly convo-
luted.) For instance, during apoptosis, phosphatidylserine translocates from the cyto-
plasmic side of the membrane to the extracellular side and can be detected with
Annexin-V (described in Section A 2.2.2 of this guide).
Figure 20: Schematic illustration of the two basic principles for detecting DNA fragmentation in single cells.

2.2.1 The TUNEL enzymatic labeling assay
Extensive DNA degradation is a characteristic event which occurs in the late stages of
apoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments
(mono- and oligonucleosomes) as well as single strand breaks (nicks) in HMW-DNA.
Those DNA strand breaks can be detected by enzymatic labeling of the free 3-OH ter-
mini with modified nucleotides (X-dUTP, X = biotin, DIG or fluorescein). Suitable label-
ing enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyl
transferase (end labeling) (Figure 21).
A
Figure 21: Schematic illustration of two enzymatic DNA labeling methods nick translation and end
labeling.
Nick translation
DNA polymerase I catalyzes the template dependent addition of nucleotides when one
strand of a double-stranded DNA molecule is nicked. Theoretically, this reaction (In Situ
Nick Translation, ISNT) should detect not only apoptotic DNA, but also the random
fragmentation of DNA by multiple endonucleases occurring in cellular necrosis.
End labeling
Terminal deoxynucleotidyl transferase (TdT) is able to label blunt ends of double-
stranded DNA breaks independent of a template. The end-labeling method has also been
termed TUNEL (TdT-mediated X-dUTP nick end labeling)18.
The TUNEL method is more sensitive and faster than the ISNT method. Cells undergo-
ing apoptosis were preferentially labeled by the TUNEL reaction, whereas necrotic cells
were identified by ISNT. Thus, experiments suggest the TUNEL reaction is more specific
for apoptosis and the combined use of the TUNEL and nick translation techniques may
be helpful to differentiate cellular apoptosis and necrosis19.
Note: For a comparison of results with the TUNEL and ISNT methods, see Figure 22.
To allow exogenous enzymes to enter the cell, the plasma membrane has to be permeabi-
lized prior to the enzymatic reaction. To avoid loss of LMW DNA from the permeabilized
cells, the cells have to be fixed with formaldehyde or glutaraldehyde before permeabiliza-
tion. This fixation crosslinks LMW DNA to other cellular constituents and precludes its
extraction during the permeabilization step.

A Figure 22: Comparison of TUNEL and ISNT methods for detecting apoptosis in CD8+ T cells from
TcR transgenic mice. F5 mice are transgenic for a T cell receptor (TcR) specific for a peptide derived from a
nucleoprotein of influenza virus ANT/1968. In this experiment, the nucleopeptide protein was injected into F5
mice to activate T cells in vivo. After 4 days, mice were sacrificed and lymphoid organs were removed. Cell sus-
pensions were prepared and incubated 4 h at 37C. To allow detection of T cells which were dying after the in vivo
immune response [Pihlgren, M., Thomas, J. and Marvel, J. (1996) Biochemica, No. 3, 1214], cells were stained for
CD8 (with a fluorescent antibody), fixed, permeabilized, and then labeled by either the TUNEL (TdT-mediated
dUTP Nick End Labeling) or the ISNT (In Situ Nick Translation) method. Labeled and control cells were analyzed
by flow cytometry, with CD8+ cells gated. Spleen cells from a control (not immunized) mouse (red) and from two
mice immunized 4 days earlier (green) are shown.
Result: The TUNEL method detected approximately 15% apoptotic cells among CD8+ T cells from the immunized
mice. No positive cells were found in the control mouse. In contrast, the ISNT method was unable to detect any
apoptotic cells, possibly due to the lower sensitivity of the technique.
If free 3 ends in DNA are labeled with biotin-dUTP or DIG-dUTP, the incorporated
nucleotides may be detected in a second incubation step with (strept)avidin or an anti-
DIG antibody. The immunocomplex is easily visible if the (strept)avidin or an anti-DIG
antibody is conjugated with a reporter molecule (e.g., fluorescein, AP, POD).
In contrast, the use of fluorescein-dUTP to label the DNA strand breaks allows the detec-
tion of the incorporated nucleotides directly with a fluorescence microscope or a flow
cytometer20. Direct labeling with fluorescein-dUTP offers several other advantages.
Direct labeling produces less nonspecific background with sensitivity equal to indirect
labeling (Figure 23) and, thus, is as powerful as the indirect method in detecting apopto-
sis. Furthermore, the fluorescence may be converted into a colorimetric signal if an anti-
fluorescein antibody conjugated with a reporter enzyme (Table 5) is added to the sample.
Although the enzymatic labeling methods are time-consuming (due to multiple incuba-
tion and washing steps), they are very sensitive and specific21.
Caution: One has to keep in mind that these methods are based on the detection of DNA
strand breaks. There are rare situations when apoptosis is induced without DNA degrada-
tion. Conversely, extensive DNA degradation, even specific to the internucleosomal linker
DNA, may accompany necrosis. Thus, one should always use another independent assay,
along with the TUNEL method, to confirm and characterize apoptosis.
Roche Applied Science offers four kits for the detection of DNA strand breaks in individ-
ual cells using the TUNEL method. Each is described on the following pages.
Note: For technical tips on the TUNEL method, see page 113 of the Appendix.

Figure 23: Comparison of direct and indirect labeling of DNA strand breaks in apoptotic cells. PBL
A
were incubated with 1 M dexamethasone for 24 h at 37C and then labeled by TUNEL. Recordings were made at
the same photomultiplier settings.
(Data were kindly provided by R. Sgonc, University of Innsbruck, Austria).
Result: Direct labeling is as sensitive as indirect labeling, but produces less non-specific background.
Method/RAS prod- Label Indirect (secondary) Analysis by

uct detection system
In Situ Cell Death Fluorescein-dUTP None (direct detection) Flow cytometry
Detection Kit, Fluorescence micros-
Fluorescein copy
In Situ Cell Death TMR-dUTP None (direct detection) Fluorescence micros-
Detection Kit, copy
TMR red
In Situ Cell Death Fluorescein-dUTP Anti-Fluorescein-AP Light microscopy
Detection Kit, AP
In Situ Cell Death Fluorescein-dUTP Anti-Fluorescein-POD Light microscopy
Detection Kit, POD
Table 5: Four different kits for the enzymatic labeling of DNA and the secondary detection systems
required.

In Situ Cell Death Detection Kit, Fluorescein

Cat. No. 11 684 795 001 50 tests
In Situ Cell Death Detection Kit, TMR red

Cat. No. 12 156 792 001 50 tests
Type Direct TUNEL labeling assay
A
Useful for Detection of DNA strand breaks in apoptotic cells by flow cytometry or
fluorescence microscopy
Samples Cells in suspension, adherent cells, cell smears, frozen or paraffin-embedded
tissue sections
Method End-labeling of DNA with fluorescein-dUTP or tetramethylrhodamine-
dUTP (TMR-dUTP), followed by direct analysis of fluorescent cells
Time 12 h (+ sample preparation, permeabilization, etc.)
Significance of kit: This two In Situ Cell Death Detection Kits, measure and quantitate
cell death (apoptosis) by labeling and detection of DNA strand breaks in individual cells
by flow cytometry or fluorescence microscopy. The kits offer a direct TUNEL detection
method, for maximum sensitivity and minimal background.
Test principle: The assays use an optimized terminal transferase (TdT) to label free 3OH
ends in genomic DNA with fluorescein-dUTP or TMR-dUTP. The procedure involves:
Fixing and permeabilizing apoptotic cells.
Incubating the cells with the TUNEL reaction mixture containing TdT and fluorescein-
dUTP or TMR-dUTP. During this incubation step, TdT catalyzes the attachment of flu-
orescein-dUTP or TMR-dUTP to free 3OH ends in the DNA.
Visualizing the incorporated fluorescein with a flow cytometer and/or a fluorescence

microscope (fluorescein/TMR red).
Figure 24: Schematic showing the principle of the In Situ Cell Death Detection Kits,
Fluorescein and TMR red.
For a detailed overview of the steps in the procedure, see Flow Chart 7.
Sensitivity: The enzymatic labeling allows the detection of an apoptotic event that
occurs, prior to changes in morphology and even before DNA fragments become detect-
able in the cytoplasm22. It detects early stage of DNA fragmentation in apoptotic cells.
This is especially important if apoptosis is studied in vivo, e.g., in tissue sections, since
apoptotic cells are rapidly and efficiently removed in vivo.
Specificity: The amount of DNA strand breaks in apoptotic cells is so large that the
degree of cell labeling in these assays is an adequate discriminator between apoptotic and
necrotic cells19.

Cell suspensions Adherent cells, cell Frozen tissue section Formalin-fixed, paraffin-
smears, cytospins embedded tissue sections
Fix samples (1 h, RT) Dewax, rehydrate, and

treat with protease
Wash samples
Permeabilize samples (2 min, on ice)
Analyze by flow
Wash samples
Incubate with TUNEL reaction mixture [enzyme solution + labeling solution] (60 min, 37C)
Wash samples
Analyze by fluorescence microscopy

A
cytometry or fluores-
cence microscopy
Flow Chart 7: Assay procedure, In Situ Cell Death Detection Kits (Fluorescein or TMR red).

Cells in suspension (permanent cell lines, normal and tumor cells ex vivo)
Cytospins, cell smears
Adherent cells cultured on chamber slides
Frozen tissue sections
Formalin-fixed, paraffin-embedded tissue sections
Kit contents
1. Enzyme solution (TdT), 5 tubes
2. Labeling solution (nucleotide mix), 5 tubes
Typical results: See Figures 2528.
Technical tips: For more information on the use of the kit for flow cytometric analysis,
see page 119 in the Appendix of this guide.
Other applications: For more examples of how the In Situ Cell Death Detection Kits (Flu-
orescein or TMR red) can be used in the lab, see Appendix, pages 134136.

A Figure 25: Detection of apoptotic cells by flow cytometry using the In Situ Cell Death Detection Kit,
Fluorescein. HL60 cells were cultured in the absence (A) or presence (B) of 2 g/ml Camptothecin for 3 h at
37C. Cells were incubated either with TUNEL reaction mixture () or label solution as negative control () or
PBS for autofluorescence ().
Figure 26: Detection of apoptotic cells (green)

by fluorescence microscopy in a tissue section
from rat. A tissue section from a rat spinal cord was
prepared and assayed with the In Situ Cell Death Detec-
tion Kit, Fluorescein. The treated section was viewed
under a fluorescence microscope. (Photomicrograph
was kindly provided by R. Gold, University of Wrzburg,
Germany.)
Result: A subpopulation of apoptotic cells, scattered
throughout the tissue section, are intensely stained
(green) by the TUNEL treatment and are easily visible
under the microscope.
Figure 27: Cell suspension stained with the In

Situ Cell Death Detection Kit, Fluorescein. U937
cells induced with 4 g/ml camptothecin, showing posi-
tive staining of apoptotic nuclei.
Note: This figure shows a high number (>80%) of
apoptotic cells. To avoid detecting cells that are under-
going secondary necrosis, analyze cells earlier in the
process after induction of apoptosis.
Figure 28: Rabbit endometrium, stained with

the In Situ Cell Death Detection Kit, TMR red, and
viewed under a fluorescence microscope. Apoptotic
nuclei stain bright red, limited fluorescence is visible in
background tissue.

In Situ Cell Death Detection Kit, AP

Cat. No. 11 684 809 001 50 tests
In Situ Cell Death Detection Kit, POD

Cat. No. 11 684 817 001 50 tests
Type Indirect TUNEL labeling assay
A
Useful for Detection of DNA strand breaks in apoptotic cells under a light microscope
Samples Cell smears, adherent cells, cytospins, frozen or fixed tissue sections
Method End-labeling of DNA with fluorescein-dUTP, followed by detection of
incorporated fluorescein with an antibody and visualization of the antibody
Time Approx. 3 h (+ sample preparation, permeabilization, etc.)
Significance of kits: These two In Situ Cell Death Detection Kits measure cell death (apo-
ptosis) by detecting DNA strand breaks in individual cells by light microscopy. The AP
and POD kits offer an indirect TUNEL detection method, which is a fast, sensitive, and
specific light microscopic assay.
Test principle: The assays use an optimized terminal transferase (TdT) to label free 3OH
ends in genomic DNA with fluorescein-dUTP. The procedure involves:
Fixing and permeabilizing apoptotic cells or tissue sections.
Incubating the cells with the TUNEL reaction mixture containing TdT and fluorescein-
dUTP. During this incubation step, TdT catalyzes the attachment of fluorescein-dUTP
to free 3OH ends in the DNA.
Detecting the incorporated fluorescein with an anti-fluorescein antibody AP

conjugate (In Situ Cell Death Detection Kit, AP) or an anti-fluorescein antibody POD
conjugate (In Situ Cell Death Detection Kit, POD).
Visualizing the immunocomplexed AP or POD with a substrate reaction.
Figure 29: Schematic showing the

principle of the In Situ Cell Death Detec-
tion Kits, AP and POD.
Sensitivity: The enzymatic labeling allows the detection of an apoptotic event that
occurs, prior to changes in morphology and even before DNA fragments become detect-
able in the cytoplasm22. It detects early stage of DNA fragmentation in apoptotic cells.
This is especially important if apoptosis is studied in vivo, e.g., in tissue sections, since
apoptotic cells are rapidly and efficiently removed in vivo.
Specificity: The amount of DNA strand breaks in apoptotic cells is so large that the
degree of cell labeling in these assays is an adequate discriminator between apoptotic and
necrotic cells19.

Remove tissue or organ and use standard methods to process for:
Frozen sectioning Paraffin embedding
Fix sections in formalin (30 min, RT) Dewax, rehydrate sections by standard methods
Optional: Inactivate endogenous POD/AP activity
Wash slides
A Fix section (30 min, RT) Add protease and incubate (30 min, 37C)
Permeabilize sections (2 min, on ice)

Wash slides
Add TUNEL-reaction mixture and incubate (60 min, 37C)
Wash slides
Optional: Analyze by fluorescence microscopy
Add Anti-Fluorescein-AP or -POD and incubate (30 min, 37C)
Wash slides
Add substrate and incubate (520 min, RT)
Analyze by lightmicroscopy
Flow Chart 8: Assay procedure In Situ Cell Death Detection Kits (AP or POD).

Cells smears, adherent cells
Cytospins
Tissue sections (frozen or paraffin-embedded).
Kit contents
In Situ Cell Death Detection Kit, AP
3. Anti-Fluorescein-AP conjugate, ready to use
In Situ Cell Death Detection Kit, POD

3. Anti-Fluorescein-POD conjugate, ready to use
Note: For added flexibility and convenience, the components of these kits, as well as several
AP and POD precipitating substrates are also available as single reagents (Table 6).
Typical results: see Figures 30 and 31.
Technical tips: For more information on the use of the kits for light microscopic analysis,
see pages 113116 in the Appendix, of this guide.

Figure 30: Detection of apoptotic cells by TUNEL and peroxidase staining in rabbit endometrium.
A tissue section from rabbit endometrium was prepared and assayed with the In Situ Cell Death Detection Kit,
POD. Slide was counterstained with hematoxylin and viewed under a light microscope.
Result: A subpopulation of apoptotic cells, scattered throughout the tissue section, are intensely stained (brown)
by the TUNEL treatment and subsequent peroxidase immunostaining.
A
A
Figure 31: Detection of apoptotic cells by TUNEL and alkaline phosphatase staining in rat spinal
cord. A tissue section from rat spinal cord was prepared and assayed with the In Situ Cell Death Detection Kit,
AP. The slide was viewed under a light microscope (Panel A). After viewing, the same slide was stained with pro-
pidium iodide and viewed by fluorescence microscopy (Panel B).
Result: A few apoptotic cells (red) are clearly visible after TUNEL treatment and subsequent alkaline phos-
phatase immunostaining (Panel A). However, the apoptotic cells are not visible in the same slide after staining
with propidium iodide (Panel B).

Other applications: For more examples of how the In Situ Cell Death Detection Kits (AP
or POD) can be used in the lab, see Appendix, pages 136138.
For your convenience, we offer a number of additional single reagents to optimize your
TUNEL reaction (Table 6)
Product Cat. No. Pack Size

TUNEL Label Mix 11 767 291 001 3 x 550 l (30 tests)
TUNEL Enzyme 11 767 305 001 2 x 50 l (20 tests)
A
TUNEL POD (Anti-Fluorescein, POD conjugate) 11 772 465 001 3.5 ml (70 tests)
TUNEL AP (Anti-Fluorescein, AP conjugate) 11 772 457 001 3.5 ml (70 tests)
TUNEL Dilution Buffer 11 966 006 001 2 x 10 ml
DAB Substrate, metal enhanced, precipitating 11 718 096 001 1 pack
(Peroxidase (POD) substrate)
NBT/BCIP Stock Solution (AP substrate) 11 681 451 001 8 ml
Fast Red Tablets (AP substrate) 11 496 549 001 20 tablets
Table 6: Single reagents available for the TUNEL technique.

2.2.2 Assays that measure membrane alterations
In contrast to necrosis, apoptosis occurs without inflammation. In the end stages of

apoptosis, apoptotic bodies are engulfed by macrophages and other phagocytic cells2
in vivo. Thus, apoptotic cells are removed from the population without spilling their con-
tents and eliciting an inflammatory response.
The exact mechanism by which the apoptotic cell becomes a target for phagocytes is
unclear. However, it has been shown that a number of changes in cell surface (mem-
brane) markers occur during apoptosis, any one of which may signal remove now to
A
the phagocytes. These membrane changes include:
Loss of terminal sialic acid residues from the side chains of cell surface glycoproteins,
exposing new sugar residues23,24.
Emergence of surface glycoproteins that may serve as receptors for macrophage-
secreted adhesive molecules such as thrombospondin25.
Loss of asymmetry in cell membrane phospholipids, altering both the hydrophobicity
and charge of the membrane surface26.
In theory, any of these membrane changes could provide an assay for apoptotic cells. In
fact, one of them has the alteration in phospholipid distribution.
In normal cells (Figure 32, left diagram), the distribution of phospholipids is asymmet-
ric, with the inner membrane containing anionic phospholipids (such as phosphati-
dylserine) and the outer membrane having mostly neutral phospholipids. In apoptotic
cells (Figure 32, right diagram) however, the amount of phosphatidylserine (PS) on the
outer surface of the membrane increases, exposing PS to the surrounding liquid27.
Annexin-V, a calcium-dependent phospholipid-binding protein, has a high affinity for

PS27. Although it will not bind to normal living cells, Annexin-V will bind to the PS
exposed on the surface of apoptotic cells (Figure 33, 34). Thus, Annexin-V has proved
suitable for detecting apoptotic cells28, 29. Roche Applied Science supplies a number of
products for the detection of PS translocation by Annexin-V.
Figure 32: Detection of surface morphology changes during apoptosis. During apoptosis, the distribu-
tion of neutral phospholipids (black symbols) and anionic phospholipids such as phosphatidylserine (red sym-
bols) in the cell membrane changes. Phosphatidylserine is present in the outer membrane of apoptotic cells, but
not of normal cells. An exogenously added molecule specific for phosphatidylserine, such as Annexin-V-FLUOS,
will bind to phosphatidylserine on the outer membrane of apoptotic cells, but cannot react with the phosphati-
dylserine of normal cells.

Annexin-V-FLUOS
Cat. No. 11 828 681 001 250 tests
Annexin-V-FLUOS Staining Kit

Cat. No. 11 858 777 001 50 tests
11 988 549 001 250 tests
A Annexin-V-Alexa 568
Cat. No. 03 703 126 001 250 tests
Type
Useful for
Direct fluorescence staining for flow cytometric or microscopic analysis
Detection of apoptotic cells with membrane alterations (phosphatidylserine
translocation); differentiation of apoptotic from necrotic cells
Samples Cell lines (adherent or suspensions), freshly isolated cells
Method Simultaneous staining of cell surface phosphatidylserine [with Annexin-V-
FLUOS (green dye) or Annexin-V-Alexa 568 (red dye)] and necrotic cells
(with propidium iodide)
Time Approx. 15 min (after induction of apoptosis)
Significance of reagent and kit: Annexin-V is a phospholipid-binding protein with a high

affinity for phosphatidylserine (PS). Detection of cell-surface PS with annexin-V thus
serves as a marker for apoptotic cells. Analysis may be by flow cytometry or by fluores-
cence microscopy.
Test principle: Annexin-V-FLUOS (green dye) and Annexin-V-Alexa 568 (red dye) serves
as a fluorescent probe for apoptotic cells. They will not bind normal, intact cells. However,
since necrotic cells are leaky enough to give Annexin-V-FLUOS and Annexin-V-Alexa 568
access to inner membrane PS, apoptotic cells have to be differentiated from necrotic cells.
Thus, the assay involves simultaneous staining with both Annexin-V-FLUOS (green) and
the DNA stain propidium iodide (red) or Annexin-V-Alexa 568 (red) and BOBO-1
(green). Exclusion of propidium iodide or BOBO-1, coupled with binding of Annexin-
V-FLUOS or Annexin-V-Alexa 568, indicates an apoptotic cell (Table 7). The procedure
(Flow Chart 9) involves:
Washing suspended cells, then pelleting the cells.
Resuspending cells in a staining solution containing Annexin-V-FLUOS and

propidium iodide or Annexin-V-Alexa 568 and BOBO-1.
Note: Cells may also be labeled with other membrane stains, such as a fluorescein-,
phycoerythrin- or TRITC-labeled monoclonal antibody simultaneously.
Analyzing samples in a flow cytometer or under a fluorescence microscope.

Normal cells Apoptotic cells Necrotic cells

Annexin-V staining + +
Propidium iodide +
staining
BOBO-1 +
Table 7: Distinguishing apoptosis from necrosis using Annexin-V, propidium iodide, or BOBO-1.
Treat sample (106 cells) with apoptosis-inducing agent (124 h)
Wash treated cells with PBS and centrifuge (200 x g) (5 min, RT)
Incubate cells in binding buffer containing Annexin-V-FLUOS and propidium iodide or Annexin-V-
Alexa 568 and BOBO-1. (1015 min, RT)
A
Add binding buffer to stained cells Analyze by fluorescence microscopy
Analyze by flow cytometry
Flow Chart 9: Assay procedure, Annexin-V-FLUOS Staining Kit and Annexin-V-Alexa 568.
Specificity: Annexin-V-FLUOS and Annexin-V-Alexa 568 bind apoptotic cells and leaky
necrotic cells. Propidium iodide and BOBO-1 are excluded from apoptotic and normal
cells, but is taken up by necrotic cells.

Cell lines (adherent or suspensions)
Freshly isolated cells
Reagent contents:
Annexin-V-FLUOS solution, 50 x concentrated
Annexin-V-Alexa 568, 50 x concentrated
Kit contents
Annexin-V-FLUOS Staining Kit
1. Annexin-V-FLUOS, 50 x concentrated
2. Propidium iodide solution, 50 x concentrated
3. Binding buffer for flow cytometry, ready to use
Other applications: For more examples of how the Annexin-V-FLUOS, Annexin-V-

FLUOS Staining Kit and Annexin-V-Alexa 568 can be used in the lab, see Appendix,
pages 138141.

A Figure 33: Apoptotic and necrotic U937 cells identified in FACS analysis after staining with Annexin-
V-FLUOS and propidium iodide (PI). Cells were then stained with the components of the Annexin-V-FLUOS
Staining Kit and analyzed.
Panels A (upper and lower), single parameter analysis, Annexin-V-FLUOS only;
Panels B, single parameter analysis, propidium iodide (PI) only;
Panels C, dual parameter analysis, Annexin-V-FLUOS and propidium iodide. FL1, Annexin-V-FLUOS; FL2,
propidium iodide.
Result: Flow cytometric analysis clearly differentiates normal (living) cells with low Annexin and low PI staining,
apoptotic cells with high Annexin and low PI staining, and necrotic cells with high Annexin and high PI staining.
necrotic apoptotic
apoptotic
Figure 34: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin.
Early-stage apoptosis detected with Annexin-V-FLUOS (green), and counterstained with propidium iodide (red
cells).
Results: The apoptotic cells are visible in green and can be differentiated from necrotic cells by the propidium
staining in figure 34. Necrotic cells take up propidium iodide and stain orange/green, while apoptotic cells stain
green only.
apoptotic
necrotic
necrotic
Figure 35: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin
(CAM) and stained with Annexin-V-Alexa 568 (red) and BOBO-1 (green).
Results: The apoptotic cells are visible in red and can be differentiated from necrotic cells by the BOBO-1 stain-
ing in figure 35. Necrotic cells take up BOBO-1 and stain green/red, while apoptotic cells stain red only.

Annexin-V-Biotin
Cat. No. 11 828 690 001 250 tests
Type Indirect fluorescence staining for flow cytometric, fluorescence or light

microscopic analysis
Useful for Detection of apoptotic cells with membrane alterations (phosphatidylserine
translocation); differentiation of apoptotic from necrotic cells
Samples Cell lines (adherent and suspensions), freshly isolated cells
A
Method Simultaneous staining of cell surface phosphatidylserine (with Annexin-V-
Biotin) and necrotic cells (with propidium iodide), followed by detection of
biotin (with streptavidin/avidin conjugate)
Time Approx. 75 min (after induction of apoptosis)
Significance of reagent: Annexin-V is a phospholipid-binding protein with a high affinity

for phosphatidylserine (PS). During apoptosis, PS translocates to the outer surface of
apoptotic cells. Detection of cell-surface PS with annexin-V thus serves as a marker for
apoptotic cells. Labeling of cells with the Biotin-conjugate of Annexin-V allows fixation
after Annexin-V binding for further analysis of additional cellular parameters in com-
bination with detection of apoptosis (van Engeland, M., Ramaekers FCS, Schutte, B &
Reutelingsperger, CPM (1996): A Novel Assay to Measure Loss of Plasma Membrane
Asymmetry During Apoptosis of Adherent Cells in Culture. Cytometry 24: 131139). For
distinguishing apoptosis using Annexin-V, see Table 7, page 45.
Test principle: Annexin-V-Biotin serves as a probe for apoptotic cells. It will not bind
normal, intact cells. However, since necrotic cells are leaky enough to give Annexin-V-
Biotin access to inner membrane PS, apoptotic cells have to be differentiated from
necrotic cells. Thus, the assay involves simultaneous staining with both Annexin-V-
Biotin, Avidin-Fluorescein and propidium iodide. Exclusion of propidium iodide, cou-
pled with binding of Annexin-V-Biotin, indicates an apoptotic cell. Annexin-V-Biotin is
visualized with a streptavidin/avidin conjugate. Analysis may be by flow cytometry, by
fluorescence microscopy, or by light microscopy. The procedure (Flow Chart 10)
involves:
Washing suspended cells, then pelleting the cells.
Resuspending cells in a staining solution containing Annexin-V-Biotin and propidium

iodide.
Note: Cells may also be labeled with other membrane stains, such as a fluorescein-,
phycoerythrin- or TRITC-labeled monoclonal antibody simultaneously.
Washing labeled cells.
Incubating cells with a streptavidin (SA)/avidin conjugate (Table 8).
Analyzing samples in a flow cytometer, under a fluorescence microscope, or under a

light microscope (depending on the SA/avidin conjugate).
Specificity: Annexin-V-Biotin binds apoptotic cells and leaky necrotic cells

Treat sample (106 cells) with apoptosis-inducing agent (124 h)
Wash treated cells with PBS and centrifuge (200 x g) (5 min, RT)
Incubate cells in incubation buffer containing Annexin-V-Biotin and propidium iodide (1015 min, RT)
Centrifuge stained cells (200 x g) and wash once with incubation buffer (approx. 10 min, RT)
Incubate washed cells with streptavidin/avidin conjugate (20 min, 4C)
A Centrifuge stained cells (200 x g) and wash once with incubation buffer (approx. 10 min, RT)
Add incubation buffer to stained

cells
Analyze by flow cytometry

Analyze by fluorescence
microscopy
Flow Chart 10: Assay procedure, Annexin-V-Biotin.

Add substrate (515 min, RT)
and wash
Analyze by light microscopy
Can be used to assay: Reagent contents:

Cell lines (adherent/suspensions) Annexin-V-Biotin solution, 50 x concen-
Freshly isolated cells trated.
Typical results: see Figure 36.
Other applications: For more examples of how the Annexin-V-Biotin can be used in the
lab, see Appendix, pages 141142.
Figure 36: Flow cytometric analysis of apoptotic

U937 cells stained with Annexin-V-Biotin, Avidin-
FLUOS and propidium iodide. U937 cells (a leukemic
cell line) were cultivated for 4 h with 4 g/ml
campothecin. Cells were stained with Annexin-V-Biotin
and propidium iodide (PI), then incubated with Avidin-
fluorescein and analyzed. Single parameter histograms
are shown at the top (Annexin-V-Biotin/Avidin-FLUOS)
and on the right side (PI) of the diagram. Two parameter
histograms are shown in quadrants 14. PI, propidium
iodide; FLUOS, fluorescein.
Result: Flow cytometric analysis clearly differentiates
normal cells (quadrant 3) with low FLUOS and low PI
staining, apoptotic cells (quadrant 4) with high FLUOS
and low PI staining, and necrotic cells (quadrant 2) with
high FLUOS and high PI staining.
Product Application Cat. No. Pack Size

Avidin-Fluorescein fluorescence microscopy, 11 975 595 001 1 mg
flow cytometry
Avidin-Rhodamine fluorescence microscopy, 11 975 609 001 1 mg
flow cytometry
SA-Peroxidase light microscopy 11 089 153 001 500 U (1 ml)
SA-Alkaline Phos- light microscopy 11 089 161 001 1000 U (1 ml)
phatase
SA--Galactosidase light microscopy 11 112 481 001 500 U
Table 8: Streptavidin (SA) /Avidin conjugates available for the indirect assay of apoptotic cells with
Annexin-V-Biotin.
Note: Additional substrates can be found in Table 6.

2.2.3 Assays that use DNA stains
One can differentiate between three methods for studying cell death that use DNA stains:
dye exclusion method, profile of DNA content, morphological changes.
Dye exclusion method

Viable (intact plasma membrane) and dead (damaged plasma membrane) cells can be
discriminated by differential staining. Cells with disturbed plasma membrane perme-
ability are stained, whereas undamaged (viable) cells are not stained with dyes that do not
penetrate the plasma membrane (exclusion dyes). The most frequently used dye for
A
exclusion tests is trypan blue. In addition, the fluorescent dye, propidium iodide (PI)
which becomes highly fluorescent after binding to DNA, can be used in the same man-
ner. The stained and unstained cells are counted with a standard light microscope (try-
pan blue), or flow cytometer (PI) (Table 9).
Profile of DNA content

If cells are permeabilized, the LMW DNA inside the cytoplasm of apoptotic cells leaks out
during the subsequent rinse and staining procedure. The lower DNA content of these
cells means they contain less DNA stained by the fluorochrome. Thus, cells with lower
DNA staining than that of G1 cells (the so-called sub-G1 peaks, A0 cells) have been
considered apoptotic. The reduction in staining/DNA content of these cells is measured
by flow cytometry (Figure 37). The major disadvantage of this technique is that apoptotic
G2-Phase cells exhibit a reduced DNA content, which could represent the DNA content
of a G1-cell. Therefore it may not be detected as apoptotic. This would result in an under-
estimation of the apoptotic population.
DNA-binding Dye enters Dye stains

dyes (Fluoro-
chromes)
Viable cells Non viable cells Nucleus (DNA) Cytoplasm (RNA)
Acridine orange Yes Yes Green Red-orange
Hoechst 33342 Yes Yes Blue No
Hoechst 33258 No Yes Blue No
DAPI No Yes Bright blue No
Ethidium No Yes Orange Slightly red
bromide
Propidium No Yes Red No
iodide
Table 9: Common fluorochromes used to stain the genomic DNA of viable and/or non-viable cells.

A Figure 37: Typical flow cytometric profile of the DNA content in normal (A) and apoptotic cells (B),
stained with PI.
Result: A prominent sub-G1 peak (earliest peak) appears in apoptotic cells, but not in normal cells.
Morphological changes
On the other hand, the bisbenzimidazole dye, Hoechst 33342 (and also acridine orange),
penetrates the plasma membrane and stains DNA in cells; without permeabilization. In
contrast to normal cells, the nuclei of apoptotic cells have highly condensed chromatin
that is uniformly stained by Hoechst 33342. This can take the form of crescents around
the periphery of the nucleus, or the entire nucleus can appear to be one or a group of fea-
tureless, bright spherical beads. These morphological changes in the nuclei of apoptotic
cells may be visualized by fluorescence microscopy. They are also visible in permeabilized
apoptotic cells stained with other DNA binding dyes like DAPI (Figure 38).
Dive et al.30 have reported that during a short exposure to Hoechst 33342, apoptotic cells
have stronger blue fluorescence compared to non-apoptotic cells. Co-staining of the cells
with propidium iodide (PI) allows the discrimination of dead cells from apoptotic cells. If
7-amino-actinomycin is used instead of PI, cell surface antigens immunostained with flu-
orescein and phycoerythrin may be quantitated simultaneously31.
One drawback of using any vital staining method for measuring apoptosis is the variabil-
ity of active dye uptake in different cells and its possible change during certain treat-
ments. Therefore, the ability of Hoechst 33342 to discriminate apoptotic cells from
normal cells by increased uptake of dye has to be tested for each new cell system31.
Reagent Cat. No. Pack Fluorescence Typical results

size
Propidium iodide* 11 348 639 001 20 ml red orange See Table 9,
Figure 33 and 34
DAPI 10 236 276 001 10 mg blue See Table 9 and
4,6-Diamidine-2-phenylindole Figure 38
dihydrochloride
Table 10: Fluorescent dyes that stain double-stranded DNA

*Only sold in the US

A
Figure 38: Fluorescent microscopic analysis of apoptotic cells stained with DAPI. DAPI stains the
nucleic of all cells (blue).
Result: The characteristic condensed nuclei of apoptotic cells are clearly visible here.
Figure 39: Fluorescent microscopic analysis of mitotic cells stained with ethidium bromide. DNA was
stained with ethidium bromide (orange). Mitotic spindles were stained with anti-tubulin antibody (green).
Result: Mitotic cells (with condensed DNA) are brightly stained. Without the double stain, mitotic cells could be
mistaken for apoptotic cells, since both have condensed DNA.

Apoptosis Assay Methods Apoptosis Assay Methods

Methods for studying apoptosis in individual cells Methods for studying apoptosis in individual cells
2.2.4 Summary of methods for studying apoptosis in individual cells
Method/Roche Applied Science Parameter analyzed Assay principle Advantages Limitations For
product product
informa-
tion, see
Staining of chromosomal DNA DNA fragmentation Apoptotic cells are permeabilized with ethanol or deter- Quick and cheap Degree of extraction of LMW DNA during the washing page 49
after permeabilization (DNA (HMW DNA, DNA content) gent. During this procedure the LMW DNA inside the Minimal overlap between the peak representing apoptotic and staining procedure not always reproducible of this guide
content)22, 32 apoptotic cell leaks out and is removed during the sub- (sub G1) and normal G1 cells Not specific for apoptosis: Sub G1 peak can also
A A
sequent washing steps. Estimation of position in cell cycle allows cell cycle speci- represent mechanically damaged cells, cells with
DAPI, propidium iodide* The HWM DNA retained in the cells is stained with a ficity of apoptosis to be studied different chromatin structure, or normal cells with lower
DNA binding dye such as propidium iodide. Applicability to any DNA fluorochrome and instrument DNA content in heterogeneous cell populations
The amount of HMW DNA is quantified by flow cyto- May not detect cells induced to apoptosis in G2
metry (sub G1 or A0 peak). No discrimination of apoptotic cells from dead cells
which have lost their membrane integrity
Staining of chromosomal DNA Chromatin morphology Apoptotic cells are stained by the addition of DNA Quick and cheap No quantitative measurement
fluorochromes which are able to cross the intact plasma Discrimination between viable and dead cells when Subjective: no clear cut-off point between normal and
membrane, such as acridine orange. Can be done with counterstained with propidium iodide using vital dyes apoptotic cells
DAPI on fixed cells. (acridine orange, Hoechst 33342) Clear morphologically distinct apoptotic nuclei appear
The stained DNA allows the altered morphology of the Simultaneous staining of cell surface antigens with late during apoptosis: May lead to an underestimation of
nuclear chromatin to be visualized by fluorescence standard fluorescein and phycoerythrin conjugates apoptotic cells
microscopy. possible if Hoechst 33342 is combined with 7-amino-
actinomycin D
Active labeling of cells by nick DNA strand breaks (nicks) and DNA Apoptotic cells are fixed with formaldehyde and Counterstaining with DNA fluorochrome (profile of DNA Labor-intensive and time-consuming; only a few tests
translation (ISNT)34 fragmentation (staggered DNA ends) subsequently permeabilized. content) allows cell cycle specificity of apoptosis to be may be performed simultaneously
DNA strand breaks are labeled with modified studied (only by flow cytometry) Undefined cell loss during fixation procedure (loss of
nucleotides using exogenous DNA polymerase (nick Identification of apoptosis at a molecular level (DNA specific cell population?)
translation). strand breaks) Many cells (25 x 106/test) required
The incorporated nucleotides are visualized with a sec- Suitable for tissue sections
ondary detection system which has a reporter molecule
(e.g. fluorescein, AP, POD).
Active labeling of cells by end DNA strand breaks (nicks) and DNA Apoptotic cells are fixed with formaldehyde and Advantages like in situ nick translation; in addition: Labor-intensive and time-consuming; only a few tests pages 36-42
labeling (TUNEL)20, 35 fragmentation (staggered DNA ends) subsequently permeabilized. More sensitive: maximum intensity of labeling (cell may be performed simultaneously of this guide
DNA strand breaks are labeled with modified staining) of apoptotic cells is higher compared to ISNT Many cells (25 x 106/test) required
In Situ Cell Death Kit, Fluorescein, nucleotides using exogenous terminal transferase Fast kinetics of dUTP incorporation in comparison with the Undefined cell loss during fixation procedure
TMR, AP, POD (end labeling). DNA polymerase method: 30 min is sufficient for the (loss of specific cell population?)
The incorporated nucleotides are visualized directly labeling reaction
(e.g. Fluorescein-dUTP) or with a secondary detection Higher sensitivity for apoptosis: TUNEL preferentially
system which has a reporter molecule (e.g. fluorescein, labels apoptosis in comparison to necrosis
AP, POD). Direct detection possible by the use of Fluorescein-dUTP
(without secondary detection system) for maximum
sensitivity and minimal background
With the direct TUNEL labeling assay less working steps
are required than with the indirect assay.
Detection of translocated Detection of phosphatidylserine on Apoptotic cells are incubated with an assay protein (e.g. Unique marker for apoptosis related plasma membrane Not specific for apoptosis: Annexin-V can stain inner pages 44-48
membrane component27 surface of apoptotic cells annexin-V) conjugated to a reporter molecule. changes membrane of ruptured cells; must distinguish apoptotic of this guide
Assay protein binds a membrane component (e.g. Allows analysis by flow cytometry fluorescence from necrotic cells with an additional DNA stain
Annexin-V-FLUOS phosphatidylserine) found on the outer surface of microscopy, or light microscopy Many cells (106/test) required
Annexin-V-Biotin apoptotic cells only. Allows simultaneous labeling of other cell surface anti- Cannot be used on tissue sections or any fixed samples
Annexin-V-Staining Kit A DNA strain is added to distinguish necrotic cells gens
Annexin-V-Alexa 568 (permeable) from apoptotic cells (impermeable). Annexin-V-Biotin allows fixation following Annexin-V
Apoptotic cells are made visible by assay of reporter binding for further analysis of additional cellular
molecule in flow cytometer or under a microscope. parameters
Trypan Blue Exclusion Assay Damage/leakage of plasma Cells are incubated with dye. The classical standard method to distinguish viable from Each individual sample has to be counted: only a few
membrane Dead cells take up dye; living cells do not. dead cells by light microscopy tests may be performed simultaneously
Stained (dead or damaged) cells are determined under Quick and cheap Subjective evaluation
a light microscope. Only a small fraction of total cells from a cell population is Not suitable for detection of apoptosis
Dye binds to intracellular proteins of leaky cells. required Stains only necrotic cells or very late apoptotic cells
(secondary necrosis)
Propidium Iodide Exclusion Assay Damage/leakage of plasma Cells are incubated with fluorescent dye. The standard method to distinguish viable from dead cells Each individual sample has to be counted: only a few page 49
membrane Dead cells take up dye; living cells do not. by fluorescence microscopy and flow cytometry tests may be performed simultaneously of this guide
Propidium iodide solution* Stained (dead or damaged) cells are determined under Double labeling procedures possible: simultaneous Not specific for apoptosis
a microscope or by flow cytometry. detection of e.g. surface antigens
Dye binds to DNA of leaky cells. Quick and cheap
Only a small fraction of total cells from a cell population is
required
*Sold only in the US
Table 11: Methods for studying apoptosis in individual cells.
52 Apoptosis, Cell Death, and Cell Proliferation Manual Cell Death Apoptosis and Necrosis 53
Detection of apoptosis-related proteins
2.3 Detection of apoptosis-related proteins

There are a number of genes that regulate apoptosis. That is, the products of these genes
interfere with apoptotic pathways. Assays to detect the proteins encoded by these genes
can complement the assays described in the previous sections.
The study of apoptosis-regulating genes and gene products is still evolving. The picture
so far is complex (as summarized in Section A 1.3 of this guide). For instance, in some
cases, the same gene has an effect on both the survival of normal cells and the develop-
ment of cancers by preventing apoptosis36. A detailed discussion of the field is beyond
the scope of this guide, but is covered in a number of reviews36, 37. As an introduction to
A the field, we discuss the characteristics of a few of these apoptosis-regulating proteins.
The relationship of the ced (caenorhabditis elegans cell death) genes to apoptosis in the
nematode Caenorhabditis elegans has been extensively studied38. Of these, the ced-3 and
ced-4 genes39 encode proteins that must be active to initiate apoptosis. In contrast, the
ced-9 gene product protects cells from apoptotic cell death, ensuring their survival40. In
other words, apoptosis is more likely when levels of ced-3 and ced-4 protein are high or
when levels of ced-9 protein are low.
In mammalian systems, the Bcl-2 protooncogene serves much the same function as ced-
9, blocking the induction of cell death41. The Bcl-2 oncoprotein also protects against the
cytotoxic effects of certain drugs42.
The Bcl-2 protein can dimerize with itself or with the product of the bax gene43. The
presence of the bax protein seems to counteract the anti-apoptotic activity of Bcl-2. In
summary, apoptosis is more likely when bax protein levels are high or when Bcl-2 protein
levels are low.
Another mammalian gene product, p53, is a tumor suppressor because it induces apop-
tosis in potentially malignant cells44. Absence or mutation of the p53 gene product led to
malignant transformation and immortalization of the cell.
Increases in expression of a growth stimulating gene, the c-myc proto-oncogene, actually

induces apoptosis in the absence of other growth factors45, 46. High levels of the Bcl-2
protein can counteract the effect of the c-myc protein.
For the analysis of apoptosis-regulating proteins, Roche Applied Science offers an ELISA
kit for the detection of p53 in fluids or extracts and antibodies to detect c-myc and Fas
(CD95/Apo-1).
Cell surface receptor genes (APO-1/Fas/CD95), other growth-stimulating genes (e.g.,

Ras), and other tumor-suppressing genes (e.g., Rb) have also been implicated in the regu-
lation of apoptosis2, 37. The Fas (CD95/APO-1) molecule has originally been identified as
a cell surface receptor that could mediate apoptotic cell death of transformed cells and
cause regression of experimental tumors growing in nude mice. The function of Fas was
assessed by establishment of mouse cell transformants that constitutively expressed
human Fas. When the transformed cells were treated with the antibody to human Fas, the
cells died by apoptosis within 5 hours, which indicated that Fas can transduce an apop-
totic signal and that anti-Fas works as an agonist. The subsequent purification of human
APO-1 antigen and molecular cloning of its cDNA established the identity of APO-1 and
Fas. Meanwhile, numerous reports have shown a pivotal role of Fas in various physiolog-
ical and pathological processes. The Anti-Fas provided by Roche Applied Science is suit-
able for the induction of apoptosis as well as for the detection of the Fas receptor.

Anti-Fas (CD95/Apo-1)
Cat. No. 11 922 432 001 100 g
Type Monoclonal antibody, clone 2R2, IgG3, mouse

Useful for Apoptosis induction in Fas expressing cells
Samples Cell suspensions, adherent cells
Method Direct induction of apoptosis by adding antibody to cell cultures
A
Time Approx. 35 h (induction of apoptosis)
Significance of reagent: The antibody may be used for the induction of apoptosis in cell
cultures through Fas by imitating the Fas-ligand. The Fas (CD95/Apo-1) molecule has
been identified as a cell surface receptor that could induce apoptotic cell death of trans-
formed cells upon activation by its ligand and cause regression of experimental tumors in
mice.
Test principle: The antibody may be used for induction of apoptosis:
Add antibody (1 g/ml) into culture medium of Fas-bearing cells
Incubation for 35 hours
Detection of apoptosis by various assays
Antibody supplied as: Mouse monoclonal antibody (clone 2R2, IgG3) in cell culture
supernatant; sterile filtered.
Sensitivity: The antibody is suitable for induction of apoptosis at 0.5 g/ml in SKW6.4
and Jurkat cells. If secondary crosslinking with an anti-mouse IgG is used, the apoptosis
inducing concentration could be reduced to 100 ng/ml. In Fas transfectants apoptosis is
induced without crosslinking at 100 ng/ml. It has to be mentioned, that some Jurkat sub-
clones do not or only in high doses respond to Anti-Fas induction of apoptosis.
Specificity: The antibody was generated by immunizing mice with transformed murine
L-cells bearing recombinant human Fas-receptor. On Western blots, Anti-Fas binds the
human Fas/Apo-1 (CD95).
Can be used for:

Induction of apoptosis through the Fas-receptor

p53 pan ELISA

Cat. No. 11 828 789 001 96 tests
Type One-step sandwich ELISA, colorimetric

Useful for Quantitation of p53 protein, both wild-type and mutant forms
Samples Tissue homogenates, cell lysates, serum, or plasma
Method Lysis/homogenization of sample, followed by immunochemical
determination of p53 in a microplate
A Time Approx. 2.5 h
Significance of kit: This kit uses antibodies to quantitate the levels of human, mouse, or
rat p53 in smear/plasma, in tumor tissue, or in tumor cell lines. Wild type p53 is a protein
that suppresses malignant transformation by inducing apoptosis. Mutations of the p53
gene which increases its stability allow transformation (immortalization) of the cell and
are quite commonly found in malignancies. The p53 protein seems also to be involved in
cell death induced by cytotoxic drugs.
Test principle: The assay uses a one-step sandwich immunoassay (Figure 40) to detect
wild-type and mutant p53. The procedure (Flow Chart 11) involves:
Preparing sample (e.g., detergent lysis of cells, homogenization of tissue), followed by

centrifugation.
Transferring an aliquot of the sample supernatant to streptavidin-coated well of a

microplate (precoated with anti-p53-biotin).
Binding p53 in the supernatant with two antibodies, a biotin-labeled monoclonal anti-
p53 (capture antibody), which is pre-bound to the streptavidin-coated plate, and a
peroxidase-labeled polyclonal anti-p53 (detection antibody).
Washing the immobilized antibody-p53 complexes five times to remove sample

Incubating sample with peroxidase substrate (tetramethylbenzidine, TMB).
Determining the amount of colored product (and thus, of immobilized antibody-p53

complexes) spectrophotometrically.
Figure 40: How the p53 pan ELISA works.

Prepare sample (for example, cell lysate or tissue homogenate)
Centrifuge sample (10,000 x g) (10 min, RT)
Transfer aliquot of supernatant to Anti-p53 precoated microplate
Incubate supernatant with Anti-p53-peroxidase (1:15 diluted) (2 h, RT)
Wash microplate wells five times with washing buffer (5 x 1 min, RT)
Add substrate solution to wells and incubate (approx. 1020 min, RT)
Flow Chart 11: Assay procedure, p53 pan ELISA.

Add stop reagent to wells (1 min, RT)

A
Sensitivity: In 4 independent assays, the lower limit of detection for the assay was deter-
mined to be 9 pg/ml.
Specificity: The biotin-labeled capture antibody from mouse recognizes a conserved,

pantropic, denaturation stable antigenic determinant of the p53 protein (human, mouse,
rat). The peroxidase-labeled detection antibody is highly specific for wild-type and
mutant p53 from different species.

Tissue homogenates
Cell lysates
Serum or plasma
Kit contents
1. Anti-human-p53 pan, polyclonal, peroxidase-labeled
2. Human p53 standards (six)
3. Incubation buffer/Sample diluent, ready-to-use
4. Washing buffer, 10 x concentrated
5. TMB substrate solution
6. TMB stop solution
7. Streptavidin-coated microplate, precoated with monoclonal Anti-p53 (biotin-labeled)
8. Adhesive plate cover foils

Cytotoxicity Assay Methods
Relationship between cytotoxicity, apoptosis and necrosis
3. Cytotoxicity Assay Methods

3.1 Relationship between cytotoxicity, apoptosis and
necrosis
As discussed in Section A 1.1 of this guide, there are two experimentally distinguishable
mechanisms of cell death: necrosis, the accidental cell death that occurs when cells are
exposed to a serious physical or chemical insult, and apoptosis, the normal cell death
that removes unwanted or useless cells.
A In contrast to these two cell death processes, cytotoxicity does not define a specific cellu-
lar death mechanism. Rather, cytotoxicity is simply the cell-killing property of a chemical
compound (such as a food, cosmetic, or pharmaceutical) or a mediator cell (such as a
cytotoxic T cell), independent from the mechanisms of death.
Note: Cytotoxicity may also be used, as it is in this guide, to denote a laboratory method for
detecting dead cells, regardless of the mechanism of their death.
Figure 41: Schematic illustration of the three basic principles to assess plasma membrane leakage.
Example of cytotoxicity
A common example of cytotoxicity is cell-mediated cytotoxicity. Cells of the immunesys-
tem [such as cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated (LAK)
cells] can recognize and destroy damaged, infected and mutated target cells. Although the
recognition machinery used by these cells is very different, their mechanism of target cell
destruction may be very similar.
Two possible cytocidal mechanisms have been proposed for cell-mediated cytotoxicity:
(i) the apoptotic mechanism, in which the effector cell triggers an autolytic cascade in the
target cell and the genomic DNA fragments before cell lysis; and (ii) the lytic mechanism,
in which lytic molecules, notably perforin, are secreted by the effector cell into the inter-
cellular space and polymerize to form pores in the target cell membrane, leading to cell
lysis3, 47. These two mechanisms are not mutually exclusive and, quite possibly, are com-
plementary.

Methods for studying cytotoxicity
3.2 Methods for studying cytotoxicity

Most current assays for measuring cytotoxicity are based on alterations of plasma mem-
brane permeability and the consequent release (leakage) of components into the super-
natant or the uptake of dyes, normally excluded by viable cells (Figure 41) (see also
A 2.2.3, on page 49 Dye exclusion method). A serious disadvantage of such permeabil-
ity assays is that the initial sites of damage of many, if not most cytotoxic agents are intra-
cellular. Therefore, cells may be irreversibly damaged and committed to die and the
plasma membrane is still intact. Thus, these assays tend to underestimate cellular damage
when compared to other methods. Despite this fact, some permeability assays have been
A
widely accepted for the measurement of cytotoxicity.
Alternatively, dead cells are unable to metabolize various tetrazolium salts (see also Sec-
tion A 2.1.1). This allows the use of the colorimetric assays MTT, XTT, or WST-1 to mea-
sure cell survival. Apoptosis, however, is an active mode of cell death requiring the
metabolism of cells. Thus, like the permeability assays mentioned above, the colorimet-
ric assays may underestimate cellular damage and detect cell death only at the later stages
of apoptosis when the metabolic activity of the cells is reduced.
Regardless of this disadvantage, the colorimetric assays are very useful for quantitating
factor-induced cytotoxicity within a 24 to 96 h period of cell culture. However, these col-
orimetric assays are of limited value for measuring cell mediated cytotoxicity, since most
effector cells become activated upon binding to the target cells. This activation results in
an increased formazan production by the effector cell, which tends to mask the decreased
formazan production resulting from target cell death.
Note: Assays for cytotoxicity can be, and frequently are, used to measure cell necrosis.
3.2.1 Assays that measure plasma membrane leakage
Widely used standard methods for measuring plasma membrane leakage are based on the
uptake or exclusion of molecules with special light-absorbing or fluorescent properties.
Viable (intact plasma membrane) and dead (damaged plasma membrane) cells can be
discriminated by differential staining. Because dyes stain individual cells, each sample has
to be analyzed by flow cytometry or microscopy. This kind of single cell analysis is not
suitable if many different samples have to be measured. In contrast, assays which quanti-
tate plasma membrane disintegration in cell populations allow many different samples to
be handled simultaneously in a single MTP.
One group of standard assays performed in a MTP is based on the release of radioactive
isotopes ([51Cr], [3H]-thymidine, [3H]-proline, [35S]- or [75Se]-methionine, 5-[125I]-2-
deoxy-uridine) or fluorescent dyes (bis-carboxyethyl-carboxyfluorescein (BCECF) or cal-
cein-AM) from prelabeled target cells48, 49, 50. The disadvantages of such assays however,
are (i) the use of radioactive isotopes in most of them, (ii) the necessity for prelabeling of
the target cells, and (iii) the high spontaneous release of most labels from the prelabeled
target cells.
Another group of assays is based on the measurement of cytoplasmic enzymes released by

damaged cells. The amount of enzyme activity detected in the culture supernatant corre-
sponds to the proportion of lysed cells51, 52. Enzyme release assays have been described
for alkaline and acid phosphatase, for glutamate-oxalacetate transaminase, for glutamate
pyruvate transaminase, and for argininosuccinate lyase. However, their use has been
hampered by the low amount of those enzymes present in many cells and by the elaborate
kinetic assays required to quantitate most enzyme activities.

In contrast to the above mentioned cytoplasmic enzymes, lactate dehydrogenase (LDH) is

a stable cytoplasmic enzyme present in all cells. It is rapidly released into the cell culture
supernatant when the plasma membrane is damaged. With the Cytotoxicity Detection
Kit (see page 61), LDH activity can easily be measured in culture supernatants by a single
point assay. The use of a spectrophotometric microplate reader (ELISA plate reader)
allows the simultaneous measurement of multiple probes and thereby guarantees the easy
processing of a large number of samples (Figure 42).
Figure 42: Measurement of LDH activity () using the microplate format (see also Flow Chart 12).

Cytotoxicity Detection Kit (LDH)

Cat. No. 11 644 793 001 2000 tests
Type Colorimetric assay, microplate format

Useful for Quantitation of LDH activity released from damaged/dying cells
Samples Cell-free supernatants from cells in culture
Method Preparation of cell-free supernatant, followed by incubation of supernatant
with INT to form colored formazan, a product which may be quantitated
A
colorimetrically
Time 0.51 h (+ induction of cell death)
Significance of kit: The Cytotoxicity Detection Kit measures cytotoxicity and cell lysis by
detecting LDH activity released from damaged cells. The assay is performed in a 96-well
microplate. The kit can be used in many different in vitro cell systems where damage of
the plasma membrane occurs. Examples are:
Detection and quantification of cell mediated cytotoxicity.
Determination of mediator-induced cytolysis.
Determination of the cytotoxic potential of compounds in environmental and medical
research, and in the food, cosmetic, and pharmaceutical industries.
Determination of cell death in bioreactors.
Test principle: The assay is based on the cleavage of a tetrazolium salt when LDH is
present in the culture supernatant. The procedure involves:
Incubating the cells in culture to allow cell death to occur. An increase in the amount
of dead or plasma membrane-damaged cells during the assay results in an increase
of LDH in the culture supernatant.
Collecting the cell-free culture supernatant.
Adding the substrate mixture from the kit to the culture supernatant. Any LDH
released into the supernatant during Step 1 will reduce the tetrazolium salt INT to
formazan by a coupled enzymatic reaction. Thus, release of LDH into the supernatant
directly correlates to the amount of formazan formed in this step.
Quantitating the formazan dye formed in an ELISA plate reader. The formazan dye
formed is water-soluble and shows a broad absorption maximum at about 500 nm.
For a detailed overview of the steps involved in the procedure, see Figures 42 and 43 and
Flow Chart 12.
Incubate cells in a microplate or batch culture for a certain period of time

(optional: induction of death by treatment with compounds or test substances)
Centrifuge cells
Remove 100 ml supernatant and transfer it to another microplate
Add 100 l/well reaction mixture to each supernatant and incubate for 0.5 h
Measure absorbance using an ELISA plate reader
Flow Chart 12: Assay procedure, Cytotoxicity Detection Kit (LDH).

A Figure 43: Biochemistry of the Cyto-toxicity Detection Kit (LDH): In the first enzymatic reaction LDH
reduces NAD+ to NADH + H+ by oxidation of lactate to pyruvate; in the second enzymatic reaction the catalyst
(diaphorase) transfers H/H+ from NADH + H+ to the tetrazolium salt INT.

Cell-free supernatants obtained from cells cultured in 96-well microplates or batch
cultures.
Kit contents
1. Catalyst (Diaphorase/NAD + mixture)
2. Dye solution (INT and sodium lactate)
Note: To prepare the reaction mixture, mix catalyst with dye solution prior to use.
Other applications: For more examples of how the Cytotoxicity Detection Kit (LDH) can
be used in the lab, see Appendix, pages 142143.

A B
Figure 44. Determination of the cytolytic activity of allogen-stimulated cytotoxic T lymphocytes

(CTLs) using the Cytotoxicity Detection Kit (LDH). A) Spleen cells of C57/BI 6 mice (H-2b) were stimulated in
vitro with P815 cells (H-2d). Viable CTLs were purified and titrated in the microplate as described in the package
insert. Target cells (1 x 104 cells/well) were incubated in the presence or absence (effector cell controls) of effec-
tor cells for 4 hours. Culture supernatant samples (100 l/well) for effector controls ( and the effector-target
cell mix () were assayed for LDH activity. The middle curve is generated when the background control values
A
are subtracted from the effector-target cell values (
). B) Calculated percentage of cell-mediated lysis.
Figure 45: Correlation of cell death (defined by increased plasma membrane permeability) and LDH
release. Ag8 cells (starting cell concentration: 2 x 105 /ml) were cultured and after 1, 2, 3 and 5 days, aliquots
were removed. The amount of viable () and dead () cells was determined by trypan blue exclusion. LDH activ-
ity in cell free culture supernatant was determined using the Cytotoxicity Detection Kit ().
Result: Increased LDH release clearly correlated with the increase of dead cells.

Cellular DNA Fragmentation ELISA

Cat. No. 11 585 045 001 500 tests
Type Sandwich ELISA, colorimetric

Useful for Quantitation of BrdU-labeled DNA fragments either released from cells
during necrosis or cell-mediated cytotoxicity, or within the cytoplasm of
apoptotic cells
Samples Cell-free supernatants from cultured cells or cytoplasmic lysates of cells,
A
prelabeled with BrdU
Method Prelabeling of cells with BrdU, followed by immunodetection of BrdU-
labeled DNA fragments in sample
Time 4.55.5 h (+ BrdU labeling and induction of cell death)
Significance of kit: The Cellular DNA Fragmentation ELISA measures apoptosis, necro-
sis, or cell mediated cytotoxicity by quantitating the fragmentation and/or release of
BrdU-labeled DNA. The kit detects DNA fragments:
In the cytoplasm of apoptotic cells, thus providing a non-radioactive alternative to the
[3H]-thymidine-based DNA fragmentation assay.
Released into the culture supernatant during cell mediated cytotoxicity, thus providing
a non-radioactive alternative to the [3H]-thymidine- and [51Cr]-release assays.
Test principle: The assay is a sandwich enzyme-linked immunosorbent assay (ELISA). It

uses two mouse monoclonal antibodies: one directed against DNA the other against
BrdU (Figure 46). The procedure (Flow Chart 13) involves:
Prelabeling of cells with BrdU.
Incubating the labeled cells in the presence of either an apoptosis inducing agent or
effector cells (for cell mediated cytotoxicity). At the end of the incubation, cells are
centrifuged and either supernatant is analyzed (for cell mediated cytotoxicity or
necrosis) or cellular lysate is analyzed for apoptosis. The supernatant, containing
LMW-DNA is used for the assay. If desired, both sample types can be prepared and
assayed (see Flow Chart 13).
Adsorbing the Anti-DNA antibody onto the wells of a microplate.
Adding the supernatant of Step 2 to the microplate. BrdU-labeled DNA fragments in

the sample bind to the immobilized Anti-DNA antibody.
Denaturing the immunocomplexed BrdU-labeled DNA-fragments by microwave

irradiation or nuclease treatment. This procedure is necessary for the accessibility of
the BrdU antigen.
Reacting Anti-BrdU antibody peroxidase conjugate (Anti-BrdU-POD) with the

BrdU-labeled DNA to form an immunocomplex.
Quantitating the bound Anti-BrdU-POD in the immunocomplex with a peroxidase

substrate (TMB).

Figure 46: How the Cellular DNA Fragmentation ELISA works.
Sample preparation:
Label cells in tissue culture flask with BrdU (218 h, 37C)
A
Aliquot BrdU-labeled cells into the microplate
Measurement of apoptosis Measurement of cell mediated cytotoxicity

or necrosis
Add cell death-inducing agent Add effector cells
Incubate cells (124 h, 37C)
Add lysis buffer and incubate (30 min, RT)
Centrifuge microplate (10 min, 250 x g)
Remove supernatant and analyze by ELISA
ELISA
Coat microplate with Anti-DNA antibody (1 h, RT)
Recoat microtiter plate with blocking solution (30 min, RT)
Wash microplate, then add supernatant and incubate (90 min, RT)
Option 1 Option 2
Wash microplate, then denature/fix sample by Wash microplate, then add nuclease and incubate
microwave radiation (5 min) (30 min, 37C)
Wash MTP
Add Anti-BrdU-POD and incubate (90 min, RT)
Wash microplate, then add substrate and incubate (1030 min, RT)
Measure absorbance using an ELISA plate reader (2 min, RT)
Flow Chart 13: Assay procedure, Cellular DNA Fragmentation ELISA.

Label cells with BrdU
Add cell death-inducing agent
Split sample into two parts
Centrifuge cells
Remove supernatant totally Transfer 100 ml supernatant into Anti-DNA
A
precoated microplate
Lyse cells Assay supernatant for BrdU-labeled DNA
Centrifuge lysate (to pellet HMW DNA) Result: Measurement of necrosis
Assay supernatant for oligonucleosomes containing

BrdU-labeled LMW DNA
Result: Measurement of apoptosis
Flow Chart 14: Simultaneous analysis of apoptosis and necrosis in the same sample with the
Cellular DNA Fragmentation ELISA.
Sensitivity
Apoptosis: When HL60/CAM is used as a model system for apoptosis, the ELISA can
detect BrdU-labeled DNA fragments in the cytoplasm of 1 x 103 cells/well (Figure 42).
Cell mediated cytotoxicity: When allogeneic-stimulated cytotoxic T cells are used as

effector cells to lyse P815 target cells in a cell mediated cytotoxicity assay, the ELISA can
detect BrdU-labeled DNA fragments from 2 x 103 target cells/well.
Note: The ability to detect a minimum number of dying/dead cells in a particular sample
strongly depends on the kinetics of cell death, the cytotoxic agent or the effector cells used to
induce cell death, and the amount of BrdU incorporated into the target cells.
Specificity
The Anti-DNA antibody binds to single-and double-stranded DNA. It shows no cross-
reactivity with BrdU.
The conjugated antibody (Anti-BrdU-POD, Fab fragments) will bind to BrdU-labeled

DNA after the DNA is partially denatured. The antibody specifically recognizes
5-bromo-2-deoxy-uridine. The antibody conjugate shows no cross-reactivity with
any endogenous cellular components such as thymidine or uridine.
The ELISA specifically detects BrdU-labeled DNA fragments in culture supernatant

and cytoplasm. The ELISA can detect BrdU-labeled DNA from any species, so the
assay is not species-restricted.

Culture supernatant and cytoplasmic fractions (lysates) of cells whose DNA have been
metabolically prelabeled with BrdU (e.g., cell lines and other in vitro proliferating
cells). Thus, only cells which proliferate in vitro can be used.

Kit contents
1. Anti-DNA antibody (clone M-CA-33)
2. Anti-BrdU-POD, Fab fragments (clone BMG 6H8)
3. Coating buffer
4. Washing buffer
5. Incubation buffer
6. Substrate solution
7. BrdU labeling reagent
8. Adhesive cover foils
A
Figure 47: Kinetics of camptothecin (CAM) induced cell death in HL60 cells. Cells were prelabeled
with BrdU overnight. Then, cells (1 x 104/well) were incubated either in the presence of 200 ng/ml CAM (, ) or
without CAM (, ) for 18 h. Supernatant (100 l/well) was removed, then cells were lysed and both superna-
tant (, ) and lysate (, ) were analyzed by Cellular DNA Fragmentation ELISA.
Result: Apoptosis clearly occurs after 34 h incubation. After 68 h, secondary necrosis begins to be seen.
Figure 48: Kinetics of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity in P815 target cells quan-
tified with the Cellular DNA Fragmentation ELISA. 2 x 104 BrdU-labeled target cells/well were incubated with
CTLs at different effector-to-target ratios (E/T) for varying times. After incubation for 1 h (), 2 h (), 4 h (), and
6 h (), culture supernatant samples (100 l/well) were assayed for DNA fragments.
Other applications: For more examples of how the Cellular DNA Fragmentation ELISA
can be used in the lab, see Appendix, page 143.

3.2.2 Assays that measure metabolic activity
Living (metabolically active) cells reduce tetrazolium salts to colored formazan com-
pounds; dead cells do not. Thus, tetrazolium salt-based colorimetric assays detect viable
cells exclusively. Because they are sensitive, these assays can readily be performed in a
microplate with relatively few cells.
Since a cytotoxic factor will reduce the rate of tetrazolium salt cleavage by a population of
cells, these metabolic activity assays are frequently used to measure factor-induced cyto-
toxicity or cell necrosis53, 54. Applications include:
A
Assessment of growth-inhibitory or cytotoxic effects of physiological mediators
(Figure 49).
Analysis of the cytotoxic and cytostatic effects of potential anti-cancer and other drugs
(Figure 50).
Analysis of cytopathic effects of viruses and screening of compounds with potential
anti-viral activity.
Screening of antibodies for growth-inhibiting potential.
Roche Applied Science offers three microplate-based metabolic activity assays. All may be
used to assay factor-induced cytotoxicity or necrosis. They are:
Cell Proliferation Kit I (MTT), Cat. No. 11 465 007 001, in which metabolically active
cells cleave the tetrazolium salt MTT to a water-insoluble formazan that can be solubi-
lized and quantitated with an ELISA plate reader (for a more detailed description of
this kit, see page 85 in this guide).
Cell Proliferation Kit II (XTT), Cat. No. 11 465 015 001, in which metabolically active
cells cleave the modified tetrazolium salt XTT to a water-soluble formazan, which may
be directly quantitated with an ELISA plate reader (for a more detailed description of
this kit, see page 86 in this guide).
Cell Proliferation Reagent WST-1, Cat. No. 11 644 807 001, a modified tetrazolium
salt that can be cleaved by metabolically active cells to a water-soluble formazan, which
may be directly quantitated with an ELISA plate reader (for a more detailed descrip-
tion of this reagent, see page 87 in this guide).
Note: Since proliferating cells are metabolically more active than non-proliferating (resting)
cells, these tetrazolium salt-based assays are also frequently used to measure cell activation
and proliferation. For a full discussion of this application, see Section B 2.1.1 on page 82 of
this guide.
For a more complete discussion of the principles behind these metabolic assays, see the
topic, Biochemical and cellular basis of cell proliferation assays that use tetrazolium
salts (Appendix, page 121) in this guide.

A
Figure 49: Measurement of the cytotoxic effects of human Tumor Necrosis Factor alpha (hTNF-)
on the mouse fibrosarcoma cell line WEHI-164. Cells in culture (106 cells/ml) were preincubated with actino-
mycin C (1 g/ml) for 3 h. Aliquots of these pretreated cells were transferred to a microtiter plate (5 x 104 cells/
well) and incubated with actinomycin C and various amounts of hTNF-alpha for 24 h. Cellular response to TNF
was measured with the Roche Applied Science Cell Proliferation Kit II (XTT) and plotted against TNF concentra-
tion.
Result: Under the assay conditions, 50% of the WEHI-164 cells were killed by a TNF concentration of
3540 pg/ml.
Figure 50: Differentiation of cytotoxic and cytostatic effects of actinomycin D. A549 cells were added
to a microtiter plate (104 cells/well) and incubated with () or without () actinomycin D (10 ng/ml) for 20 h.
Graph A: Some aliquots of actinomycin-treated cells were assayed for cytotoxic effects (changes in metabolic
activity). These cells were assayed with either the Cell Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT), or
Cell Proliferation Reagent WST-1 (WST). Cells were incubated with each tetrazolium salt for 4 h, then analyzed on
an ELISA plate reader.
Graph B: Other aliquots of actinomycin-treated cells were assayed for cytostatic effects (suppression of DNA
synthesis). These cells were incubated with either non-radioactive bromodeoxyuridine (BrdU) or tritiated thymi-
dine ([3H]-TdR). Incorporation of BrdU into DNA was determined with the Cell Proliferation ELISA, BrdU (colori-
metric). Incorporation of [3H]-TdR into DNA was determined by liquid scintillation counting.
Result: Although actinomycin D is not significantly cytotoxic (as indicated by graph A) under these conditions, it
does have a profound cytostatic (proliferation-inhibiting) effect (as indicated by graph B).

Cytotoxicity Assay Methods Cytotoxicity Assay Methods

Methods for studying cytotoxicity Methods for studying cytotoxicity
3.2.3 Summary of methods for studying cytotoxicity
Method/Roche Applied Science Label Parameter Assay principle Advantages Limitations For
product analyzed product
informa-
tion, see
[51Cr] Release Assay55 [51Cr] prelabel Damage/ Viable cells are incubated with Na2[51Cr]O4, which Labeling of proteins by [51Cr] generally independent of the Radioactive isotope
leakage of binds tightly to most intracellular proteins (prelabeling). rate of protein synthesis Requires prelabeling and extensive washing of the target
plasma After washing, cells are incubated with cytotoxic agent. Generally not restricted to target cell type: non-proli- cells
A A
membrane During this period, labeled proteins are released into ferating or slow turn-over populations can be studied High spontaneous release: assay limited to cytotoxic
the culture supernatant (SN) due to plasma membrane Quantitative measurement over a large logarithmic range events causing high cell lysis over short period of time
damage. Measurement of cell death in mixed cell populations; may (25 h)
The radioactivity in the SN is determined with a be used to quantitate cell-mediated cytotoxicity For proper intracellular binding [51Cr]6+ has to be
gamma-counter. converted to [51Cr]3+: cells with low metabolic activity
may not label sufficiently
[3H]-Thymidine ([3H]-TdR) [3H]-TdR prelabel Damage/ Cells proliferating in vitro are incubated with [3H] TdR, Quantitative measurement over a large logarithmic range Radioactive isotope
Release Assay56 leakage of which is incorporated into the genomic DNA. Low spontaneous release: cytotoxic events causing low Requires prelabeling and extensive washing of the target
plasma After they are washed, cells are incubated with a cell lysis over a prolonged period of time (824 h) can be cells
membrane and cytotoxic agent. During this period, [3H] labeled DNA is studied Limited to target cells proliferating in vitro
DNA fragmenta- released into the culture SN due to plasma membrane Measurement of cell death in mixed cell populations; may
tion damage. be used to quantitate cell-mediated cytotoxicity
The radioactivity in the SN and in the pellet is
determined with a scintillation -counter.
DNA Release Assay, BrdU prelabel Damage/ Cells proliferating in vitro are incubated with BrdU, Sensitive (103104 cells/test required) Prelabeling of the target cells required page 64
nonradioactive leakage of which is incorporated into the genomic DNA. Labeled cells do not have to be washed Can only assay target cells proliferating in vitro of this guide
plasma Cells are incubated with a cytotoxic agent or non- Optimal for microplate format Narrow range of quantitative measurement
Cellular DNA Fragmentation membrane and labeled effector cells (for cell-mediated cytotoxicity). Non-radioactive (only one order of magnitude)
ELISA DNA fragmenta- During this period, BrdU-labeled DNA is released into Measurement of cell death in mixed cell populations; may
tion the cytoplasm of apoptotic cells or into the culture SN be used to quantitate cell-mediated cytotoxicity
due to plasma membrane leakage of damaged cells. Possible to measure apoptosis and necrosis in parallel
The BrdU-labeled DNA in the SN or cytoplasm is deter-
mined with an enzyme linked immunosorbent assay.
LDH Release Assay51 none Damage/ Target cells are incubated with cytotoxic agent. During Constitutively expressed ubiquitous protein: assay gener- LDH activity in serum: special assay medium (reduced page 61 of
leakage of this period, cytoplasmic LDH is released into the culture ally not restricted by target cell type serum concentrations or BSA instead of serum) required this guide
Cytotoxicity Detection Kit, LDH plasma SN due to plasma membrane damage. Does not require prelabeling and extensive washing of the Spontaneous release of LDH by target cells and effector
membrane The LDH activity in the culture SN is measured by a target cells cells: assay limited to cytotoxic events causing high cell
substrate reaction and quantitated with an ELISA plate lysis over short period of time (28 h)
reader.
Metabolic activity assays BrdU Reduced Dye solution is added to cells cultured in MTP and cells No cell type restriction Increased metabolic activity of effector cells may mask pages
metabolic activity are incubated. Viable (metabolically active) cells cleave Does not require prelabeling and washing of the cells target cell death during cell-mediated cytotoxicity 8587 of this
Cell Proliferation Kit I (MTT), Kit II tetrazolium salts to colored formazan compounds; dead Optimal for microplate format No changes in the metabolic activity during the early guide
(XTT), Reagent (WST-1) cells do not. phases of apoptosis
Amount of formazan is quantitated with an ELISA plate
reader.
Table 12: Methods for studying cytotoxicity.
70 Apoptosis, Cell Death, and Cell Proliferation Cell Death Apoptosis and Necrosis 71
Seite 72 - Vakat
B
Cell Proliferation
and Viability
Introduction
Terminology of cell proliferation and viability
1. Introduction
Rapid and accurate assessment of viable cell number and cell proliferation is an impor-
tant requirement in many experimental situations involving in vitro and in vivo studies.
Examples of where determination of cell number is useful include the analysis of growth
factor activity, serum batch testing, drug screening, and the determination of the cyto-
static potential of anti-cancer compounds in toxicology testing. In such toxicological
studies, in vitro testing techniques are very useful to evaluate the cytotoxic, mutagenic,
and carcinogenic effects of chemical compounds on human cells.
1.1 Terminology of cell proliferation and viability

Usually, one of two parameters is used to measure the health of cells: cell viability or cell
proliferation. In almost all cases, these parameters are measured by assaying for vital
functions that are characteristic of healthy cells.
Cell Viability
Cell viability can be defined as the number of healthy cells in a sample. Whether the cells
are actively dividing or are quiescent is not distinguished. Cell viability assays are often
useful when non-dividing cells (such as primary cells) are isolated and maintained in cul-
ture to determine optimal culture conditions for cell populations.
The most straightforward method for determining viable cell number is a direct counting
of the cells in a hemocytometer. Sometimes viable cells are scored based on morphology
alone; however, it is more helpful to stain the cells with a dye such as trypan blue. In this
case, viability is measured by the ability of cells with uncompromised membrane integ-
B
rity to exclude the dye.
Alternatively, metabolic activity can be assayed as an indication of cell viability. Usually

metabolic activity is measured in populations of cells by incubating the cells with a tetra-
zolium salt (MTT, XTT, WST-1) that is cleaved into a colored formazan product by met-
abolic activity.
Cell Proliferation
Cell proliferation is the measurement of the number of cells that are dividing in a culture.
One way of measuring this parameter is by performing clonogenic assays. In these assays,
a defined number of cells are plated onto the appropriate matrix and the number of colo-
nies that are formed after a period of growth are enumerated. Drawbacks to this type of
technique are that it is tedious and it is not practical for large numbers of samples. In
addition, if cells divide only a few times and then become quiescent, colonies may be too
small to be counted and the number of dividing cells may be underestimated. Alterna-
tively, growth curves could be established, which is also time-consuming and laborious.
Another way to analyze cell proliferation is the measurement of DNA synthesis as a

marker for proliferation. In these assays, labeled DNA precursors (3H-thymidine or bro-
modeoxyuridine) are added to cells and their incorporation into DNA is quantified after
incubation. The amount of labeled precursor incorporated into DNA is quantified either
by measuring the total amount of labeled DNA in a population, or by detecting the
labeled nuclei microscopically. Incorporation of the labeled precursor into DNA is
directly proportional to the amount of cell division occurring in the culture.
Cell proliferation can also be measured using more indirect parameters. In these tech-
niques, molecules that regulate the cell cycle are measured either by their activity (e.g.,
CDK kinase assays) or by quantifying their amounts (e.g., Western blots, ELISA, or
immunohistochemistry).

Introduction
Cell Cycle
1.2 Cell Cycle

In an organism, the rate of cell division is a tightly regulated process that is intimately
associated with growth, differentiation and tissue turnover. Generally, cells do not
undergo division unless they receive signals that instruct them to enter the active seg-
ments of the cell cycle. Resting cells are said to be in the G0 phase (quiescence) of the cell
cycle (Figure 51). The signals that induce cells to divide are diverse and trigger a large
number of signal transduction cascades.
A thorough discussion of the types of signals and the variety of responses they can elicit
are beyond the scope of this guide (Table 13). Generally, signals that direct cells to enter
the cell cycle are called growth factors, cytokines, or mitogens.
Figure 51: Cell cycle: A schematic overview.
Abbreviation
RTK
RAS
RAF
Description
Receptor Tyrosine Kinase
GTP exchange protein
MAP kinase kinase kinase

Reference
Marshall, (1995) Specificity of receptor tyrosine kinase signaling: transient
versus sustained extracellular signal-regulated kinase activation. Cell 80:
179185.
White, M. A. et al. (1995) Multiple Ras functions can contribute to mamma-
lian cell transformation. Cell 80: 533541.
Avruch, J. et al. (1994) Raf meets Ras-Completing the framework of a sig-
B
nal transduction pathway. Trends Biochem. Sci. 19: 279283.
MEK MAP kinase kinase or Marshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and
MAPk/Erk kinase MAP kinase. Curr. Opin. Genet. Dev. 4: 8289.
MAPK Mitogen activated protein Marshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and
kinase or Erk MAP kinase. Curr. Opin. Genet. Dev. 4: 8289.
PKC Protein Kinase C Blobe, G. et al. (1996) Protein Kinase C isoenzymes: regulation and func-
tion. Cancer Surveys 27: 213248.
JAK Just Another Kinase or Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks
Janus Kinase and STATs. TIBS 19: 222227.
STAT Signal Transducers and Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks
Activators of Transcription and STATs. TIBS 19: 222227.
Cyclins Marx, J. (1994) How cells cycle toward cancer. Science 263: 319321.
CDK Cyclin Dependent Kinase MacLachlan, T. K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-depen-
dent kinases and cdk inhibitors: implications in cell cycle control and can-
cer. Crit. Rev. Eukaryot. Gene Expr. 5: 127156.
CDC2 Cell division cycle mutant MacLachlan, T. K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-depen-
dent kinases and cdk inhibitors: implications in cell cycle control and can-
cer. Crit. Rev. Eukaryot. Gene Expr. 5: 127156.
CAK CDK Activating Kinase Morgan, D. O. (1995) Principles of CDK Regulation. Nature 374: 131134.
Table 13: Published sources that contain more information about cell proliferation.
Cell Proliferation and Viability 75

Introduction
Cell Cycle
Signal Transduction Pathways

Three major types of signal transduction pathways are activated in cells in response to
growth factors or mitogenic stimuli. The response to these stimuli varies from cell type to
cell type and the pathways continue to grow more and more complex. These types of
pathways continue to be the focus of a great deal of research and, considering the impor-
tance of cell cycle regulation in biology, the pathways will continue to grow in complexity
for some time to come.
The MAP kinase (MAPK) type of pathways are triggered through a cascade of phos-
phorylation events that begins with a growth factor binding to a tyrosine kinase recep-
tor at the cell surface. This causes dimerization of the receptor and an intermolecular
cross-phosphorylation of the two receptor molecules. The phosphorylated receptors
then interact with adaptor molecules that trigger downstream events in the cascade.
The cascade works through the GTP exchange protein RAS, the protein kinase RAF
(MAPKKK), the protein kinase MEK (MAPKK), and MAP kinase (Erk). MAPK then
phosphorylates a variety of substrates that control transcription, the cell cycle, or rear-
rangements of the cytoskeleton.
The protein kinase C (PKC) pathways consist of a family of phospholipid dependent
protein kinases. PKC is regulated by a large variety of metabolic pathways involving
phospholipids and calcium levels within a cell. The main regulator of the pathway is
diacylglycerol (DAG) which appears to recruit PKC to the plasma membrane and
cause its activation. The activity of DAG is mimicked by the phorbol-ester tumor pro-
moters. Once activated, PKC can phosphorylate a wide variety of cellular substrates
that regulate cell proliferation and differentiation. Responses to PKC appear to vary
with the types of PKCs expressed and the types of substrates available within a cell.
Some evidence shows that the PKC pathway may interact with and exert effects
through the MAPK pathway.
The JAK/STAT pathway is activated by cytokine interaction with a family of receptors
B
called the cytokine receptor superfamily. These receptors do not contain a protein
kinase domain themselves, but they associate with and activate a family of protein
kinases called the JAK (Just Another Kinase or JAnus Kinase) family. JAK family mem-
bers are recruited to receptor complexes that are formed as a result of ligand binding.
The high concentration of JAK in the complex leads to a cross-phosphorylation of
JAK and thus activation. JAK then phosphorylates members of another protein family
called STAT (signal transducers and activators of transcription). These proteins then
translocate to the nucleus and directly modulate transcription.
Control of the Cell Cycle
Once the cell is instructed to divide, it enters the active phase of the cell cycle, which can
be broken down into four segments:
During G1 (G = gap), the cell prepares to synthesize DNA. In the latter stages of G1,
the cell passes through a restriction point (R) and is then committed to complete the
cycle.
During S phase the cell undergoes DNA synthesis and replicates its genome.
During G2 the cell prepares to undergo division and checks its replication using DNA
repair enzymes.
During M phase, the cell undergoes division by mitosis or meiosis and then re-enters
G1 or G0.

Introduction
Cell Cycle
In most instances, the decision for a cell to undergo division is regulated by the passage of
a cell from G1 to S phase. Progression through the cell cycle is controlled by a group of
kinases called cyclin-dependent kinases (CDKs), (see Figure 51). CDKs are thought to
phosphorylate cellular substrates, such as the retinoblastoma gene, that are responsible
for progression into each of the phases of the cell cycle. CDKs are activated by associating
with proteins whose levels of expression change during different phases of the cell cycle.
These proteins are called cyclins. Once associated with cyclins, CDKs are activated by
phosphorylation via CDK-activating kinase (CAKs) or by dephosphorylation via a phos-
phatase called CDC25.
D-types cyclins are the primary cyclins that respond to external cellular factors. Their lev-
els start off low during G1 and increase towards the G1/S boundary. Cyclin D regulates
CDK4 and CDK6. Cyclin E is expressed transiently during the G1/S transition and is rap-
idly degraded once the cell enters S. Cyclin E regulates CDK2 and perhaps CDK3. When S
phase begins, levels of cyclin A increase and activate CDK2. The cyclin A/CDK2 complex
is thought to have a direct role in DNA replication. The progression through mitosis
is regulated by the presence of cyclin B. Cyclin B associates with CDC2 and forms the pri-
mary kinase present during mitosis (MPF = M-phase/maturation promoting factor).
During anaphase cyclin B is degraded. This degradation of cyclin B appears to regulate the
cells progression out of mitosis and into G1.

Cell proliferation/viability assay methods
2. Cell proliferation/viability assay methods

A variety of methods have been devised that measure the viability or proliferation of cells
in vitro and in vivo. These can be subdivided into four groups:
Reproductive assays can be used to determine the number of cells in a culture that are
capable of forming colonies in vitro. In these types of experiments, cells are plated at
low densities and the number of colonies is scored after a growth period. These clono-
genic assays are the most reliable methods for assessing viable cell number57, 58, 59.
These methods, however, are very time-consuming and become impractical when
many samples have to be analyzed.
Permeability assays involve staining damaged (leaky) cells with a dye and counting via-
ble cells that exclude the dye. Counts can either be performed manually using a
hemocytometer and for example trypan blue (Figure 52). This method is quick, inex-
pensive, and requires only a small fraction of total cells from a cell population. There-
fore, this method is generally used to determine the cell concentration (cell number/
ml) in batch cell cultures. This is helpful in ensuring that cell cultures have reached the
optimal level of growth and cell density before routine sub-culture, freezing, or any
experiment60.
Or counts can be performed mechanically using for example a flow cytometer and pro-
pidium iodide. Alternatively, membrane integrity can be assayed by quantifying the
release of substances from cells when membrane integrity is lost, e. g., Lactate dehydroge-
nase (LDH) or 51Cr (described in section A 3.2.1 starting on page 59 of this guide).
Metabolic activity can be measured by adding tetrazolium salts to cells. These salts are
converted by viable cells to colored formazan dyes that are measured spectrophoto-
B
metrically (described in section B 2.1.1 starting on page 82 of this guide).
Direct proliferation assays use DNA synthesis as an indicator of cell growth. These
assays are performed using either radioactive or nonradioactive nucleotide analogs.
Their incorporation into DNA is then measured (nonradioactive assays are described
in section B 2.1.2 starting on page 89 of this guide).
Figure 52: Measurement of proliferation by counting the cells with a hemocytometer. The addition of
trypan blue helps to distinguish viable, unstained cells ( ) from non-viable, blue-stained cells ().

The first section describes those assays designed to study cell proliferation in whole pop-
ulations of cells, followed by a section covering proliferation assays designed to measure
proliferation in individual cells (in situ).
For a discussion of the advantages and limitations of all types of cell proliferation assays,
read Sections B 2.1.3 and B 2.2.2 of this guide.
For discussions of particular assays, turn to the pages indicated in the following method
selection guide.

Start
Are you studying

Cell Proliferation
or Cytotoxicity*
Are you studying

Cell Proliferation
in vivo in vitro
Are you studying
cell populations single cells
B
Do you want to measure
presence of cell
cycle associated DNA synthesis single
antigens cells
Are you studying
cell
populations

microscopy flow cytometry
Do you use
light microscopy fluorescence

microscopy
BrdU Labeling and Detection Kit II, AP 11 299 964 001 In Situ Cell Proliferation Kit, FLUOS 11 810 740 001
BrdU Labeling and Detection Kit I, 11 296 736 001
Fluorescein
80 Figure 53: Product Selection Guide Apoptosis, Cell Death, and Cell Proliferation Manual
yes
Do your cells proliferate in vitro ?
no Cytotoxicity Detection Kit (LDH) 11 644 793 001

cytotoxitic
substances
Are you studying Cell Proliferation Kit II (XTT) 11 465 015 001
cell-mediated Cell Proliferation Kit I (MTT) 11 465 007 001
cytotoxicity
yes
Do your cells proliferate in vitro ?
no
Cytotoxicity Detection Kit (LDH) 11 644 793 001
In Situ Cell Proliferation Kit, FLUOS 11 810 740 001

BrdU Labeling and Detection Kit I, 11 296 736 001
flow
cytometry
Fluorescein
microscopy
DNA fluorescence
synthesis microscopy
Do you want to measure Do you use
presence of cell
light
B
cycle associated
microscopy
antigens
BrdU Labeling and Detection Kit II, AP 11 299 964 001
cell cycle
associated
antigens
metabolic activity
of living cells
other Cell Proliferation Kit I (MTT) 11 467 007 001
cellular changes
DNA synthesis Cell Proliferation ELISA, BrdU 11 669 915 001

(chemiluminescent)
chemilumi-
nescent assay
Do you want to use a
colorimetric
assay
Cell Proliferation ELISA, BrdU 11 647 229 001
(colormetric)
BrdU Labeling and Detection Kit III, 11 444 611 001
POD
Methods for studying cell proliferation and viability in cell populations
2.1 Methods for studying cell proliferation and viability in

cell populations
A number of methods have been developed to study cell viability and proliferation in cell
populations. The most convenient modern assays have been developed in a microplate
format (96-well plates). This miniaturization allows many samples to be analyzed rapidly
and simultaneously. The microplate format also reduces the amount of culture medium
and cells required as well as cost of plasticware. Colorimetric assays allow samples to be
measured directly in the microplate with an ELISA plate reader.
Microplate assays have been developed based on different parameters associated with cell
viability and cell proliferation. The most important parameters used are metabolic activ-
ity and DNA synthesis for microplate format.
Cellular damage will inevitably result in loss of the ability of the cell to maintain and
provide energy for metabolic cell function and growth. Metabolic activity assays are
based on this premise. Usually they measure mitochondrial activity. The cells are
incubated with a colorometric substrate (MTT, XTT, WST-1) (described on pages 82-
87 of this guide.
As outlined above, during the S phase the cell undergoes DNA synthesis and replicates
its genome. If labeled DNA precursors, in our case BrdU, are added to the cell culture,
cells that are about to divide incorporate BrdU into their DNA. The incorporated
BrdU can then be detected by a quantitative cellular enzyme immunoassay using mon-
oclonal antibodies against BrdU (described on pages 88-107 of this guide).
In the following sections we will describe details of each of these cell viability and prolif-
eration assays.
2.1.1 Assays that measure metabolic activity
B
One parameter used as the basis for colorimetric assays is the metabolic activity of viable
cells. For example, a microtiter plate assay which uses the tetrazolium salt MTT is now
widely used to quantitate cell proliferation and cytotoxicity53, 65.
Because tetrazolium salts are reduced to a colored formazan only by metabolically active
cells, these assays detect viable cells exclusively. For instance, in the MTT assay, MTT is
reduced by viable cells to a colored, water-insoluble formazan salt. After it is solubilized,
the formazan formed can easily and rapidly be quantitated in a conventional ELISA plate
reader at 570 nm (maximum absorbance).
[Authors note: MTT is cleaved to formazan by the succinate-tetrazolium reductase system

(EC 1.3.99.1) which belongs to the mitochondrial respiratory chain and is active only in via-
ble cells. Interestingly however, recent evidence suggests that mitochondrial electron transport
may play a minor role in the cellular reduction of MTT. Since most cellular reduction occurs
in the cytoplasm and probably involves the pyridine nucleotide cofactors NADH and
NADPH, the MTT assay can no longer be considered strictly a mitochondrial assay.]
More recently, modified tetrazolium salts like XTT62, 67, MTT68, and WST-1 (Figure 54)
have become available. The major advantage of these compounds is that viable cells con-
vert them to a water-soluble formazan. Thus, a metabolic assay with any of these com-
pounds requires one less step (solubilization of product) than an assay with MTT. In
addition, WST-1 is stable enough to be packaged as a ready-to-use solution.

B
Figure 54: Molecular structure of MTT, XTT, WST-1 and their corresponding reaction products.
Since proliferating cells are metabolically more active than non-proliferating (resting)
cells, the assays are suitable not only for the determination of cell viability and factor-
mediated cytotoxicity (see Section A 3.2.2.) but also for the determination of cell activa-
tion and proliferation. However, one has to keep in mind that under non-ideal cell cul-
ture conditions (such as the pH and D-glucose concentration in culture medium), the
MTT response may vary greatly in viable cells due to the metabolic state of the cells (e.g.,
cellular concentration of pyridine nucleotides)65, 69.
These colorimetric assays are very rapid and convenient. Because this technique needs no
washing or harvesting of the cells, the complete assay from the start of the microculture
to data analysis in an ELISA plate reader is performed in the same microplate. In addi-
tion, an ELISA plate reader linked with a computer allows rapid and automated data pro-
cessing (Figure 55).

Roche Applied Science offers three microplate-based assays similar to the ones described
in this section. All three assays are suitable for measurement of cell proliferation in
response to growth factors, cytokines, mitogens and nutrients.
One of these assays uses MTT, which forms an insoluble formazan product; the other two
use tetrazolium salts (XTT and WST-1) that form soluble formazan products. All three
assays are described on the following pages.
Note: For a more detailed discussion of the principles behind these metabolic assays, see the
topic, Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts
(Appendix, page 121) in this guide.
B
Figure 55: Measurement of metabolic activity using the tetrazolium salts MTT, XTT and WST-1.

Cell Proliferation Kit I (MTT)

Cat. No. 11 465 007 001 2500 tests
Type Colorimetric, microplate format

Useful for Quantitation of cell viability and proliferation as well as cytotoxicity
Samples Adherent or suspension cell cultures
Method Incubation of cells with MTT, followed by solubilization and spectrophoto-
metric assay of colored product
Time 528 h
Significance of kit: The Cell Proliferation Kit I (MTT) measures the metabolic activity of
viable cells. The assay is nonradioactive and can be performed entirely in a microplate. It
is suitable for measuring cell proliferation, cell viability or cytotoxicity.
Test principle: The assay is based on the reduction of the tetrazolium salt MTT by viable
cells. The reaction produces a water-insoluble formazan salt which must be solubilized.
The procedure involves:
Culturing the cells in a 96-well microplate, then incubating them with MTT solution
for approx. 4 h. During this incubation period, viable cells convert MTT to a water-
insoluble formazan dye.
Solubilizing the formazan dye in the microplate.
Quantitating the dye with an ELISA plate reader. The absorbance directly correlates
with the cell number.

Adherent and suspension cells cultured in microplate.
Kit contents
1. MTT labeling reagent
2. Solubilization solution
B
Figure 56: Measurement of human Interleukin 6

(hIL-6) activity on the mouse hybridoma cell line
7TD1. Cells (2 x 103/well) were incubated in the pres-
ence of various amounts of hIL-6. After 4 days incuba-
tion, cell proliferation was analyzed by Cell Proliferation
Kit I (MTT).
Other applications: For more examples of how the Cell Proliferation Kit I (MTT) can be
used in the lab, see Appendix, page 144.

Cell Proliferation Kit II (XTT)

Cat. No. 11 465 015 001 2500 tests

Useful for Quantitation of cell viability, proliferation, or cytotoxicity
Method Incubation of cells with XTT, followed by spectrophotometric assay of
colored product
Time 4h
Significance of kit: The Cell Proliferation Kit II (XTT) measures the metabolic activity of
viable cells. The assay is nonradioactive and can be performed entirely in a microplate. It
is suitable for measuring cell proliferation, cell viability, or cytotoxicity.
Test principle: The assay is based on the reduction of the tetrazolium salt XTT by viable
cells in the presence of an electron coupling reagent. The reaction produces a soluble for-
mazan salt. The procedure involves:
Culturing the cells in a 96-well microplate, then incubating them with XTT solution for
approx. 4 h. During this incubation period, viable cells convert XTT to a water-soluble
formazan dye.
Quantitating the formazan dye in the microplate with an ELISA plate reader. The
absorbance directly correlates with the cell number.
B
Kit contents
1. XTT Labeling reagent
2. Electron-coupling reagent
Note: To prepare XTT labeling mixture, mix XTT labeling reagent with electron-coupling
reagent prior to use.
Typical results: see Figure 57 and 58.
Figure 57: Measurement of human tumor necrosis fac-

tor (hTNF) activity on the mouse fibrosarcoma cell
line WEHI-164. After preincubation of the cells (1 x 106/ml)
with actinomycin C (1 g/ml) for 3 h, cells (5 x 104/well) were
incubated in the presence of actinomycin C and various
amounts of hTNF for 24 h. The cellular response was ana-
lyzed by Cell Proliferation Kit II (XTT).
Other applications: For more examples of how the Cell Proliferation Kit II (XTT) can be

Cell Proliferation Reagent WST-1

Cat. No. 11 644 807 001 2500 tests

Useful for Quantitation of cell viability, proliferation, or cytotoxicity
Method Incubation of cells with WST-1, followed by spectrophotometric assay of
colored product
Time 0.54 h
Significance of reagent: The Cell Proliferation Reagent WST-1 is a ready-to-use substrate

which measures the metabolic activity of viable cells. The WST-1 assay is nonradioactive
and can be performed entirely in a microplate. It is suitable for measuring cell prolifera-
tion, cell viability or cytotoxicity.
Test principle: The assay is based on the reduction of WST-1 by viable cells. The reaction
produces a soluble formazan salt. The procedure involves:
Culturing the cells in a 96-well microplate, then incubating them with WST-1 for
approx. 0.54 h. During this incubation period, viable cells convert WST-1 to a water-
soluble formazan dye.
Quantitating the formazan dye in the microplate with an ELISA plate reader. The
absorbance directly correlates with the cell number.
For a detailed comparison of the WST-1 assay procedure with the MTT and XTT assays,
B
see Flow Chart 15 and Figure 58.

Figure 58: Comparison of the sensitivity of various tet-

razolium salts. P815 cells were preincubated at various con-
centrations for 20 h before MTT (), XTT () or Cell
Proliferation Reagent WST-1 () was added. After 4 h sub-
strate reaction, the absorbance was determined at the
respective wavelength with an ELISA plate reader.

Figure 59: Combined use of the Cell Proliferation

Reagent WST-1 and the Cell Proliferation ELISA, BrdU
(colorimetric) for the simultaneous measurement of
cell viability and cell proliferation. A549 cells (1 x 104/well
in 100 l) were incubated in the presence of various amounts
of actinomycin D for 20 h. After labeling the cells with BrdU
for 2 h, additionally Cell Proliferation Reagent WST-1 () was
added and cells were reincubated for another 2 h. Thereafter,
the formazan formed was quantitated at 450 nm with an
ELISA plate reader. Subsequently, BrdU incorporation was
determined using the Cell Proliferation ELISA, BrdU (colori-
metric) ().
Result: Actinomycin D inhibits DNA synthesis (), but it
does not inhibit the metabolic activity of the cell (). Thus,
actinomycin D is cytostatic (inhibition of DNA synthesis) but
not cytotoxic (no inhibition of metabolic activity).
Other applications: For more examples of how the Cell Proliferation WST-1 can be used
in the lab, see Appendix, pages 146147.
Culture cells in a MTP for a certain period of time (37C)
Kit I (MTT) Kit II (XTT) WST-1
Prepare XTT labeling mixture
B
Add MTT labeling reagent Add XTT labeling mixture Add Cell Proliferation Reagent
WST-1
Incubate cells (0.54 h, 37C)
Add solubilized solution and

incubate (124 h, 37C)
Flow Chart 15: Assay procedures for Cell Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT), and Cell Proli-
feration Reagent WST-1.

2.1.2 Assays that measure DNA synthesis
During cell proliferation the DNA has to be replicated before the cell is devided into two
daughter cells.
This close association between DNA synthesis and cell doubling (Figure 60) makes the
measurement of DNA synthesis very attractive for assessing cell proliferation. If labeled
DNA precursors are added to the cell culture, cells that are about to divide incorporate the
labeled nucleotide into their DNA. Traditionally, those assays involve the use of radiola-
beled nucleosides, particularly tritiated thymidine ([3H]-TdR). The amount of [3H]-TdR
incorporated into the cellular DNA is quantitated by liquid scintillation counting
(LSC)63, 70.
Figure 60: Cell proliferation, a close association between DNA synthesis and cell doubling.
Experiments have shown that the thymidine analogue 5-bromo-2-deoxy-uridine (BrdU)

is incorporated into cellular DNA like thymidine (Figure 61). The incorporated BrdU
could be detected by a quantitative cellular enzyme immunoassay using monoclonal anti-
bodies directed against BrdU64. The use of BrdU for such proliferation assays circum-
vents the disadvantages associated with the radioactive compound [3H]-TdR.
The first report of this technique involved the extraction and partial purification of DNA
from BrdU-labeled proliferating cells, followed by an enzyme immunoassay in a separate
B
assay71. Because this method was relatively laborious, the entire BrdU-based procedure
was adapted to a 96 well microplate72. This adaptation required no harvesting of the cells;
the complete assay from the start of the microculture to data analysis by an ELISA plate
reader was performed in the same microplate (Figure 62).
Figure 61: Molecular structure of thymidine and BrdU.

B Figure 62: Measurement of DNA synthesis using modified nucleotides [3H]-TdR and BrdU.
Roche Applied Science offers three kits that use the convenient BrdU-based assay and the
microplate format. The BrdU Labeling and Detection Kit III is a first generation assay.
The colorimetric and chemiluminescence Cell Proliferation ELISAs, are second genera-
tion assays that offer fewer steps, a faster assay, and greater sensitivity than the first gener-
ation assay (Table 14). These three kits are described on the following pages.

5-Bromo-2-deoxy-uridine Labeling and

Detection Kit III
Cat. No. 11 444 611 001 1000 tests
Type 1st generation ELISA with colorimetric detection

Useful for Quantitation of DNA synthesis during cell activation and proliferation
Sample Adherent or suspension cell cultures
Method Incubation of cells with BrdU, followed by partial digestion of DNA and
immunodetection of incorporated BrdU label
Time 2.56 h (+ cell labeling)
Significance of kit: The BrdU Labeling and Detection Kit III measures cell proliferation
by quantitating BrdU incorporated into the newly synthesized DNA of replicating cells. It
offers a nonradioactive alternative to the [3H]-thymidine-based cell proliferation assay.
Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal anti-
body directed against BrdU (Figure 63 and Flow Chart 16).
Note: This kit belongs to the first generation of kits used to measure DNA synthesis. The same
assay procedure has been optimized and improved in the second generation Cell Proliferation
ELISA, BrdU (colorimetric) kit (see Table 14).
B
Figure 63: How the BrdU Labeling and Detection Kit III works.

Label cells with BrdU (218 h, 37C)
Suspension cells Adherent cells
Remove labeling medium using a cannula Remove labeling medium by inverting the micro-
plate
Air dry cells (180 min, 60C)
Add fixative and incubate (30 min, 20C)
Wash microplate 3 times (15 min, RT)
Add Nuclease and incubate (30 min, 37C)
Add Anti BrdU-POD and incubate (30 min, 37C)
B
Flow Chart 16: Assay procedure, BrdU Labeling and Detection Kit III.
Sensitivity: The BrdU Labeling and Detection Kit III is almost as sensitive as the [3H]-
thymidine-based cell proliferation assay. The ability to detect a minimum number of pro-
liferating cells in a certain sample strongly depends on the amount of BrdU incorporated
into the cells and thus on the labeling period. In most cases, detection requires a labeling
period of 2 to 4 h.
Specificity: The antibody conjugate (Anti-BrdU-POD, Fab fragments) will bind to BrdU-
labeled DNA after the DNA is denatured. The antibody is conjugated to peroxidase and
specifically recognizes 5-bromo-2-deoxyuridine; it shows no cross-reactivity with any
endogenous cellular components such as thymidine or uridine.


Adherent cells as well as cells cultured in suspension in 96-well microplates (e.g., cell
lines, activated peripheral blood lymphocytes and other in vitro proliferating cells).
Kit contents
1. BrdU labeling reagent (1000 x), sterile 5. Nucleases
2. Anti-BrdU-POD Fab fragments 6. Substrate buffer
3. Incubation buffer (ready-to-use) 7. ABTS substrate tablets
4. Washing buffer (10 x) 8. Substrate enhancer
Typical results:
Figure 64: Proliferation of AKR-2B cells Figure 65: Proliferation of 7TD1 cells
(mouse fibroblast cell line) in response to (mouse-mouse hybridoma) in response to
recombinant human epidermal growth recombinant mouse interleukin-6 (mlL-6)
factor (hEGF) using the BrdU Labeling and using the BrdU Labeling and Detection Kit
B
Detection Kit III (), or the [3H]-thymi- III (), or the [3H]-thymidine method (),
dine method (), respectively. respectively.
Result: Figures 64 and 65 illustrate the equivalent sensitivity of the BrdU and [3H]-thymidine methods in measur-
ing proliferation, as shown in hEGF and mIL-6 stimulation assays.
Other applications: For examples of how the BrdU Labeling and Detection Kit III can be
Parameter BrdU Labeling and Cell Proliferation ELISA BrdU (colorimetric)

Detection Kit III Cell Proliferation ELISA, BrdU (chemilumines-
cence)
Incubation 3 2
steps
Washing steps 34 1
Working 6 (4 included in the kit) 4 (all included in the kit)
solutions
Assay time 2.56 h 1.52.5 h
Incubation 20C: Fixation For Cell Proliferation ELISA, BrdU (colorimetric) each
temperatures RT: Substrate reaction step at RT
37C: Nuclease treatment
60C: Air drying
Measuring Absorbance: 0.12.5 U Same as BrdU Kit III
range (factor 25) For Cell Proliferation ELISA, BrdU (chemiluminescence):
rlu/s: 103106 (factor 1000)
Sensitivity Almost as sensitive as As sensitive as [3H]-TdR
[3H]-TdR
Table 14: Improvements of the assay procedure using the Cell Proliferation ELISA, BrdU (colori-
metric) and Cell Proliferation ELISA, BrdU (chemiluminescence) described on the following pages.

Cell Proliferation ELISA, BrdU (colorimetric)

Cat. No. 11 647 229 001 1000 tests
Cell Proliferation ELISA, BrdU

(chemiluminescence)
Cat. No. 11 669 915 001 1000 tests
Type 2nd generation ELISAs with colorimetric or chemiluminescent detection

Useful for Quantitation of DNA synthesis during cell activation and proliferation
Method Incubation of cells with BrdU, followed by immunodetection of incorpo-
rated BrdU label
Time 1.52.5 h (+ cell labeling)
Note: These two kits belong to the second, improved generation of kits for measuring DNA
synthesis (see Tables 14 and 15)
Significance of the kits: The two Cell Proliferation ELISAs measure cell proliferation by
quantitating BrdU incorporated into the newly synthesized DNA of replicating cells.
They offer a nonradioactive alternative to the [3H]-thymidine-based cell proliferation
assay with comparable sensitivity.
Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal anti-
body directed against BrdU. The procedure (Figures 66 and 67, Flow Chart 17) involves:.

Culturing the cells in a 96-well microtiterplate and pulse-labeling them with BrdU.
Only proliferating cells incorporate BrdU into their DNA.
Fixing the cells with FixDenat solution. This FixDenat solution also denatures the
genomic DNA, exposing the incorporated BrdU to immunodetection.
Locating the BrdU label in the DNA with a peroxidase-conjugated anti-BrdU antibody
(anti-BrdU-POD).
Quantitating the bound anti-BrdU-POD with a peroxidase substrate. TMB is used as

a substrate in the Cell Proliferation, BrdU (colorimetric). Luminol/4-iodophenol is
used as a substrate in the Cell Proliferation, BrdU (chemiluminescence).
Figure 66: Principle of the Cell Proliferation ELISA, Figure 67: Principle of the Cell Proliferation ELISA, BrdU
BrdU (colorimetric). (chemiluminescent).

Label cells with BrdU (218 h, 37C)
Suspension cells Adherent cells
Remove labeling medium using a needle Remove labeling medium by inverting the
microplate
Air dry cells (10 min, hairdryer)
Add FixDenat solution and incubate (30 min, RT)
Tap microplate to remove FixDenat then add Anti-BrdU-POD and incubate (3090 min, RT)
Cell Proliferation ELISA, BrdU Cell Proliferation ELISA, BrdU

(colorimetric) (chemiluminescence)
Measure absorbance using an ELISA plate reader Measure light emission using a luminometer
(2 min, RT) (2 min, RT)
Flow Chart 17: Assay procedures for Cell Proliferation ELISA, BrdU (colorimetric) and Cell Proliferation
ELISA, BrdU (chemiluminescence).
Sensitivity: The Cell Proliferation ELISA BrdU (colorimetric) and Cell Proliferation
B
ELISA, BrdU (chemiluminescence) are as sensitive as the [3H]-thymidine-based cell pro-
liferation assay.
Note: The ability to detect a minimum number of proliferating cells in a certain sample
depends on the amount of BrdU incorporated into the cells and thus on the labeling period.
In most cases, detection requires a labeling period of 2 to 24 h.
The use of a chemiluminescence substrate allows the measurement of cell proliferation

over a broad range. This range is directly comparable to the measuring range of the [3H]-
thymidine-based cell proliferation assay.
Specificity: The anti-BrdU antibody peroxidase-conjugate (anti-BrdU-POD, Fab frag-

ments) will bind to BrdU-labeled DNA after the DNA is denatured. The antibody specifi-
cally recognizes 5-bromo-2-deoxyuridine; it shows no cross-reactivity with any
endogenous cellular components such as thymidine or uridine.

Adherent cells as well as cells in suspension cultured in 96-well microplates (e.g., cell
lines, activated peripheral blood lymphocytes and other in vitro proliferating cells).
Kit contents
Cell Proliferation ELISA, BrdU (colorimetric):
1. BrdU labeling reagent (1000 x), sterile
2. Anti-BrdU-POD Fab fragments
3. Antibody dilution solution (ready-to-use)
4. Washing buffer (10 x)
5. FixDenat (ready-to-use)
6. TMB substrate solution (ready-to-use)

Cell Proliferation ELISA, BrdU (chemiluminescence):

2. Anti-BrdU-POD Fab fragments
3. Antibody dilution solution (ready-to-use)
4. Washing buffer (10 x)
5. FixDenat (ready-to-use)
6. Substrate component A (luminol/4-iodophenol)
7. Substrate component B (peroxide)
Note: The FixDenat solution (included in the kits) is also available as a separate reagent
(Cat. No. 11 758 764 001, 4 x 100 ml [enough for 2000 tests]). This ready-to-use solution sim-
plifies detection of BrdU-labeled DNA in ELISA applications, since it simultaneously fixes
cells and denatures DNA to expose BrdU epitopes.
Typical results: see Figures 68 71.
Figure 68: Comparison of the sensitivity of the Figure 69: Comparison of the Cell Prolifera-
Cell Proliferation ELISA, BrdU (colorimetric) and tion ELISA, BrdU (colorimetric) and the radioac-
the radioactive thymidine incorporation assay for tive thymidine incorporation assay for measuring
measuring proliferation in various concentrations stimulation of various concentrations of mitogen.
B
of cells. Various concentrations of L929 cells were Human peripheral blood lymphocytes were cultured in
cultured in the wells of a microtiter plate. Duplicate the presence of varying concentrations of phytohe-
cultures of each cell concentration were labeled for 4 magglutinin (PHA) in the wells of a microtiter plate.
h with either bromodeoxyuridine (BrdU) or tritiated Duplicate cultures from each PHA concentration were
thymidine ([3H]-TdR). The cells were assayed for cell labeled for 4 h with either bromodeoxyuridine (BrdU)
proliferation with either the Cell Proliferation ELISA, or tritiated thymidine ([3H]-TdR). The cells were
BrdU (BrdU labeling, ) or a standard filtration/liquid assayed for cell proliferation with either the Cell Prolif-
scintillation counting protocol ([3H]-TdR labeling, ). eration ELISA, BrdU (BrdU labeling, ) or a standard
Result: The Cell Proliferation ELISA, BrdU (colori- filtration/liquid scintillation counting protocol ([3H]-
metric) measures proliferation with a sensitivity com- TdR labeling, ).
parable to the radioactive thymidine assay at all cell Result: The Cell Proliferation ELISA, BrdU (colori-
concentrations. metric) is able to detect mitogen-stimulation with a
sensitivity comparable to the radioactive thymidine
assay.
Figure 70: Reduced PBL proliferation of an immuno-

suppressed patient in response to various mitogens.
Cells (1 x 105/well) from a healthy volunteer () or an immu-
nosuppressed individual ( ) were incubated in the presence
of various mitogens for 56 h. Cells were labeled with BrdU for
16 h, then cell proliferation was analyzed by Cell Proliferation
ELISA, BrdU (colorimetric). The error bars indicate the maxi-
mum and minimum values of triplicate microcultures (data
from T. Brning, [1994] Klin. Lab. 40, 917927, Figure 3). Mito-
gens used were: PHA (phytohemagglutinin), OKT3 (anti-CD3
monoclonal antibody), Con A (concanavalin A), PWM
(pokeweed mitogen), and SAC (Staphylococcus aureas
Cowan I).
Result: The BrdU ELISA clearly detected the difference in
response between the healthy and immunosuppressed sub-
jects.

Figure 71: Measurement of the proliferation of antigen-activated PBL. Cells (1 x 105/well) were incu-
bated in the presence of various viral antigens on culture medium alone for 5 days. After labeling with BrdU ( )
or [3H]-TdR () for 16 h, cell proliferation was analyzed by Cell Proliferation ELISA BrdU (chemiluminescence)
() or LSC (). Antigens used were: INV-KA (influenza control antigen), INV-A (influenza virus A), INV-B, (nflu-
enza virus B), RUV (Rubella virus), and HSV (herpes simplex virus type I).
Result: The Cell Proliferation ELISA, BrdU (chemiluminescence) detected antigen stimulation with a sensitivity
comparable to the radioactive thymidine assay.
Other applications: For more example of how the Cell Proliferation ELISAs can be used
in the laboratory, see Appendix, pages 150151.

2404 A poptosis K ap_B Tab_15_A K 1.fm Seite 1 D onnerstag,26.A ugust2004 12:23 12
Cell proliferation/viability assay methods Cell proliferation/viability assay methods

Methods for studying cell proliferation and viability in cell populations Methods for studying cell proliferation and viability in cell populations
2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations
DNA Synthesis
Method/Roche Applied Science Label Assay Principle Advantages Limitations For

product product
informa-
tion, see
[3H]-TdR Proliferation Assay [3H]-TdR [3H]-TdR is added to cells cultured in MTP and the cells are incubated Sensitive (103104 cells/test required) Radioactive isotope handling and storage problems
(usually for 224 h). During this labeling period, [3H]-TdR is incorporated Linear measurement of cell proliferation over a broad, Long half life
into the DNA of proliferating cells. logarithmic range Radioactive waste disposal costs
Cells are harvested by vacuum aspiration onto glass fiber filters. While Low background
free [3H]-TdR is washed through the filters, the [3H]-TdR incorporated in
the DNA is retained.
The radioactivity retained on the filters is measured by liquid scintillation
counting (LSC).
BrdU incorporation assay BrdU BrdU is added to cells cultured in MTP and the cells are incubated (usu- No transfer of the cells; the entire assay is performed in a Assay is not linear over a broad logarithmic range of cell page 91-93
ally for 224 h). During this labeling period BrdU is incorporated into the single MTP proliferation (limitation of the ELISA plate reader) of this guide
BrdU Labeling and Detection Kit III DNA of proliferating cells. Non-radioactive 3 washing and incubation steps
After the culture supernatant is removed, the cells are fixed and then Longer assay time
incubated with an anti-BrdU antibody conjugated with peroxidase (anti-
BrdU-POD).
This antibody binds to BrdU which has been incorporated into the DNA.
Bound anti-BrdU-POD is detected by a substrate reaction and quantified
by an ELISA plate reader.
BrdU incorporation assay BrdU See above (BrdU incorporation assay) No transfer of the cells; the entire assay is performed in a Assay is not linear over a broad logarithmic range of cell page 94-97
single MTP proliferation (limitation of the ELISA plate reader) of this guide
Cell Proliferation ELISA, BrdU 1 washing and 2 incubation steps only
(colorimetric) Short assay time
Robust system: low standard deviation
Sensitive (103104 cells/test required)
BrdU incorporation assay BrdU See above (BrdU incorporation assay) [see also Cell Proliferation ELISA, BrdU (colorimetric)] For chemiluminescence measurement special MTP page 94-97
Linear measurement of cell proliferation over a broad, (Black with clear, flat bottom) required of this guide
B B
Cell Proliferation ELISA, BrdU logarithmic range
(chemiluminescence)
Table 15: Summary of methods to study DNA synthesis in cell populations.
Metabolic activity
Method/Roche Applied Science Label Assay Principle Advantages Limitations For

product product
informa-
tion, see
MTT Assay61 Non-isotopic MTT solution is added to cells cultured in MTP and the cells are incubated No transfer of the cells; the entire assay is performed in a Assay is not linear over a broad logarithmic range of cell page 82-88
(usually for 4 h). During this period, MTT is converted into a colored, single MTP proliferation due to the ELISA plate reader of this guide
Cell Proliferation Kit I (MTT) water-insoluble formazan salt by the metabolic activity of viable cells. MTT is metabolized by all cells; the assay can be used with Insoluble reaction product; resolubilization of the reac-
The insoluble formazan is solubilized. all cell types tion product required
The amount of formazan is quantified by an ELISA plate reader at Inexpensive Connot take multiple time points in a single assay
550600 nm. Cells with low metabolic activity (e.g., lymphocytes) must
be used in high numbers
XTT Assay62 Non-isotopic XTT solution is added to cells cultured in MTP and the cells are incubated No transfer of the cells; the entire assay is performed in a Assay is not linear over a broad logarithmic range of cell page 82-88
(usually for 24 h). During this period, XTT is converted into a colored, single MTP proliferation due to the ELISA plate reader of this guide
Cell Proliferation Kit II (XTT) soluble formazan salt by the metabolic activity of viable cells. Soluble reaction product XTT working solution has to be prepared shortly before
The amount of formazan is quantified by an ELISA plate reader at Can take multiple time points in a single assay use
450500 nm. XTT is not metabolized by all cell types
WST-1 Assay Non-isotopic WST-1 solution is added to cells cultured in MTP and the cells are incu- No transfer of the cells; the entire assay is performed in a Assay is not linear over a broad logarithmic range of cell page 82-88
bated (usually for 0.52 h). During this period, WST-1 is converted into a single MTP proliferation due to the ELISA plate reader of this guide
Cell Proliferation Reagent (WST-1) colored, soluble formazan salt by the metabolic activity of viable cells. Soluble reaction product WST-1 is not metabolized by all cell types
The amount of formazan is quantified by an ELISA plate reader at Repeated measurement of the assay
420480 nm. Ready-to-use solution
Table 16: Summary of methods to study metabolic activity in cell populations.
2.1.4 Single reagents for the measurement of DNA synthesis
Product Cat. No. Pack Size Table 17: Single reagents available Product Cat. No. Pack Size
for detection of DNA fragmentation.
FixDenat 11 758 764 001 4 x 100 ml (2000 tests) Anti-Bromodeoxyuridine formalin grade 11 170 376 001 50 g (250 tests*)
* Flow cytometry
Anti-Bromodeoxyuridine-Peroxidase, 11 585 860 001 15 U** ** 0.2 U/ml (ELISA), 1.5 U/ml Anti-Bromodeoxyuridine-Fluorescein 11 202 693 001 50 g (100 tests**)
Fab fragments, formalin grade (Immunochemistry) formalin grade
98 Apoptosis, Cell Death, and Cell Proliferation Manual Cell Proliferation and Viability 99
Methods for studying cell proliferation and viability in individual cells
2.2 Methods for studying cell proliferation and viability in

individual cells
As mentioned in Section B 1, the viability as well as proliferation of individual cells can be
assessed by standard microscopic methods. For instance, cells may be treated with a vital
stain or exclusion dye and counted directly in a hemocytometer. The same cell parame-
ters may be determined by flow cytometry if the cells are differentially stained with fluo-
rescent dyes that bind DNA (DNA fluorochromes), see also section A 2.2.3 on page 49 of
this guide.
In the following section we will describe details of the following proliferation assays:
Assays that measure DNA synthesis: As outlined above, if labeled DNA precursors are
added to the cell culture, cells that are about to divide incorporate this precursor into
their DNA (described on the following pages of this guide)
2.2.1 Assays that measure DNA synthesis
Studies of cell proliferation in vivo as well as on individual cells in vitro frequently employ
[3H]-TdR to label the DNA of replicating cells and autoradiography to reveal the radioac-
tive label. As a nonradioactive alternative, bromodeoxyuridine (BrdU) can be used to
label proliferating cells in vivo and in vitro. Incorporated BrdU can be detected by
immunohistochemistry, immunocytochemistry or flow cytometry.
Immunochemical techniques allow both the visualization of dividing cells and the detec-
tion of tissue morphology by counterstaining (e.g., with hematoxylin and/or eosin).
Thus, it is possible to visualize cells which have incorporated BrdU into DNA in its natu-
ral environment and to localize cell position in the tissue73, 74.
B
As only those cells which are actually in the S-phase (DNA-synthesis) of the cell cycle will
be labeled, the so-called labeling index can be determined if the labeled nucleotide
([3H]-TdR or BrdU) is present for only short periods of time (e.g., 1560 minutes). The
labeling index (proportion of S-phase cells in an asynchronously growing population)
is calculated by dividing the number of labeled cells by the total number of cells in the
entire population.
While short labeling periods (pulse labeling) are suitable to quantify the percentage of S-
phase cells within a cellular population, longer labeling periods (e.g., for a whole cell cycle
transition) can be used to determine a replicating population.
Roche Applied Science offers several kits and reagents for measuring proliferating cells by
BrdU incorporation. These products are described on the following pages.


Detection Kit I
Cat. No. 11 296 736 001 100 tests

Detection Kit II
Cat. No. 11 299 964 001 100 tests
Type 1st generation immunostaining assays for fluorescence (Kit I)

or light (Kit II) microscopy
Useful for Detection of BrdU-labeled DNA in proliferating individual cells
Samples Cultured or freshly isolated cells, tissue explants or sections
Method Incubation of cells with BrdU, or injection into an animal, followed
by nuclease digestion of DNA of cells or tissue sections and indirect
immunodetection (with anti-BrdU and a secondary antibody) of incorpo-
rated BrdU label
Time approx. 23 h (+ BrdU labeling)
Significance of kits: The BrdU Labeling and Detection Kits I and II offer an indirect
immunostaining method for visualizing proliferating cells under a fluorescence micro-
scope (Kit I) or under a light microscope (Kit II). The kits detect BrdU-labeled DNA with
an anti-BrdU antibody, then make the antibody-labeled DNA visible with either a fluo-
rescein-labeled (Kit I) (Figure 72) or an alkaline phosphatase-labeled anti-mouse sec-
B
ondary antibody (Kit II) (Figure 73).
Note: These kits belong to the first generation of kits used to measure DNA synthesis. The
same assay procedure has been optimized and improved in the second generation of kits,
namely the In Situ Cell Proliferation Kit, FLUOS (for flow cytometry and fluorescence
microscopy) and the In Situ Cell Proliferation Kit, AP (for light microscopy). For a detailed
description of these second generation kits, see the following pages.
Other applications: For examples of how the BrdU Labeling and Detection Kits I and II
can be used in the laboratory, see Appendix, pages 147149.
Figure 72: Principle of the BrdU Labeling and Figure 73: Principle of the BrdU Labeling and
Detection Kit I (Fluorescein). Detection Kit II (AP).

In Situ Cell Proliferation Kit, FLUOS

Cat. No. 11 810 740 001 100 tests
Type Direct immunofluorescence staining for flow cytometry or fluorescence

microscopy
Method Incubation of cells with BrdU, or injection of BrdU into an animal followed
by denaturation of DNA of cells or tissue sections and direct immunodetec-
tion of incorporated BrdU label
Time approx. 2 h (+ 0.54 h BrdU labeling)
Significance of kit: Bromodeoxyuridine (BrdU) is only incorporated into the DNA of

proliferating cells. Short periods (1560 min) of incubation in vitro with BrdU will tag
only cells actually going through the S phase of the cell cycle. Alternatively, BrdU can be
injected into an animal to label growing cells in vivo. The In Situ Cell Proliferation Kit,
FLUOS can detect proliferating cells in culture or in tissues which have been tagged by in
vitro or in vivo BrdU labeling. Analysis can be done by flow cytometry or by fluorescence
microscopy.
Test principle: The BrdU solution and fluorescein-conjugated anti-BrdU antibody sup-
plied in the kit allow BrdU labeling and detection of proliferating cells. The procedure
(Figure 74 and Flow Chart 18) involves:
A: Incubating growing animal tissue or cells in vitro with BrdU

or
B
B: Injecting BrdU into whole animals for in vivo labeling, then sacrificing the animal
and preparing tissue sections.
Note: Only proliferating cells incorporate BrdU into their DNA.
Fixing BrdU-labeled tissue or cells.
Denaturing cellular DNA with acid.
Detecting incorporated BrdU with fluorescein-labeled anti-BrdU monoclonal

antibody.
Analyzing the antibody-labeled samples with a flow cytometer or a fluorescence

microscope.
Figure 74: Principle of the In Situ Cell Proliferation Kit, FLUOS.

Specificity: The antibody conjugate (anti-BrdU-fluorescein, F(ab)2 fragments) will bind

to BrdU-labeled DNA after the DNA is denatured and partially degraded with acid. The
antibody specifically recognizes 5-bromo-2-deoxyuridine; it shows no cross-reactivity
with any endogenous cellular components such as thymidine or uridine.

Cell lines (in adherent or suspension cell culture)
Freshly isolated cells
Tissue explants labeled with BrdU in vitro
Frozen or paraffin-embedded tissue sections from animals labeled with BrdU in vivo.
Kit contents
2. Anti-BrdU-fluorescein, monoclonal, F(ab)2 fragments
3. Antibody incubation buffer
Typical results: see Figures 7577.
Other applications: For examples of how the In Situ Cell Proliferation Kit, FLUOS can be
Figure 75: Flow cytometric measurement of

total DNA and incorporated BrdU with the In Situ
Cell Proliferation Kit, FLUOS. Exponentially growing
U937 cells were incubated with BrdU for 30 min.
Incorporated BrdU was measured flow cytometrically
with the fluorescein-conjugated anti-BrdU antibody
(<BrdU>fluos) from the In Situ Cell Proliferation Kit,
FLUOS. Total DNA was counterstained with 1 g/ml
propidium iodide (PI). The phase of the cell cycle
B
represented by each population of cells is indicated
on the flow cytometric histogram. FL1-H, fluorescein
intensity (relative BrdU content); FL3-H, propidium
iodide intensity (relative DNA content).
Result: BrdU labeling is confined exclusively to the
S-phase (DNA synthesis) of the cell cycle.

Figure 76: In vivo labeling and analysis of dorsal, hyperproliferative epidermis tissue from mouse
with the In Situ Cell Proliferation Kit, FLUOS. Undiluted BrdU labeling solution from the kit was injected intra-
peritoneally into a mouse (1 ml BrdU solution/100 g body weight). After 2 h of in vivo BrdU labeling, the mouse
was sacrificed and 5 m thick, paraffin-embedded tissue sections were prepared. Sections were deparaffinized
and rehydrated according to standard methods, then digested with trypsin (15 min). DNA was partially denatured
with HCl (20 min) and detected with anti-BrdU-fluorescein. Each section was analyzed by differential interference
microscopy (upper photo) and epifluorescence microscopy (lower photo). Magnification, 530 x. (Data kindly pro-
vided by S. Kaiser and M. Blessing, I. Med. Klinik der Universitt Mainz, Germany.)
Result: Proliferating cells (green spots) are clearly visible throughout the tissue under epifluorescence
microscopy.
B
Figure 77: In vitro labeling and analysis of proliferating HeLa cells with the In Situ Cell Proliferation
Kit, FLUOS. HeLa cells in culture were labeled with BrdU and the BrdU-labeled DNA detected with anti-BrdU-
fluorescein, according to the package insert of the In Situ Cell Proliferation Kit, FLUOS. The labeled cell prepara-
tion was analyzed under a light microscope (upper photo) and a fluorescence microscope (lower photo).
Result: Proliferating cells (bright green nuclei) within the HeLa preparation are clearly visible under the fluores-
cence microscope.

Anti-BrdU, formalin grade

Cat. No. 11 170 376 001 50 g
Anti-BrdU-Fluorescein
Cat. No. 11 202 693 001 50 g
Anti-BrdU-Peroxidase, Fab fragment

Cat. No. 11 585 860 001 15 units
Type Monoclonal antibodies, from mouse

Method Incubation of samples with BrdU, followed by denaturation of DNA, detec-
tion of BrdU label with anti-BrdU antibody, and (if necessary) visualization
of anti-BrdU antibody with secondary antibody
Time Variable (depending on sample and antibody used)
Significance of antibodies: Bromodeoxyuridine (BrdU) is only incorporated into the DNA

of proliferating cells. Short periods (1560 min) of incubation in vitro with BrdU will tag
only cells going through the S phase of the cell cycle. Alternatively, BrdU can be injected
into an animal to label growing cells in vivo. Conjugated or unconjugated anti-BrdU anti-
body may be used to detect proliferating cells or tissues which have been tagged by in
B
vitro or in vivo BrdU labeling. Depending on the sample and the antibody used, analysis
can be by flow cytometry, fluorescence microscopy, or light microscopy.
Test principle: The anti-BrdU antibodies may be used to detect BrdU-labeled DNA in
proliferating cells. The procedure involves (Flow Chart 19):
A: Incubating growing animal tissue or cells in vitro with BrdU

or
B: Injecting BrdU into whole animals for in vivo labeling, then sacrificing the animal
and preparing tissue sections.
Note: Only proliferating cells (cells in S-phase) incorporate BrdU into their DNA.
Fixing BrdU-labeled tissue or cells.
Denaturing cellular DNA.
Detecting incorporated BrdU with conjugated or unconjugated anti-BrdU monoclonal

antibody.
(Option) A: Localizing unconjugated anti-BrdU antibody with a secondary antibody

detection system
or
(Option) B: Localizing enzyme-conjugated anti-BrdU antibody with an enzyme
substrate.
Analyzing the antibody-labeled samples with a flow cytometer, a fluorescence

microscope, or a light microscope.

Inject the animal with BrdU labeling reagent intraperitoneally
Sacrifice the animal (approx. 14 h later) and remove tissue samples or organ
Process tissue samples or organ for:
Frozen sectioning Paraffin embedding
Freeze tissue samples/ Fix tissue samples/organ in formalin immediately

organ immediately after removal after removal
Store sample frozen until required Use standard dehydration and paraffin embed-
for sectioning ding procedures to process fixed sample
Cut sections of frozen sample in a cryostat Cut sections of embedded sample on a microtome
Transfer sections onto a glass slide and fix Transfer sections onto a glass slide and use stand-
ard procedures to dewax and rehydrate sections
Proceed with the immunostaining procedure
Flow Chart 18: Assay procedure, in vivo labeling of proliferating cells with BrdU.
Fixed, BrdU-labeled sample
B
Denature sample DNA e.g., with HCl (1020 min, RT) or microwaves (15 min, 100 C)
Incubate sample with Incubate sample with Incubate sample with

Anti-BrdU-Fluorescein Anti-BrdU Anti-BrdU-POD
Analyze sample by flow Incubate sample with Add peroxidase substrate and incubate
cytometry or fluores- anti-mouse-fluores- until color forms
cence microscopy. cein or anti-mouse-
enzyme
(+ enzyme substrate)
Analyze sample by flow Analyze sample by light microscopy

cytometry, fluores-
cence or light micros-
copy
Flow Chart 19: Immunostaining procedure, Anti-BrdU antibody and conjugates.
Specificity: Conjugated or unconjugated anti-BrdU antibody will bind to BrdU-labeled

DNA after the DNA is denatured and partially degraded (e.g., with DNase, acid or micro-
waves). The antibody specifically recognizes 5-bromo-2-deoxyuridine; it shows no cross-
reactivity with any endogenous cellular components such as thymidine or uridine.

Cell lines (in adherent or suspension cell culture)
Freshly isolated cells, or tissue explants labeled with BrdU in vitro
Frozen or paraffin-embedded tissue sections from animals labeled with BrdU in vivo.

Typical results: The anti-BrdU antibody has been used to determine the cell cycle posi-
tion of apoptotic cells76.
Briefly, the experimental procedure was as follows: Cultured mouse thymocytes were
treated with 0.5 M ionomycin (2 h or 12 h) to induce apoptosis. After treatment, the
cells were harvested, fixed in paraformaldehyde and ethanol (two-step fixation), and ana-
lyzed for apoptosis and cell cycle position by flow cytometry. As a measure of apoptotic
cells, fragmented DNA content was quantitated with the In Situ Cell Death Detection Kit,
Fluorescein (TUNEL method, according to the kit package insert). Either of two flow
cytometric techniques was used to determine the cell cycle position of the cells: 1) Rela-
tive DNA content was determined by treating the cells with 5 g/ml propidium iodide
and 200 g/ml ribonuclease (30 min, room temperature). 2) Cells going through S-phase
were identified by labeling with BrdU (10 M BrdU, 30 min), detection of BrdU-labeled
cells with anti-BrdU monoclonal antibody (30 min, 37C), and visualization of those
cells with R-phycoerythrin-conjugated goat anti-mouse antibody (30 min, 37C). For
results, see Figure 78.
Other applications: For examples of how the Anti-BrdU conjugates and the antibodies
may be used in the lab, see Appendix, pages 152 and 153.
Figure 78: Concomitant flow cytomet-

ric analysis of apoptosis and cell
cycle position with the anti-BrdU
antibody, propidium iodide, and the In
Situ Cell Death Detection Kit, Fluores-
cein. Cultured mouse thymocytes were
treated with ionomycin (2 h or 12 h) to
induce apoptosis. After treatment, the
cells were harvested, fixed, and analyzed
for apoptosis and cell cycle position by
flow cytometry. Histograms A, C, and E
B
show data obtained from cells after 2 h
treatment with ionomycin. Histograms B,
D, and F show data obtained from cells
after 12 h treatment with ionomycin. His-
tograms A and B show fluorescein inten-
sity (green fluorescence) alone, a
measure of DNA fragmentation. Histo-
grams C and D show a two-parameter
analysis of fluorescein intensity (green
fluorescence, DNA fragmentation) and
propidium iodide intensity (red fluores-
cence, DNA content). Histograms E and F
show a two-parameter analysis of fluo-
rescein intensity (green fluorescence,
DNA fragmentation) and phycoerythrin
intensity (orange fluorescence, BrdU
content). The percentage of positive cells
is indicated in each panel. [Data from
Hanon, E., Vanderplasschen, A. and Pas-
toret, P.-P. (1996) Biochemica No. 2, 25
27.]
Result: The ionomycin-treated cells con-
tained about 13% apoptotic cells (histo-
gram A) after 2 h and about 29%
apoptotic cells (histogram B) after 12 h
exposure. Concomitant analysis of apop-
tosis and total DNA content (histograms
C and D) showed that apoptotic cells
contained about as much DNA as cells in
G0/G1 or early S-phase. Concomitant
analysis of apoptosis and BrdU content
after 12 h ionomycin treatment (histo-
gram F) showed that 6% of the apoptotic
cells went through S phase (that is, were
positive for BrdU) while 21% of apoptotic
cells remained in G0/G1 (that is, were
negative for BrdU).

2404 A poptosis K ap_B Tab_18_A K 1.fm Seite 1 M ontag,23.A ugust2004 12:28 12
Cell proliferation/viability assay methods Cell proliferation/viability assay methods

Methods for studying cell proliferation and viability in individual cells Methods for studying cell proliferation and viability in individual cells
2.2.2 Summary of methods for studying cell proliferation and viability in individual cells
DNA Synthesis
Method/ Assay principle Advantages Limitations For

Roche Applied Science product product
informa-
tion, see
Autoradiography The samples are incubated with [3H]-TdR for a certain period of time. If Quantitative detection of S phase cells: Determination of Long exposure time (days) required
[3H]-TdR is present for 1 h or less, only those cells which are in the growing fraction in population Radioactive isotope, handling and storage problems
S-phase (DNA synthesis) of the cell cycle will be labeled.
The samples are fixed and immersed in emulsion.
The radiolabel is visualized as black grains on the film.
Immunocytochemistry (fluorescence microscopy) The samples are incubated with BrdU for a certain period of time. If BrdU is Quantitative detection of S-phase cells: Determination of Stained samples cannot be stored for long periods of pages 101-
present for 1 h or less, only those cells which are in the S-phase (DNA growing fraction in population time 104 of this
In Situ Cell Proliferation Kit, FLUOS synthesis) of the cell cycle will be labeled. Results within a few hours Histological tissue organization cannot be observed guide
The samples are fixed and the DNA is denatured. Can counterstain the tissue simultaneously to reveal tissue simultaneously
BrdU Labeling and Detection Kit I Incorporated BrdU is bound by a fluorescein-conjugated monoclonal morphology
antibody against BrdU.
Bound Anti-BrdU-Fluorescein is detected by fluorescence microscopy or
flow cytometry.
Immunocyto/histochemistry (light microscopy) The samples are incubated with BrdU for a certain period of time. If BrdU is Quantitative detection of S-phase cells: Determination of page 101 of
present for 1 h or less, only those cells which are in the S-phase (DNA growing fraction in population this guide
BrdU Labeling and Detection Kit II synthesis) of the cell cycle will be labeled. Results within a few hours
The samples are fixed and the DNA is denatured. Can counterstain the tissue simultaneously to reveal tissue
Incorporated BrdU is bound by an alkaline phosphate (AP)-conjugated morphology
monoclonal antibody against BrdU.
Bound anti-BrdU AP is detected by a substrate reaction and visualized by
light microscopy.
Table 18: Summary of methods to study DNA synthesis in individual cells.
B B
108 Apoptosis, Cell Death, and Cell Proliferation Cell Death Apoptosis and Necrosis 109
Seite 110 - Vakat
Appendix
Technical tips
Selected frequently asked questions (FAQs) about cell death assays
1. Technical tips
1.1 Selected frequently asked questions (FAQs) about cell
death assays
The questions below were chosen from those received by our Technical Services represen-
tatives. Wherever possible, the answers will direct you to pages and sections of this guide
which can provide more information.
Can I determine the number of apoptotic cells using the

Cell Death Detection ELISAPLUS?
A: No. The ELISA data is interpreted as a change in the level of death in an apoptotic
population compared to an uninduced control population. It does not provide data on
individual cells.
What is the best way to get rid of non-specific (false-positive) background in the
TUNEL (In Situ Cell Death Detection) kits?
A: The best approach to reducing background depends on the results you obtain with the
controls:
If cells incubated with fluorescein-dUTP but without terminal transferase are false
positive, try washing the cells more thoroughly, reducing the concentration of fluo-
rescein-dUTP, or using an alternative permeabilization procedure.
If false positives are produced only in reactions which include both fluorescein-
dUTP and terminal transferase, the best means of reducing false positives is a
reduction in enzyme concentration or a change in permeabilization procedure.
Note: For further tips on obtaining the best results with the TUNEL method, see page 113
of this Appendix.
What types of sample can be assayed with the TUNEL method?

A: Tissue sections, adherent cell cultures, cytospins and cell smears have all been used
with this assay (page 33, Section A 2.2.1). Note, however, that the sample material
must be preserved with a cross-linking fixative (such as paraformaldehyde).
Why isnt substrate included in the TUNEL kits (In Situ Cell Death Detection Kits,
AP or POD)?
A: These kits will work with a variety of common alkaline phosphatase or peroxidase
substrates. Since many laboratories already have these substrates, and know how these
substrates work in their system we decided to leave them out. In addition this gives
the researcher the flexibility for secondary staining.
How long and at what temperature can I store my samples before analyzing them with
the various kits that you offer?
A: Table 19 gives some general guidelines for sample storage. Note however that some
samples may be more or less stable than others.
C Is a special wash/stop buffer required for the TUNEL kits?

A: Our procedure does not require an equilibration buffer. Our wash buffer is PBS, a
commonly used solution.

Technical tips
Technical tips on the TUNEL method
Kit Storage of samples before assay

PLUS
Cell Death Detection ELISA Purified cytoplasmic samples can be stored at
20C for 2 weeks (with some reduction of signal)
Apoptotic DNA Ladder Kit Purified DNA can be stored at 20C for 1 year
Annexin-V-FLUOS Staining Kit; Cells must be used live, directly after induction of
Annexin V-Biotin apoptosis
Cellular DNA Fragmentation ELISA Purified cytoplasmic samples can be stored at

20C for at least 2 weeks
Table 19: Storage of samples for apoptosis assay
1.2 Technical tips on the TUNEL method

1.2.1 TUNEL: Improvement and evaluation of the method for in situ
apoptotic cell identification
[from Adrien Negoescu, Philippe Lorimier, Francoise Labat-Moleur, Laurent Azoti, Catherine Robert, Christiane
Guillermat, Christian Brambilla, and Elisabeth Brambilla; of Groupe de recherche sur le cancer du poumon, Insti-
tut Albert Bonniot, Faculte de Medecine, Domaine de la Merci, 38706 Grenoble cedex, France, and Laboratoire de
Pathologie cellulaire, CHRU, BP 217X, 38043 Grenoble cedex 09, France]
Note: This is a summary of an article that appeared in the Biochemica No. 2 (1997). For fur-
ther experimental detail and background, see the full Biochemica article.
Summary: TUNEL or terminal deoxynucleotidyl transferase-mediated dUTP nick end-

labeling, is a preferred method for rapid identification and quantification of the apop-
totic cell fraction in cultured cell preparations. However, the accessibility of DNA breaks
to enzymatic reactions is reduced by the nuclear protein environment (Kerrigan et al.,
1987) and impaired by cell fixation (Gold et al., 1994) and postfixation (Gorczyca et al.,
1994). Thus, several sample pretreatments have been devised to improve TUNEL sensi-
tivity (Desjardins and MacManus, 1995; Kerrigan et al., 1987; Lucassen et al., 1995). An
optimized TUNEL protocol for cultured cells has been developed.
C
Appendix 113
Technical tips
1.2.2 TUNEL protocol for tissues which tend to give false positives
The protocol given below has been found to eliminate the TUNEL labeling false posi-
tives seen with certain paraffin-embedded tissue sections (for example, of rabbit
endometrium). The key step is pretreatment of the slide with microwave irradiation
rather than proteinase K.
Sample: Paraffin-embedded tissue sections (e.g., of rabbit endometrium)
Reagents: In Situ Cell Death Detection Kit, POD, Cat. No. 11 684 817 001
DAB Substrate, Cat. No. 11 718 096 001
Dewax paraformaldehyde- or formalin-fixed tissue sections according to standard

procedures.
Place the slide(s) in a plastic jar containing 200 ml 0.1 M citrate buffer, pH 6.0, put the
jar in a microwave oven, and apply 750 W (high) microwave irradiation for 1 min. For
rapid cooling, immediately add 80 ml redist. water (2025C) to the jar, then transfer
the slide(s) into PBS (2025C).
Caution: DO NOT perform a proteinase K treatment!
Immerse the slide(s) for 30 min at room temperature (RT) in a blocking solution
containing 0.1 M Tris-HCl, 3% BSA, and 20% normal bovine serum, pH 7.5.
Rinse the slide(s) twice with PBS at RT. Let excess fluid drain off.
Apply 50 l of TUNEL reaction mixture to the section and incubate for 60 min at 37C
in a humidified atmosphere.
Rinse slide(s) three times in PBS (5 min for each wash).

Note: At this stage, you can evaluate the section under a fluorescence microscope.
Block endogenous peroxidase activity by incubating slides for 10 min at RT with 0.3%
H2O2 in methanol.
Repeat steps 3 and 4 to block nonspecific binding of the anti-fluorescein-antibody to

the tissue.
Add 50 l Converter-POD, pre-diluted 1:2 in blocking solution (from Step 3), and
incubate for 30 min at 37C in a humidified atmosphere.
Rinse slide(s) three times in PBS at RT for 5 min each.
Add 50 l DAB substrate solution and incubate for 13 min at RT.
Wash slide(s) extensively in tap water and counterstain if needed.
C
Technical tips
1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts
To avoid this artifact Which may be caused by Try the following

Nonspecific TUNEL DNA strand breaks induced by UV irradia- Use a different embedding material, which
labeling tion during tissue embedding (UV used to does not require UV irradiation
polymerize tissue embedding material such Use an alternate polymerization method
as methacrylate)
Acid tissue fixatives (e.g., mathacarn, Use buffered 4% paraformaldehyde as
Carnoys fixative) cause DNA strand breaks fixative
Endogenous nuclease activity which occurs Fix tissue immediately after organ harvest
soon after tissue preparation (e.g., in smooth Perfuse fixative through liver vein in intact
muscle tissue slices) animal
TdT concentration too high during TUNEL Reduce concentration of TdT by diluting it
labeling 1:2 or 1:3 with TUNEL Dilution Buffer
(Cat.No. 1966 006) containing 30 mM Tris
(pH 7.2) containing 140 mM sodium caco-
dylate and 1 mM CoCl2
Endogenous alkaline phosphatase activity Block endogenous AP activity by adding
during converter reaction 1 mM levamisole to the AP substrate
solution
Endogenous peroxidase activity during Before permeabilizing cells, block endoge-
converter reaction nous POD activity by immersing the slides in
a solution of 0.3% H202 in methanol
Nonspecific binding of anti-fluorescein anti- Block nonspecific sites with normal
body conjugate during converter reaction anti-sheep serum
Block nonspecific sites with PBS containing
3% BSA (20 min)
Use 1:2 dilution of converter solution in PBS
High background Formalin fixation, which causes yellow stain- Use methanol fixation
ing of cells containing melanin precursors Note: This fixation may lead to a reduction in
TUNEL labeling sensitivity
TUNEL labeling mix too concentrated (e.g., Reduce concentration of labeling mix by
for carcinomas) diluting it 1:2 with TUNEL Dilution Buffer
(Cat. No. 1966 006) containing 30 mM Tris
(pH 7.2) containing 140 mM sodium caco-
dylate and 1 mM CoCl2
Endogenous alkaline phosphatase activity Block endogenous AP activity by adding 1
during converter reaction mM levamisole to the AP substrate solution
Endogenous peroxidase activity during Before permeabilizing cells, block endoge-
converter reaction nous POD activity by immersing the slides in
a solution of 0.3% H202 in methanol
Nonspecific binding of anti-fluorescein Block nonspecific sites with normal
antibody conjugate during converter anti-sheep serum
reaction Block nonspecific sites with PBS containing
3% BSA (20 min)
Use 1:2 dilution of converter solution in PBS
C
Appendix 115
Technical tips
To avoid this artifact Which may be caused by Try the following

Low TUNEL labeling Ethanol and methanol fixation Use buffered 4% paraformaldehyde as
(low sensitivity) fixative
Extensive crosslinking during prolonged Reduce fixation time
fixation reactions Use buffered 2% paraformaldehyde as
fixative
Insufficient permeabilization of cells, so Pretreat with proteinase K (concentration
TUNEL reagents cannot reach nuclei and time must be optimized empirically)
Note: To avoid possible nuclease contamina-
tion, use only Proteinase K from Roche
Applied Science, Cat. No. 03 115 836 001
Pretreat with 0.01 M sodium citrate for
30 min at 70C
Increase TUNEL incubation time
Restricted access of TUNEL reagents to After dewaxing tissue sections, treat with
nuclei, caused by paraffin-embedding proteinase K (concentration, time, and tem-
perature must be optimized empirically)
Note: To avoid possible nuclease contamina-
tion, use only Proteinase K from Roche
Applied Science, Cat. No 03 115 836 001
Immerse dewaxed tissue sections in 200 ml
0.01 M citrate buffer (pH 6.0) and treat with
microwave irradiation (370 W, 5 min)
Note: Conditions must be experimentally
optimized for each tissue
No signal on positive Inadequate DNase treatment (DNase For cryosections, apply 1 g/ml DNase
control concentration too low) For paraffin-embedded tissue sections,
apply 0.5 mg/ml DNase
For many other samples, apply 1 U/ml
DNase in a solution of 10 mM Tris-HCl
(pH 7.4), 10 mM NaCl, 5 mM MnCl2, 0.1 mM
CaCl2, 25 mM KCl; incubate 30 min at 37C
As an alternative DNase buffer, use a
solution of 10 mM Tris-HCl (pH 7.5), 1 mM
MgCl2, 1 mg/ml BSA
Diminished TUNEL Quenching of fluorescein signal by Use 0.5 g/ml PI as DNA stain
staining during DNA propidium iodide (PI) Substitute TO-PRO-3 (from Molecular
counterstaining Probes) in place of PI
C
Technical tips
Technical tips on the use of Annexin-V-Biotin for light microscope detection
1.3 Technical tips on the use of Annexin-V-Biotin for light

microscope detection
The following protocol provides a method for the detection of Annexin-V-Biotin-
binding to cell culture cells with light microscopy. The percentage of necrotic cells is
determined by trypan blue staining.
Preparation of solutions
Annexin-V-Biotin working solution: Dilute 20 l. Annexin-V-Biotin labeling reagent
in 1000 l incubation buffer (sufficient for 10 samples).
HEPES buffer: Prepare according to the instructions in the Annexin-V-Biotin pack
insert.
All steps can be performed at room temperature
Incubate 1 x 106 cells in 100 l Annexin-V-Biotin working solution for 1015 min.
Wash 2 times with HEPES buffer.

For suspension cells: Continue with step 3.
For adherent cells: Continue with step 4.
Resuspend suspension cells in 1 ml HEPES buffer. Transfer 2 x 105 cells to a slide

using cytospin device.
Air dry cells. Fix with methanol/ethanol 1:1 for 90 sec.
Air dry cells. Add 100 l Streptavidin-POD (Cat. No. 11 089 153 001) working solution,
incubate for 1 h.
Rinse with HEPES buffer
Add DAB substrate solution (Cat. No. 11 718 096 001) working solution, incubate for
1015 min.
Rinse with HEPES buffer
Analyze samples under a light microscope.
C
Appendix 117
Technical tips
Technical tips on the use of the Apoptotic DNA Ladder Kit and the Cell Proliferation ELISA Kits
1.4 Technical tips on the use of the Apoptotic DNA Ladder

Kit on tissue samples
The package insert for our Apoptotic DNA-Ladder Kit, Cat. No. 11 835 246 001, des-
cribes the purification of nucleic acids from whole blood and cultured cells. By following
the modified procedure decribed here it is also possible to use tissue samples.
Preliminary Information
Weight of sample: The tissue sample should weigh between 25 and 50 mg.
Additional required solutions:
Lysis buffer: Prior to extraction of DNA, prepare a lysis buffer. 200 l of this buffer
are sufficient for one tissue sample. The lysis buffer consists of 4 M urea, 100 mM
Tris, 20 mM NaCl and 200 mM EDTA, pH 7.4 (25C).
Proteinase K solution: 20 mg/ml in 50 mM Tris-HCl (pH 8.0) and 1 mM CaCl2.
Protocol for isolation of DNA from tissue samples
Add 200 l lysis buffer and 40 l proteinase K solution to 2550 mg tissue, mix.
Incubate for 1 h at 55C.
Add 200 l binding buffer, mix.
Incubate for 10 min at 72C.
Proceed with the addition of 100 l isopropanol as described in the pack insert
(3rd. step of section 5).
Note: Be aware, that apoptosis is a single cell event, and therefore in most tissues you will not
find a sufficient number of apoptotic cells to produce a DNA ladder.
1.5 Technical tips on the Cell Proliferation ELISA Kits

How to interrupt the proliferation assay
The detection of BrdU-labeled DNA with the Cell Proliferation ELISAs does not take
more than 1.53 hours. Nevertheless, the labeling period which may vary between 2 and
24 hours can get the scientist in time trouble. Our assay can be interrupted after the label-
ing process: After the removal of the culture medium, the protocol proceeds with the dry-
ing of the labeled cells using e.g., a hair-dryer. The dry cells stay safe and sound up to one
week when stored at 4C in the microtiter plate before they are fixed and denatured
according to the provided protocol.
A tip for measuring lymphocyte proliferation
C
To study the proliferation of lymphocytes, the cells are stimulated e.g., with growth fac-
tors, cytokines or mitogens. The increase in cell numbers can (in special cases) lead to
cluster formation of the lymphocytes: Cells from the same progenitor stick together and
form aggregates in the culture plate. This effect may disturb the antibody recognition of
the ELISA system and thereby result in an underestimation of response. To avoid signal
variation: Carefully resuspend the cells after the BrdU-labeling period and before centrif-
ugation for removing the culture medium. This will enable the equal accessibility of each
cell for the antibody recognizing the BrdU-label.

Special applications of cell death and cell proliferation methods
TUNEL assays
2. Special applications of cell death and cell

proliferation methods
This section of the Appendix contains condensed versions of articles that appeared in the
Roche Applied Science Biochemica newsletter. For further experimental detail and back-
ground, see the full Biochemica articles.
2.1 TUNEL assays

2.1.1 Discrimination between dead and viable apoptotic cells using two-
color TdT assay and surface labeling as detected by flow cytometry
[from Earl A. Timm, Jr. and Carleton C. Stewart, Laboratory of Flow Cytometry, Roswell Park Cancer Institute,
Buffalo, N.Y., USA]
Note: This article appeared in Biochemica No. 1 (1996), 4447.
Summary: The TUNEL method uses terminal dideoxynucleotidyl transferase (TdT) to

incorporate hapten-tagged nucleotides into the 3-strand breaks that occur in DNA dur-
ing apoptosis (Gorczyca et al., 1993; Chapman et al., 1995). If these nucleotides are cou-
pled to a fluorescent molecule, or if the hapten can be detected by a fluorescent secondary
reagent, the apoptotic cells can be analyzed by flow cytometry.
Flow cytometry permits not only the detection of apoptotic populations, but also the
simultaneous detection and immunophenotyping of necrotic populations. The drawback
to using the current TdT method, however, is that the ethanol permeabilization of the
cells is incompatible with immunophenotyping because it denatures cellular epitopes
(Darzynkiewicz et al., 1992; Li et al., 1995).
A protocol has been developed that both preserves the surface markers and detects apop-
totic cells. In addition, it is possible to discriminate between dead apoptotic cells and via-
ble apoptotic cells with a second hapten-tagged nucleotide that labels dead cells. The
method also can distinguish dead apoptotic cells from cells that have died by other mech-
anisms (e.g., necrosis).
2.1.2 The use of flow cytometry for concomitant detection of apoptosis

and cell cycle analysis
[from E. Hanon, A. Vanderplasschen and P.-P. Pastoret, Department of Immunology/Vaccinology,
Faculty of Veterinary Medecine, University of Lige, Lige, Belgium]
Summary: Two distinct modes of cell death, apoptosis and necrosis, can be distinguished
on the basis of differences in morphological, biochemical, and molecular changes occur-
ing in the dying cells (Duvall and Wyllie, 1986).
Cells undergoing apoptosis display a characteristic pattern of structural changes in the

nucleus and cytoplasm, including rapid blebbing of the plasma membrane and nuclear
disintegration (Duvall and Wyllie, 1986). Extensive damage to chromatin and cleavage of
DNA into oligonucleosomal-length fragments both occur during apoptosis (Duvall and
Wyllie, 1986).
Several flow cytometric methods for identifying cells undergoing DNA fragmentation
have been described recently. These include DNA content analysis and in situ labeling of
C
DNA fragments with tracer-dUTP. The former is based on the accumulation of ethanol-
Appendix 119
TUNEL assays
fixed apoptotic cells in the sub-G0/G1 peak of DNA content histogram as a result of loss
of DNA fragments out of the cells and because of a reduced DNA stainability (Telford
et al.,1991, 1992). The latter uses exogenous terminal deoxynucleotidyl transferase (TdT)
to label in situ the DNA strand breaks with a tracer-dUTP (Gorczyca et al., 1993; Sgonc et
al., 1994).
Recent observations have revealed a profound regulatory interrelationship between apo-

ptosis and the cell cycle (Gorczyca et al.). The investigation of this relationship ideally
requires techniques that permit concomitant apoptosis detection and cell cycle analysis at
a single-cell level.
Two flow cytometric techniques are usually used to investigate the cell cycle: DNA quan-
tification to identify the cell cycle position (Vindelov et al., 1990) and detection of bro-
modeoxyuridine (BrdU) incorporation to reveal cells going through the S phase
(Gratzner, 1982). In this investigation, the development of flow cytometric techniques
that permit concomitant detection of apoptosis and cellular DNA content or BrdU con-
tent analysis by adapting the apoptosis detection protocol of the Roche Diagnoctics In
Situ Cell Death Detection Kit, Fluorescein is reported.
2.1.3 Comparison of two cell death detection methods: In situ nick

translation and TUNEL
[from Maria Pihlgren, Joelle Thomas, and Jaqueline Marvel, Immunologie cellulaire, Lyon Cedex, France]
Summary: Apoptosis is a form of regulated cell death characterized by specific morpho-

logical changes. These include cell shrinkage, membrane blebbing, chromatin condensa-
tion, and cell fragmentation into small apoptotic bodies. At the molecular level, the
activation of an endogenous endonuclease results in the fragmentation of cellular DNA
into oligosomal length fragments (Martin et al. 1994). These can be readily detected by
DNA gel electrophoresis. However, gel electrophoresis does not allow the detection of
apoptosis in individual cells.
In contrast, techniques that use enzymatic labeling of DNA strand breaks can provide
information regarding apoptosis at a single-cell level. The TdT-mediated dUTP Nick End
Labeling (TUNEL) technique uses terminal deoxnucleotidyl transferase (TdT) and
allows the labeling of double-stranded DNA breaks (free 3-OH DNA ends), while the In
Situ Nick Translation (ISNT) method employs DNA Polymerase I and detects single-
stranded DNA breaks. Another advantage of these techniques is that they can be used in
combination with cell surface staining or cell cycle analysis. The abilities of the TUNEL
and ISNT techniques to detect apoptosis in two types of cells: the IL-3-dependent cell line
BAF-3 and freshly isolated CD8+ lymphocytes from mouse spleen are compared.
2.1.4 Fixation of tissue sections for TUNEL combined with staining for
thymic epithelial cell marker
[from Olav Schreurs, Trond S. Halstensen, Zlatko Dembic, Bjarne Bogen, Karl Schenck, Department of Oral
Biology, University of Oslo, Oslo, Norway]
C Note: This article appeared in Biochemica No. 4 (1997), 1921.
Summary: In the thymus, positive and negative selection of thymocytes are important
forces that shape the repertoire of mature T lymphocytes in the immune system. In stud-
ies on negative selection, it is of great interest to determine whether apoptotic cells reside
in thymic cortex or medulla (Surh et al. 1994; Kisielow et al. 1995). Terminal dUTP nick
end labeling (TUNEL) is a technique, that is well suited to demonstrate apoptosis in situ,
and the method may be combined with labeling of other markers. In order to distinguish
between thymic cortex and medulla, differential expression of cytokeratin, MHC class II

Metabolic and Annexin-V assays
molecules and epithelial cell markers have been used (Surh et al. 1994; Wack et al. 1996;
Douek et al. 1996). In the course of an investigation on deletion of tumor-specific TCR-
transgenic T-cells in the thymus (Lauritzsen et al.), TUNEL has been combined with
commercially available monoclonal antibodies, that are monospecific for thymic epithe-
lial cells, to unambiguously localize T-cell deletion. During the course of these studies, we
established fixating conditions, that gave us superior results.
2.2 Metabolic assays, Annexin assays

2.2.1 Biochemical and cellular basis of cell proliferation assays that use
tetrazolium salts
[from Michael V. Berridge, An S. Tan, Kathy D. McCoy, and Rui Wang, Malaghan Institute of Medical Research,
Wellington School of Medicine, Wellington South, New Zealand]
Summary: Tetrazolium salts (such as MTT, XTT, and WST-1) are used extensively in cell
proliferation and cytotoxicity assays, enzyme assays, histochemical procedures, and bac-
teriological screening. In each, these tetrazolium salts are metabolically reduced to highly
colored end products called formazans. Yet, the nature of their cellular bioreduction is
poorly understood despite their long-time use (Stoward and Pearse, 1991).
In our laboratory, we demonstrated that most cellular reduction of MTT was dependent
on the reduced pyridine nucleotides NADH and NADPH, not on succinate as had been
previously believed (Berridge et al., 1993, 1994; Berridge and Tan, 1993). Cellular reduc-
tion of MTT was associated with enzymes of the endoplasmic reticulum and was more
related to NADH production through glycolysis than to respiration.
Recently, assays have been introduced based on tetrazolium salts (such as XTT and WST-
1) that are reduced to soluble formazans. These assays depend on intermediate electron
acceptors such as phenazine methosulfate (PMS).
The question arises: Is the cellular reduction of these new salts similar to that of MTT? In
this article, the answer to that question is attempted.
In summary, it could be shown that, unlike MTT, XTT and WST-1 are efficiently reduced
by NADH and NADPH in the absence of cells or enzymes, and their reduction involves
superoxide. Cellular reduction of WST-1 occurs at the cell surface and also involves
superoxide.
2.3 Annexin-V assays

2.3.1 The use of annexin for concomitant detection of apoptosis and
cellular phenotype
[from S. Hoornaert, E. Hanon, J. Lyaku, and P.-P. Pastoret, Department of Immunology/Vaccinology, Faculty of
Veternary Medicine, University of Lige, Lige, Belgium]
Summary: Two distinct modes of cell death, apoptosis and necrosis, can be distinguished
on the basis of differences in morphological and biochemical characteristics. Under the
elctron microscope, cells undergoing apoptosis display cell shrinkage, apoptotic body
C
formation, and chromatin condensation. Biochemically, the apoptotic process is chara-
terized by fragmentation of DNA into oligonucleosomal fragments. Furthermore, during
Appendix 121
BrdU assays
the early stages of apoptosis, changes also occur at the cell surface membrane (Andree et
al. 1990; Creutz, 1992; Fadok et al. 1992). One of these plasma membrane alterations is
the translocation of phosphatidylserine (PS) from the inner part to the outer layer of the
plasma (Vermes et al., 1995), thus exposing PS at the external surface of apoptotic cells,
where it can be specifically recognized by macrophage (Fadok et al. 1992).
Annexin-V, a Ca2+-dependent phospholipid-binding protein, possesses high affinity for

PS (Vermes et al., 1995) and can thus be used for detecting early apoptotic cells (Koop-
man et al. 1992, Verhoven et al. 1995, Vermes et al. 1995, Homburg et al. 1995). Since
annexin-V can also detect necrotic cells as a result of the loss of membrane integrity, apo-
ptotic cells have to be differentiated from these necrotic cells by the use of propidium
iodide (PI). Indeed, PI selectively labels necrotic, but not apoptotic cells.
Several studies have revealed a correlation between apoptosis and cell phenotype (Car-
bonari et al. 1995, Lewis, et al. 1994). The investigation of this relationship ideally
requires techniques that permit the concomitant detection of apoptosis and cell pheno-
type analysis at a single cell level. In this report, the development of a procedure which
permits concomitant detection of apoptosis and cell phenotype characterization by flow
cytometry is described.
2.4 BrdU assays

2.4. Detection of bromodeoxyuridine in paraffin-embedded tissue
sections using microwave antigen retrieval is dependent on the
mode of tissue fixation
[from Wesley M. Garrett and H.D. Guthrie, Germplasm and Gamete Physiology, Agricultural Research Service,
Beltsville, United States]
Summary: A simple routine microwave antigen retrieval procedure allows the sensitive
detection of incorporated BrdU in pulse labeled cells. Of the two fixatives tested, Carnoys
offers superior nuclear morphology, but with a sacrifice of immunostaining intensity. For
investigations where animals are sacrificed within several hours after pulse labeling, Car-
noys fixative may prove adequate for a general fixative, but it is not known what effect it
has on cellular antigens of interest. For our purposes, 10% neutral buffered formalin was
found to be a superior fixative, because of its ability to cross-link nuclear proteins and
associated chromatin, resulting in more intense immunostaining for BrdU. In addition,
we have found that formalin fixation coupled with microwave antigen retrieval is com-
pletely compatible with immunostaining of other antigens of interest.
C
References
Apoptosis-related parameters Abbreviations and References
3. References
3.1 Apoptosis-related parameters Abbreviations and
References
Parameter Full length name Reference Roche Applied Science

product
AIF Apoptosis inducing Susin S. A. et al. (1996) J. Exp. Med. 184, 1331.
factor
Apaf Apoptotic protease Zou H. et al. (1997) Cell 90, 405.
activating factor Li P. et al. (1997) Cell 91, 479.
APO-2 (L) Apoptosis receptor/ Masters S. A. et al. (1996) Curr. Biol. 6, 750.
ligand Pit R. M. et al. (1996) J. Biol. Chem. 271, 12687.
APO-3 (L) Apoptosis receptor/ Masters S. A. et al. (1996) Curr. Biology 6, 1669.
ligand Chinnaiyan A. M. et al. (1996) Science 274, 990.
Apopain Schlegel J. et al. (1996) J. Biol. Chem. 271, 1841.
Bad Yang E. et al. (1995) Cell 80, 285.
Bak Sattler M. et al. (1997) Science 275, 983.
Orth R. & Dixit V. M. (1997) J. Biol. Chem. 272, 8841.
Bax Bargou R. C. et al. (1995) Eur. J. Immunol. 25, 770.
Zhan Q. M. et al. (1994) Oncogene 9, 3743.
Yang E. et al. (1995) Cell 80, 285.
Bcl-2 Craig W. C. (1995) Cancer Biology 6, 35.
Yang E. et al. (1995) Cell 80, 285.
Bcl-xL Yang E. et al. (1995) Cell 80, 285.
Bcl-xS Williams G. T. & Smith C. A. (1993) Cell 74, 777.
Yang E. et al. (1995) Cell 80, 285.
bik Orth R. & Dixit V. M. (1997) J. Biol. Chem. 272, 8841.
Ca2+ McConkey D. J. et al. (1995) J. Immunology 155, 5133.
Kataoka A. et al. (1995) FEBS Letters 364, 264.
Sokolova I. A. et al. (1995) Biochimica et Biophysica
Acta Mol. Cell Res. 1266, 135.
CAD Caspase activated Enari, M. et al. (1998) Nature 391, 43.
DNase
Calpain Kikuchi H. & Imajohohmi S. (1995) Cell Death and Calpain inhibitor I,
Differentiation 2, 195. Cat. No. 11 086 090 001
Slukvin I. I. & Jerrelis T. R. (1995) Immunopharmacology Calpain inhibitor II,
31, 43. Cat. No. 11 086 103 001
Caspase Cysteine protease Cohen G. M. (1997) Biochem. J. 326, 1.
cleaving an aspartic Alnemri E. S. et al. (1996) Cell 87, 171.
acid residue Nicholson D. W. & Thornberry N. A. (1997) TIBS 22, 299.
ced-3 Caenorhabditis ele- Yuan J. et al. (1993) Cell 75, 641.
gans cell death gene Miura M. et al. (1993) Cell 75, 653.
ced-9 Caenorhabditis ele- Henegartner M. O. & Horovitz H. R. (1994) Cell 76, 665.
gans cell death gene
Ceramide Wiegmann K. et al. (1994) Cell 78, 1005.
c-Jun Grand R. J. A. et al. (1995) Exp. Cell Res. 218, 439.
Wang Y. et al. (1993) Cell Growth Differ. 4, 467.
C
c-Myc
Schwartz L. M. & Osborne B. A. (1993) Immunol.
Today 14, 582.
CPP32 Darmon A. J. et al. (1995) Nature 377, 446. Anti-PARP,
Cat. No. 11 835 238 001
crm A Cytokine response Zhou Q. et al. (1997) J. Biol. Chem. 272, 7797.
modifier A Ogasawara J. et al. (1993) Nature 364, 806.
Cytochrome C Liu X. et al. (1996) Cell 86, 147.
Krippner A. et al. (1996) J. Biol. Chem. 271, 21629.
Yang J. et al. (1997) Science 275, 1129.
Li P. et al. (1997) Cell 91, 479.
Appendix 123
References

product
D4-GDP-DI DI = dissociation Danley D. E. et al. (1996) J. Immunology 157, 500.
inhibitor
Daxx Death-domain-associ- Yang X. L. et al. (1997) Cell 89.
ated protein xx
DcR1 Decoy receptor 1 Pan G. et al. (1997) Science 277, 815.
Sheridan J. P. et al. (1997) Science 277.
DD Death Domain Muzio M. et al. (1996) Cell, 85, 817.
DED Death Effector Domain Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.
DISC Death Inducing Signal Muzio M. et al. (1996) Cell, 85, 817.
Complex
DNA- Wyllie A. H. et al. (1980) Int. Rev. of Cytol. 68, 251. Apoptotic DNA Ladder Kit,
Fragmentation Burgoyne L. A. et al. (1974) Biochem. J. 143, 67. Cat. No. 11 835 246 001
Stach R. W. et al. (1979) J. Neurochem. 33, 257. Cell Death Detection ELISAPLUS,
Cat. No. 11 744 425 001
Cell Death Detection ELISA,
Cat. No. 11 544 675 001
Cellular DNA Fragmentation
ELISA, Cat. No. 11 585 045 001
In Situ Cell Death Detection Kit,
Fluorescein,
Cat. No. 11 684 795 001
TMR, Cat. No. 12 156 792 001
AP, Cat. No. 11 684 809 001
POD, Cat. No. 11 684 817 001
DNA-PKCS DNA-dependent pro- Casiolarosen L. et al. (1996) J. Exp. Med. 183, 1957.
tein kinase catalytic
subunit
DNA-repair De Murcia G. & De Murcia J. (1994) TIBS 19, 172. Anti-PARP,
Cat. No. 11 835 238 001
DR3 Death Receptor Chinnaiyan A. M. et al. (1996) Science 274, 990.
DR4 Death Receptor Pan G. H. et al. (1997) Science 276, 111.
DR5 Death Receptor Walczak H. et al. (1997) EMBO J. 16, 5386.
Sheridan J. P. et al. (1997) Science 277.
Endonuclease Walker P. R. & Sikorska (1994) Biochem. and Cell Nuclease S7,
Biology 72, 615. Cat. No. 10 107 921 001
Dini L. et al. (1996) Exp. Cell Res. 223, 340. Nuclease P1,
Cat. No. 10 236 225 001
Nuclease S1,
Cat. No. 10 818 348 001
DNase I, RNase free,
Cat. No. 10 776 785 001
DNase I, grade I,
Cat. No. 10 104 132 001
DNase I, grade II,
Cat. No. 10 104 159 001
FADD/ FADD = Chinnaiyan A. M. et al. (1995) Cell 81, 505.
MORT-1 Fas-associated Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.
death domain Vincenz C. & Dixit V. M. (1997) J. Biol. Chem. 272, 6578.
FAK Focal adhesion Crouch D. H. et al. (1996) Oncogene 12, 2689.
C
kinase Hungerford J. E. et al. (1996) J. Cell Biol. 135, 1383.
Fas Synonyms: Trauth et al. (1989) Science 245, 301. Anti-Fas, Cat. No. 11 922 432 001
Fas = CD 95 = Apo1
Fas-ligand Synonyms: Nagata S. & Goldstein P. (1995) Science 267, 1449.
CD 95/fas Fas = CD 95 = Apo1 Lynch D. H. et al. (1995) Immunol. Today 16, 569.
(receptor) Tanaka M. et al. (1998) Nature Medicine 4, 1, 31.
FLICE/MACH FADD like ICE Muzio M. et al. (1996) Cell 85, 817.
Boldin M. P. et al. (1996) Cell 85, 803.
Fernandes-Alnemri T. et al. (1996) Proc. Natl. Acad. Sci.
USA 93, 7464.
Scaffidi C. et al. (1997) J. Biol. Chem. 272, 43, 26953.

References

product
FLIP FLICE-inhibitory Thome M. et al. (1997) Nature 386, 517.
proteins Irmler M. et al (1997) Nature 388, 190.
Fodrin Martin S. J. et al. (1995) J. Biol. Chemistry 270, 6425.
fos Smeyne R. J. et al. (1995) Nature 363, 166 and Erratum
Nature 365, 279.
Colotta F. et al. (1992) J. Biol. Chem. 267, 18278.
G-Actin Boone D. L. & Tsang B. K. (1997) Biology and
Reproduction 57, 813.
Gas-2 Brancolini C. et al. (1997) Cell Death and Diff. 4, 247.
Gelsolin Kothakota S. et al. (1997) Science 278, 294.
Glucocorticoid/ Schwartzman R. A. & Cidlowski J. A. (1994) Int. Arch. of
Glucocorticoid- Allergy and Immunology 105, 347.
Receptor Perrinwolff M. et al. (1995) Biochem. Pharmacology 50,
103.
Kiefer J. et al. (1995) J. Immunology 155, 4525.
Granzyme Irmler M. et al. (1995) J. Exp. Med. 181, 1917.
A, B Peitsch M. C. & Tschopp J. (1994) Proteolytic Enzymes
244, 80.
Nakajima H. et al. (1995) J. Exp. Med. 181, 1037.
Smyth M. J. & Trapani J. A. (1995) Immunology Today
16, 202.
Darmon A. J. et al. (1995) Nature 377, 446.
Quan L. T. et al. (1996) Proc. Nat. Acad. Sci. 93, 1972.
hnRNPs Heteronuclear Waterhaus N. et al. (1996) J. Biol. Chem. 271, 29335.
C1/C2 Ribonucleoproteins
ICAD Inhibitor of CAD Enari M. et al. (1998) Nature 391, 43.
ICE Interleukin-1/ Whyte M. & Evan G. (1995) Nature 376, 17. Interleukin-1, human,
converting enzyme Atkinson E. A. & Bleackley R. C. (1995) Critical Reviews Cat. No. 11 457 756 001
in Immunology 15, 359. Interleukin-1, mouse,
Kumar S. & Harvey N. L. (1995) FEBS Letters 375, 169. Cat. No. 11 444 590 001
JNK Jun N-terminal Hibi M. et al. (1993) Genes Dev. 7 (11), 2135.
kinase
Lamin A, B Weaver V. M. et al. (1996) J. of Cell Science 109, 45.
MAP Mitogen activated pro- Meyer C. F. et al. (1996) J. Biol. Chem. 271, 8971.
tein kinase
MCL-1 Williams G. T. & Smith C. A. (1993) Cell 74, 777.
Mdm-2 Chen J. D. et al. (1996) Mol. and Cellular Biol. 16, 2445.
Yu K. et al. (1997) Cell Growth & Diff. 8.
MEKK-1 MAP Kinase Cardone M. H. et al. (1997) Cell 90.
Kinase 1 Meyer C. F. et al. (1996) J. Biol. Chem. 271, 8971.
MORT-1 Boldin M. P. (1995) J. Biol. Chem. 270, 7795.
(see FADD) Chinnaiyan A. M. et al. (1995) Cell 81, 505.
Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.
NEDD Gu Y. et al. (1995) J. Biol. Chemistry 270, 18715.
NF-B Nuclear factor Wiegmann K. et al. (1994) Cell 78, 1005.
kappaB
NuMa Nuclear matrix protein Guethhallonet C. et al. (1997) Exp. Cell Res. 233.
Weaver V. M. et al. (1996) J. Cell science 109, 45.
Hsu H. L. & Yeh N. H. (1996) J. Cell science 109, 277.
C
p53 Yonish-Rouach E. et al. (1993) Mol. Cell Biol. 13, 1415. p53 ELISA,
Zambetti G. P. (1993) FASEB J. 7, 855. Cat. No. 11 828 789 001
Lowe S. W. et al. (1993) Cell 74, 957.
PAK-2 p21 activated kinase Rudel T. & Bokoch G. M. (1997) Science 276.
PARP Poly-ADP-ribose- Lippke J. A. et al. (1996) J. Biol. Chem. 271, 1825.
polymerase De Murcia G. & De Murcia J. (1994) TIBS 19, 172.
Perforin Nakajima H. et al. (1995) J. Exp. Med. 181, 1037.
Schroter M. et al. (1995) Europ. J. Immunol. 25, 3509.
Lowin B. et al. (1996) Int. Immunology 8, 57.
Appendix 125
References

product
Phosphatidyl- Vermes I. et al. (1995) J. Immunol. Methods 184, 39. Annexin-V-Alexa 568,
serine Cat. No. 11 985 485 001,
(new Cat. No. 03 703 126 001)
Annexin-V-FLUOS,
Cat. No. 11 828 681001
Annexin-V-Biotin,
Cat. No. 11 828 690 001
PITSLRE Beyaert R. et al. (1997) J. Biol. Cem. 272, 11694.
PKC Protein kinase C Emoto Y. et al. (1995) EMBO J. 14, 6148.
Ghayur T. et al. (1996) J. Exp. Med. 184, 2399.
pRb Retinoblastoma Hansen R. et al. (1995) Oncogene 11, 2535.
protein Haaskogan D. A. et al. (1995) EMBO J. 14, 461.
Picksley S. M. (1994) Curr. Opinion in Cell Biology 6, 853.
Presenilin Loetscher H. et al. (1997) J. Biol. Chem. 272.
prICE Smyth M. J. et al. (1996) Biochem. Journal 316, 25.
RAIDD RIP associated Duan & Dixit (1997) Nature 385, 86.
ICH-1/CED-3
homologous protein
with a death domain
Ras Krueger G. R. F. et al. (1995) Pathologe 16, 120.

Wang H. G. et al. (1995) J. Cell Biol. 129, 1103.
Fernandez A. et al. (1995) Oncogene 10, 769.
RIP Receptor interacting Stanger B. Z. et al. (1995) Cell 81, 513.
protein Hsu H. et al. (1996) Immunity 4, 387.
Grimm S. et al. (1996) Proc. Natl. Acad. Sci. 93, 10923.
Sphingo- Heller R. A. & Kronke M. (1994) J. Cell Biol. 126, 5.
myelinase Kolesnik R. & Golde D. W. (1994) Cell 77, 325.
SREBPs Sterol-regulatory Wang X. D. et al. (1996) EMBO J. 15, 1012.
element binding
proteins
TNF- Tumor necrosis Leist M. et al. (1994) J. Immunol. 153, 1778. TNF-, human,
factor Cat. Nos. 11 371 843 001,
11 088 939 001
TNF- Nagata S. (1997) Cell, 88, 355. TNF-, mouse,
receptor Tartaglia L. A. et al. (1993) Cell 74, 845. Cat. No. 11 271 156 001
TNF- ELISA, human,
Cat. No. 11 425 943 001
TRADD TNFR1-associated Hsu H. et al. (1995) Cell 81, 495.
death domain
TRAF2 TNF receptor Liu Z.-G. et al. (1996) Cell 87, 565.
associated factor
TRAIL TNF-related Wiley S. R. et al. (1995) Immunity 3, 673.
-R1, -R2, -R3 apoptosis inducing Walczak H. et al. (1997) EMBO Journal 16, 5386.
ligand Deglli-Esposti M. A. et al. (1997) J. Exp. Med. 186, 1165.
Sheridan J. P. et al. (1997) Science 277, 818.
Trans- Zhang L.-X. et al. (1995) J. Biol. Chemistry 270, 6022.
glutaminase Melino G. et al. (1994) Mol. and Cell Biology 14, 6584.
U1-70 kDa U1 small nuclear Rosena & Casciolarosen L. (1997) J. Biol. Chem. 64, 50.
snRNP ribonucleoprotein
C
protein
YAMA Synonyms: Tewari M. et al (1995) Cell 81, 801.
CPP32, Apopain
Table 20: Published sources that contain more information about the components of the apoptosis pathways (Figure 2, page 5).

References
Synonyms
Proteases Synonyms
Caspase-1 ICE
Caspase-2 ICH-1
Caspase-3 CPP32, Yama, Apopain
Caspase-4 ICErel-II, TX, ICH-2
Caspase-5 ICErel-III, TY
Caspase-6 Mch2
Caspase-7 Mch3, ICE-LAP3, CMH-1
Caspase-8 FLICE, MACH, Mch5
Caspase-9 ICE-LAP6, Mch6
Caspase-10 Mch4 / FLICE 2
Caspase-11 ICH-3
Caspase-12
Caspase-13 ERICE
Caspase-14 MICE
Granzyme B CTL proteinase-1, Fragmentin-2, RNKP-1
Receptor Synonyms
CD95 APO-1, Fas
DcR1 TRID, LIT and TRAIL-R3
DcR2 TRAIL-R4
DcR3
DR-3 APO-3, TRAMP, WSL-1, LARD
DR-4 TRAIL-R1
DR-5 TRAIL-R2, TRICK2, KILLER
DR-6
DR-1 TNF-R1
DR-2 CD95
RANK
Ligands
CD95L Fas ligand, APO-1L
TRAIL APO-2L
TWEAK APO-3L
RANK L TRANCE
C
Apaf Synonyms
Apaf-1 (no alternative, homologue to ced-4)
Apaf-2 Cytochrome C
Apaf-3 Caspase 9 (homologue to ced-3)
Appendix 127
References
Examples for applications of Roche Applied Science products
3.2 Examples for applications of Roche Applied Science

products
Cell Death Kan Koizumi, Vassiliki Poulaki, Sven Doehmen, Gerhard
Welsandt, Sven Radetzky, Alexandra Lappas, Norbert Kociok,
Apoptotic DNA-Ladder Kit, Cat.-No. 11 835 246 001 Bernd Kirchhof, and Antonia M. Joussen
Contribution of TNF- to Leukocyte Adhesion, Vascular Leakage,
Jennifer Wessells, Shoshana Yakar, and Peter F. Johnson
and Apoptotic Cell Death in Endotoxin-Induced Uveitis In Vivo
Critical Prosurvival Roles for C/EBP and Insulin-Like Growth Invest. Ophthalmol. Vis. Sci., May 2003; 44: 2184 - 2191.
Factor I in Macrophage Tumor Cells
Mol. Cell. Biol., Apr 2004; 24: 3238 - 3250. Wendy E. Smith, Anne V. Kane, Sausan T. Campbell, David W. K.
Acheson, Brent H. Cochran, and Cheleste M. Thorpe
Renu A. Kowluru, Anjaneyulu Kowluru, Subrata Chakrabarti, Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to
and Zia Khan
p38 and JNK Activation and Induction of Apoptosis in Intestinal
Potential Contributory Role of H-Ras, a Small G-Protein, in the
Epithelial Cells
Development of Retinopathy in Diabetic Rats
Infect. Immun., Mar 2003; 71: 1497 - 1504.
Diabetes, Mar 2004; 53: 775 - 783.
Hanping Feng, Yi Zeng, Michael W. Graner, Anna Likhacheva,
Li Wen, Jian Peng, Zhenjun Li, and F. Susan Wong
and Emmanuel Katsanis
The Effect of Innate Immunity on Autoimmune Diabetes and the
Exogenous stress proteins enhance the immunogenicity of
Expression of Toll-Like Receptors on Pancreatic Islets apoptotic tumor cells and stimulate antitumor immunity
J. Immunol., Mar 2004; 172: 3173 - 3180.
Blood, Jan 2003; 101: 245 - 252.
Athanassios Argiris, Chun-Xia Wang, Steve G. Whalen, and
Lawrence Y. Lee, Yuko J. Miyamoto, Bradley W. McIntyre, Mag-
Michael P. DiGiovanna nus Hk, Kirk W. McCrea, Damien McDevitt, and Eric L. Brown
Synergistic Interactions between Tamoxifen and Trastuzumab
The Staphylococcus aureus Map protein is an immunomodula-
(Herceptin)
tor that interferes with T cellmediated responses
Clin. Cancer Res., Feb 2004; 10: 1409 - 1420. J. Clin. Invest., Nov 2002; 110: 1461 - 1471.
Renu A. Kowluru and Saiyeda Noor Abbas
Fabienne Vernejoul, Patrick Faure, Naoual Benali, Denis Calise,
Diabetes-Induced Mitochondrial Dysfunction in the Retina
Grard Tiraby, Lucien Pradayrol, Christiane Susini, and Louis
Invest. Ophthalmol. Vis. Sci., Dec 2003; 44: 5327 - 5334. Buscail
Laura Suomalainen, Jukka K. Hakala, Virve Pentikinen, Marjut Antitumor Effect of in Vivo Somatostatin Receptor Subtype 2
Otala, Krista Erkkil, Markku O. Pentikinen, and Leo Dunkel Gene Transfer in Primary and Metastatic Pancreatic Cancer
Sphingosine-1-Phosphate in Inhibition of Male Germ Cell Apo- Models
ptosis in the Human Testis Cancer Res., Nov 2002; 62: 6124 - 6131.
J. Clin. Endocrinol. Metab., Nov 2003; 88: 5572 - 5579.
William C. Gordon, Douglas M. Casey, Walter J. Lukiw, and
Katare Gopalrao Rajesh, Shiro Sasaguri, Ryoko Suzuki, and Nicolas G. Bazan
Hironori Maeda DNA Damage and Repair in Light-Induced Photoreceptor
Antioxidant MCI-186 inhibits mitochondrial permeability transi- Degeneration
tion pore and upregulates Bcl-2 expression Invest. Ophthalmol. Vis. Sci., Nov 2002; 43: 3511 - 3521.
Am J Physiol Heart Circ Physiol, Nov 2003; 285: 2171 - 2178.
Julien Davaille, Liying Li, Ariane Mallat, and Sophie Lotersztajn
R Horvth, H Lochmller, C Scharfe, B H Do, P J Oefner, Sphingosine 1-Phosphate Triggers Both Apoptotic and Survival
J Mller-Hcker, B G Schoser, D Pongratz, D P Auer, and Signals for Human Hepatic Myofibroblasts
M Jaksch J. Biol. Chem., Sep 2002; 277: 37323 - 37330.
A tRNAAla mutation causing mitochondrial myopathy clinically
Jenny M. Kim, Junjie Fang, Susan Rheingold, Richard Aplenc,
resembling myotonic dystrophy Robert Wasserman, and Stephan A. Grupp
J. Med. Genet., Oct 2003; 40: 752 - 757.
Cytoplasmic Heavy Chain Confers Sensitivity to Dexametha-
K. Erkkil, L. Suomalainen, M. Wikstrm, M. Parvinen, sone-induced Apoptosis in Early B-lineage Acute Lymphoblas-
and L. Dunkel tic Leukemia
Chemical Anoxia Delays Germ Cell Apoptosis in the Human Cancer Res., Aug 2002; 62: 4212 - 4216.
Testis
HENRIKKA AITO, KRISTIINA T. AALTO, and KARI O. RAIVIO
Biol. Reprod., Aug 2003; 69: 617 - 626. Biphasic ATP Depletion Caused by Transient Oxidative Exposure
Irina A. Vasilevskaya, Tatiana V. Rakitina, and Peter J. ODwyer Is Associated with Apoptotic Cell Death in Rat Embryonal Corti-
Geldanamycin and its 17-Allylamino-17-Demethoxy Analogue cal Neurons
Antagonize the Action of Cisplatin in Human Colon Adenocar- Pediatr. Res., Jun 2002; 52: 40 - 45.
C
cinoma Cells: Differential Caspase Activation as a Basis for
M. Otala, K. Erkkil, T. Tuuri, J. Sjberg, L. Suomalainen, A-M.
Interaction
Suikkari, V. Pentikinen, and L. Dunkel
Cancer Res., Jun 2003; 63: 3241 - 3246. Cell death and its suppression in human ovarian tissue culture
Maria Elena Dell'Aquila, Maria Albrizio, Filippo Maritato, Paolo Mol. Hum. Reprod., Mar 2002; 8: 228 - 236.
Minoia, and Katrin Hinrichs
K. Erkkil, H. Aito, K. Aalto, V. Pentikinen, and L. Dunkel
Meiotic Competence of Equine Oocytes and Pronucleus Forma- Lactate inhibits germ cell apoptosis in the human testis
tion after Intracytoplasmic Sperm Injection (ICSI) as Related to
Mol. Hum. Reprod., Feb 2002; 8: 109 - 117.
Granulosa Cell Apoptosis
Biol. Reprod., Jun 2003; 68: 2065 - 2072.

References
Virve Pentikinen, Laura Suomalainen, Krista Erkkil, Eeva Steve Gendron, Julie Couture, and Fawzi Aoudjit
Martelin, Martti Parvinen, Markku O. Pentikinen, and Leo Integrin 21 Inhibits Fas-mediated Apoptosis in T Lymphocytes
Dunkel by Protein Phosphatase 2A-dependent Activation of the MAPK/
Nuclear Factor-B Activation in Human Testicular Apoptosis ERK Pathway
Am. J. Pathol., Jan 2002; 160: 205 - 218. J. Biol. Chem., Dec 2003; 278: 48633 - 48643.
Maria Laura Garcia-Bermejo, Federico Coluccio Leskow, PART XIV. CELL DEATH AND NEURODEGENERATIVE DIS-
Teruhiko Fujii, Qiming Wang, Peter M. Blumberg, Motoi Ohba, EASES:
Toshio Kuroki, Kee-Chung Han, Jeewoo Lee, Victor E. Marquez, A CORSARO, S THELLUNG, V VILLA, D ROSSI PRINCIPE,
and Marcelo G. Kazanietz D PALUDI, S ARENA, E MILLO, D SCHETTINI, G DAMONTE,
Diacylglycerol (DAG)-lactones, a New Class of Protein Kinase C A ACETO, G SCHETTINI, and T FLORIO
(PKC) Agonists, Induce Apoptosis in LNCaP Prostate Cancer Prion Protein Fragment 106-126 Induces a p38 MAP Kinase
Cells by Selective Activation of PKC Dependent Apoptosis in SH-SY5Y Neuroblastoma Cells Inde-
J. Biol. Chem., Jan 2002; 277: 645 - 655. pendently from the Amyloid Fibril Formation
Ann. N.Y. Acad. Sci., Dec 2003; 1010: 610 - 622.
Robert X.-D. Song, Gil Mor, Fred Naftolin, Robert A. McPherson,
Joon Song, Zhenguo Zhang, Wei Yue, JiPing Wang, and Richard Nicoletta Ferrari, Monica Morini, Ulrich Pfeffer, Simona Ming-
J. Santen helli, Douglas M. Noonan, and Adriana Albini
Effect of Long-Term Estrogen Deprivation on Apoptotic Inhibition of Kaposis Sarcoma in Vivo by Fenretinide
Responses of Breast Cancer Cells to 17-Estradiol Clin. Cancer Res., Dec 2003; 9: 6020 - 6029.
J Natl Cancer Inst, Nov 2001; 93: 1714 - 1723.
PART XIII. INFECTION-INDUCED APOPTOSIS:
PACO PINO, IOANNIS VOULDOUKIS, NATHALIE DUGAS,
GERALDINE HASSANI-LOPPION, BERNARD DUGAS, and
Cell Death Detection ELISA, Cat.-No. 11 544 675 001
DOMINIQUE MAZIER
Jeffrey A. Rudolph, Julia L. Poccia, and Mitchell B. Cohen Redox-Dependent Apoptosis in Human Endothelial Cells after
Cyclic AMP Activation of the Extracellular Signal-regulated Adhesion of Plasmodium falciparum-Infected Erythrocytes
Kinases 1 and 2: IMPLICATIONS FOR INTESTINAL CELL Ann. N.Y. Acad. Sci., Dec 2003; 1010: 582 - 586.
SURVIVAL THROUGH THE TRANSIENT INHIBITION OF
Shruti Nagar, Leslie E. Smith, and William F. Morgan
APOPTOSIS Mechanisms of cell death associated with death-inducing fac-
J. Biol. Chem., Apr 2004; 279: 14828 - 14834.
tors from genomically unstable cell lines
Riichiro Abe, Tadamichi Shimizu, Sho-ichi Yamagishi, Akihiko Mutagenesis, Nov 2003; 18: 549 - 560.
Shibaki, Shinjiro Amano, Yosuke Inagaki, Hirokazu Watanabe, Barinder P. S. Kang, Arunas Urbonas, Andrew Baddoo,
Hiroshi Sugawara, Hideki Nakamura, Masayoshi Takeuchi,
Stuart Baskin, Ashwani Malhotra, and Leonard G. Meggs
Tsutomu Imaizumi, and Hiroshi Shimizu
IGF-1 inhibits the mitochondrial apoptosis program in
Overexpression of Pigment Epithelium-Derived Factor mesangial cells exposed to high glucose
Decreases Angiogenesis and Inhibits the Growth of Human
Am J Physiol Renal Physiol, Nov 2003; 285: 1013 - 1024.
Malignant Melanoma Cells in Vivo
Am. J. Pathol., Apr 2004; 164: 1225 - 1232. Jiyun Yoo, Mayshan Ghiassi, Ludmila Jirmanova, Arthur G.
Balliet, Barbara Hoffman, Albert J. Fornace, Jr., Dan A.
Ho-Seong Kim, Angela R. Ingermann, Junko Tsubaki, Stephen
Liebermann, Erwin P. Bttinger, and Anita B. Roberts
M. Twigg, Gillian E. Walker, and Youngman Oh
Transforming Growth Factor--induced Apoptosis Is Mediated
Insulin-Like Growth Factor-Binding Protein 3 Induces Caspase- by Smad-dependent Expression of GADD45b through p38
Dependent Apoptosis through a Death Receptor-Mediated
Activation
Pathway in MCF-7 Human Breast Cancer Cells
J. Biol. Chem., Oct 2003; 278: 43001 - 43007.
Cancer Res., Mar 2004; 64: 2229 - 2237.
Tsungda Hsu, Suzanne M. Hingley-Wilson, Bing Chen, Mei
W K Leung, A H C Bai, V Y W Chan, J Yu, M W Y Chan, K-F To,
Chen, Annie Z. Dai, Paul M. Morin, Carolyn B. Marks, Jeevan
J-R Wu, K-K Chan, Y-G Fu, F K L Chan, and J J Y Sung
Padiyar, Celia Goulding, Mari Gingery, David Eisenberg, Robert
Effect of peroxisome proliferator activated receptor ligands on G. Russell, Steven C. Derrick, Frank M. Collins, Sheldon L.
growth and gene expression profiles of gastric cancer cells
Morris, C. Harold King, and William R. Jacobs, Jr.
Gut, Mar 2004; 53: 331 - 338.
The primary mechanism of attenuation of bacillus Calmette
Samuel K. Kulp, Ya-Ting Yang, Chin-Chun Hung, Kuen-Feng Gurin is a loss of secreted lytic function required for invasion
Chen, Ju-Ping Lai, Ping-Hui Tseng, Joseph W. Fowble, Patrick J. of lung interstitial tissue
Ward, and Ching-Shih Chen PNAS, Oct 2003; 100: 12420 - 12425.
3-Phosphoinositide-Dependent Protein Kinase-1/Akt Signaling Nilesh M. Dagia and Douglas J. Goetz
Represents a Major Cyclooxygenase-2-Independent Target for
A proteasome inhibitor reduces concurrent, sequential, and
Celecoxib in Prostate Cancer Cells
long-term IL-1- and TNF--induced ECAM expression and
C
Cancer Res., Feb 2004; 64: 1444 - 1451. adhesion
Nathalie Mthot, JingQi Huang, Nathalie Coulombe, John P. Am J Physiol Cell Physiol, Oct 2003; 285: 813 - 822.
Vaillancourt, Dita Rasper, John Tam, Yongxin Han, John Colucci,
Alex C. Chin, Nathalie Vergnolle, Wallace K. MacNaughton,
Robert Zamboni, Steven Xanthoudakis, Sylvie Toulmond, John L. Wallace, Morley D. Hollenberg, and Andre G. Buret
Donald W. Nicholson, and Sophie Roy
Proteinase-activated receptor 1 activation induces epithelial
Differential Efficacy of Caspase Inhibitors on Apoptosis Markers
apoptosis and increases intestinal permeability
during Sepsis in Rats and Implication for Fractional Inhibition PNAS, Sep 2003; 100: 11104 - 11109.
Requirements for Therapeutics
J. Exp. Med., Jan 2004; 199: 199 - 207.
Appendix 129
References
M. Lonergan, D. Aponso, K. W. Marvin, R. J. A. Helliwell, T. A. Cell Death Detection ELISAPLUS, Cat.-No. 11 774 425 001
Sato, M. D. Mitchell, T. Chaiwaropongsa, R. Romero, and J. A.
Raffaella Spagnuolo, Monica Corada, Fabrizio Orsenigo, Lucia
Keelan
Zanetta, Ulrich Deuschle, Peter Sandy, Claudio Schneider,
Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand
(TRAIL), TRAIL Receptors, and the Soluble Receptor Osteopro- Christopher J. Drake, Ferruccio Breviario, and Elisabetta Dejana
Gas1 is induced by VE-cadherin and vascular endothelial
tegerin in Human Gestational Membranes and Amniotic Fluid
growth factor and inhibits endothelial cell apoptosis
during Pregnancy and Labor at Term and Preterm
J. Clin. Endocrinol. Metab., Aug 2003; 88: 3835 - 3844. Blood, Apr 2004; 103: 3005 - 3012.
Dharminder Chauhan, Guilan Li, Klaus Podar, Teru Hideshima,

Hideharu Tomita, Michael Nazmy, Katsuya Kajimoto, Ghassan
Reshma Shringarpure, Laurence Catley, Constantine Mitsiades,
Yehia, Carlos A. Molina, and Junichi Sadoshima
Inducible cAMP Early Repressor (ICER) Is a Negative-Feedback Nikhil Munshi, Yu Tzu Tai, Nanjoo Suh, Gordon W. Gribble,
Tadashi Honda, Robert Schlossman, Paul Richardson, Michael
Regulator of Cardiac Hypertrophy and an Important Mediator of
B. Sporn, and Kenneth C. Anderson
Cardiac Myocyte Apoptosis in Response to -Adrenergic
Receptor Stimulation The bortezomib/proteasome inhibitor PS-341 and triterpenoid
CDDO-Im induce synergistic antimultiple myeloma (MM)
Circ. Res., Jul 2003; 93: 12 - 22.
activity and overcome bortezomib resistance
GLYCOBIOLOGY AND EXTRACELLULAR MATRICES: Blood, Apr 2004; 103: 3158 - 3166.
Liliana Schaefer, Karl-Friedrich Beck, Igor Raslik, Sebastian
Walpen, Daniel Mihalik, Miroslava Micegova, Katarina Macak- Chadrick E. Denlinger, Michael D. Keller, Marty W. Mayo, R.
Michael Broad, and David R. Jones
ova, Elke Schnherr, Daniela G. Seidler, Georg Varga, Roland M.
Combined proteasome and histone deacetylase inhibition in
Schaefer, Hans Kresse, and Josef Pfeilschifter
Biglycan, a Nitric Oxide-regulated Gene, Affects Adhesion, nonsmall cell lung cancer
J. Thorac. Cardiovasc. Surg., Apr 2004; 127: 1078 - 1086.
Growth, and Survival of Mesangial Cells
J. Biol. Chem., Jul 2003; 278: 26227 - 26237. Harry A. Rogoff, Mary T. Pickering, Fiona M. Frame, Michelle E.
Arno Hueber, Michael Weller, Gerhard Welsandt, Norbert Debatis, Yolanda Sanchez, Stephen Jones, and Timothy F.
Kowalik
Kociok, Bernd Kirchhof, and Peter Esser
Apoptosis Associated with Deregulated E2F Activity Is Depen-
Characterization of Daunorubicin-Induced Apoptosis in Retinal
Pigment Epithelial Cells: Modulation by CD95L dent on E2F1 and Atm/Nbs1/Chk2
Mol. Cell. Biol., Apr 2004; 24: 2968 - 2977.
Invest. Ophthalmol. Vis. Sci., Jul 2003; 44: 2851 - 2857.
Anna Csiszar, Zoltan Ungvari, Akos Koller, John G. Edwards,
Ryuya Shimoda, William E. Achanzar, Wei Qu, Takeaki Naga-
mine, Hitoshi Takagi, Masatomo Mori, and Michael P. Waalkes and Gabor Kaley
Proinflammatory phenotype of coronary arteries promotes
Metallothionein Is a Potential Negative Regulator of Apoptosis
endothelial apoptosis in aging
Toxicol. Sci., Jun 2003; 73: 294 - 300.
Physiol Genomics, Mar 2004; 17: 21 - 30.
Rui-Dong Duan, Yajun Cheng, Gert Hansen, Erik Hertervig,
Renu A. Kowluru, Anjaneyulu Kowluru, Subrata Chakrabarti,
Jian-Jun Liu, Ingvar Syk, Hans Sjstrm, and ke Nilsson
and Zia Khan
Purification, localization, and expression of human intestinal
alkaline sphingomyelinase Potential Contributory Role of H-Ras, a Small G-Protein, in the
Development of Retinopathy in Diabetic Rats
J. Lipid Res., Jun 2003; 44: 1241 - 1250.
Diabetes, Mar 2004; 53: 775 - 783.
Lara Longobardi, Monica Torello, Caroline Buckway, Lynda
ORear, William A. Horton, Vivian Hwa, Charles T. Roberts, Jr., Ioannis P. Trougakos, Alan So, Burkhard Jansen, Martin E.
Gleave, and Efstathios S. Gonos
Francesco Chiarelli, Ron G. Rosenfeld, and Anna Spagnoli
Silencing Expression of the Clusterin/Apolipoprotein J Gene in
A Novel Insulin-Like Growth Factor (IGF)-Independent Role for
IGF Binding Protein-3 in Mesenchymal Chondroprogenitor Cell Human Cancer Cells Using Small Interfering RNA Induces
Spontaneous Apoptosis, Reduced Growth Ability, and Cell
Apoptosis
Sensitization to Genotoxic and Oxidative Stress
Endocrinology, May 2003; 144: 1695 - 1702.
Cancer Res., Mar 2004; 64: 1834 - 1842.
Li Hua Wang, Xiao Yi Yang, Xiaohu Zhang, Kelly Mihalic,
Stefan Hammerschmidt, Hartmut Kuhn, Thomas Grasenack,
Weihua Xiao, and William L. Farrar
Christian Gessner, and Hubert Wirtz
The cis Decoy against the Estrogen Response Element Sup-
presses Breast Cancer Cells via Target Disrupting c-fos not Apoptosis and Necrosis Induced by Cyclic Mechanical Stretch-
ing in Alveolar Type II Cells
Mitogen-activated Protein Kinase Activity
Am. J. Respir. Cell Mol. Biol., Mar 2004; 30: 396 - 402.
Cancer Res., May 2003; 63: 2046 - 2051.
Dong Xiao, Sanjay K. Srivastava, Karen L. Lew, Yan Zeng, Pam- Sujoy Bhattacharya, Ramesh M. Ray, and Leonard R. Johnson
Prevention of TNF--induced apoptosis in polyamine-depleted
ela Hershberger, Candace S. Johnson, Donald L. Trump, and
IEC-6 cells is mediated through the activation of ERK1/2
Shivendra V. Singh
C
Allyl isothiocyanate, a constituent of cruciferous vegetables, Am J Physiol Gastrointest Liver Physiol, Mar 2004; 286:
479 - 490.
inhibits proliferation of human prostate cancer cells by causing
G2/M arrest and inducing apoptosis
Carcinogenesis, May 2003; 24: 891 - 897.

References
Masahiko Watabe, Keiichi Hishikawa, Atsushi Takayanagi, Kimberly A. Carlson, Gary Leisman, Jenae Limoges, Garrett D.
Nobuyoshi Shimizu, and Toshio Nakaki Pohlman, Masahide Horiba, James Buescher, Howard E. Gen-
Caffeic Acid Phenethyl Ester Induces Apoptosis by Inhibition of delman, and Tsuneya Ikezu
NFB and Activation of Fas in Human Breast Cancer MCF-7 Molecular Characterization of a Putative Antiretroviral Tran-
Cells scriptional Factor, OTK18
J. Biol. Chem., Feb 2004; 279: 6017 - 6026. J. Immunol., Jan 2004; 172: 381 - 391.
Geoffrey A. Walford, Rose-Laure Moussignac, Anne W. Scribner, Salvatore Cortellino, David Turner, Valeria Masciullo, Filippo
Joseph Loscalzo, and Jane A. Leopold Schepis, Domenico Albino, Rene Daniel, Anna Marie Skalka,
Hypoxia Potentiates Nitric Oxide-mediated Apoptosis in Endot- Neal J. Meropol, Christophe Alberti, Lionel Larue, and Alfonso
helial Cells via Peroxynitrite-induced Activation of Mitochon- Bellacosa
dria-dependent and -independent Pathways From The Cover: The base excision repair enzyme MED1 medi-
J. Biol. Chem., Feb 2004; 279: 4425 - 4432. ates DNA damage response to antitumor drugs and is associ-
ated with mismatch repair system integrity
Sylvie Toulmond, Keith Tang, Yves Bureau, Helen Ashdown, PNAS, Dec 2003; 100: 15071 - 15076.
Sarah Degen, Ruth O'Donnell, John Tam, Yongxin Han,
John Colucci, Andr Giroux, Yanxia Zhu, Mathieu Boucher, George W. Small, Sivagurunathan Somasundaram, Dominic T.
Bill Pikounis, Steven Xanthoudakis, Sophie Roy, Michael Rigby, Moore, Yue Y. Shi, and Robert Z. Orlowski
Robert Zamboni, George S Robertson, Gordon Y K Ng, Donald Repression of Mitogen-Activated Protein Kinase (MAPK) Phos-
W Nicholson, and Jean-Pierre Flckiger phatase-1 by Anthracyclines Contributes to Their Antiapoptotic
Neuroprotective effects of M826, a reversible caspase-3 inhibi- Activation of p44/42-MAPK
tor, in the rat malonate model of Huntington's disease J. Pharmacol. Exp. Ther., Dec 2003; 307: 861 - 869.
Br. J. Pharmacol., Feb 2004; 141: 689 - 697.
Stephanie Brndlein, Tina Pohle, Nele Ruoff, Ewa Wozniak,
Qiang Liu and Baolu Zhao Hans-Konrad Mller-Hermelink, and H. Peter Vollmers
Nicotine attenuates -amyloid peptide-induced neurotoxicity, Natural IgM Antibodies and Immunosurveillance Mechanisms
free radical and calcium accumulation in hippocampal neuronal against Epithelial Cancer Cells in Humans
cultures Cancer Res., Nov 2003; 63: 7995 - 8005.
Maria V. Papadopoulou and William D. Bloomer
Michishige Harada, Ken-ichiro Seino, Hiroshi Wakao, Sakura NLCQ-1 (NSC 709257): Exploiting Hypoxia with a Weak DNA-
Sakata, Yuko Ishizuka, Toshihiro Ito, Satoshi Kojo, Toshinori Intercalating Bioreductive Drug
Nakayama, and Masaru Taniguchi Clin. Cancer Res., Nov 2003; 9: 5714 - 5720.
Down-regulation of the invariant V14 antigen receptor in NKT
Erding Hu, Edward Dul, Chiu-Mei Sung, Zunxuan Chen, Robert
cells upon activation
Kirkpatrick, Gui-Feng Zhang, Kyung Johanson, Ronggang Liu,
Int. Immunol., Feb 2004; 16: 241 - 247.
Amparo Lago, Glenn Hofmann, Ricardo Macarron, Maite De
Masahito Shimizu, Masumi Suzui, Atsuko Deguchi, Jin T. E. Lim, Los Frailes, Paloma Perez, John Krawiec, James Winkler, and
and I. Bernard Weinstein Michael Jaye
Effects of Acyclic Retinoid on Growth, Cell Cycle Control, Epi- Identification of Novel Isoform-Selective Inhibitors within Class I
dermal Growth Factor Receptor Signaling, and Gene Expression Histone Deacetylases
in Human Squamous Cell Carcinoma Cells J. Pharmacol. Exp. Ther., Nov 2003; 307: 720 - 728.
Clin. Cancer Res., Feb 2004; 10: 1130 - 1140.
Chia-Ron Yang, Shie-Liang Hsieh, Che-Ming Teng, Feng-Ming
M30 CytoDEATH, Cat.-No. 12 140 322 001
Ho, Wen-Ling Su, and Wan-Wan Lin
Soluble Decoy Receptor 3 Induces Angiogenesis by Neutraliza- M30 CytoDEATH, Fluorescein, Cat.-No. 12 156 857 001
tion of TL1A, a Cytokine Belonging to Tumor Necrosis Factor
Superfamily and Exhibiting Angiostatic Action Leonieke Terpstra, Jose Prud'homme, Alice Arabian, Shu
Cancer Res., Feb 2004; 64: 1122 - 1129. Takeda, Grard Karsenty, Shoukat Dedhar, and Ren St-Arnaud
Reduced chondrocyte proliferation and chondrodysplasia in
Pak-Shan Leung, William J. Aronson, Tung H. Ngo, Lawrence A. mice lacking the integrin-linked kinase in chondrocytes
Golding, and R. James Barnard J. Cell Biol., Jul 2003; 162: 139 - 148.
Exercise alters the IGF axis in vivo and increases p53 protein in
prostate tumor cells in vitro Ingrid Herr, Esat Ucur, Kerstin Herzer, Stella Okouoyo, Rdiger
J Appl Physiol, Feb 2004; 96: 450 - 454. Ridder, Peter H. Krammer, Magnus von Knebel Doeberitz, and
Klaus-Michael Debatin
Ulrich Laufs, Nikos Werner, Andreas Link, Matthias Endres, Glucocorticoid Cotreatment Induces Apoptosis Resistance
Sven Wassmann, Kristina Jrgens, Eckart Miche, Michael toward Cancer Therapy in Carcinomas
Bhm, and Georg Nickenig Cancer Res., Jun 2003; 63: 3112 - 3120.
Physical Training Increases Endothelial Progenitor Cells, Inhibits
C
Neointima Formation, and Enhances Angiogenesis Kay-Uwe Wagner, Andrea Krempler, Yongyue Qi, KyungRan
Circulation, Jan 2004; 109: 220 - 226. Park, MaLinda D. Henry, Aleata A. Triplett, Gregory Riedlinger,
Edmund B. Rucker III, and Lothar Hennighausen
Catherine Kern, Jean-Franois Cornuel, Christian Billard, Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Sur-
Ruoping Tang, Danielle Rouillard, Virginie Stenou, Thierry vival of Embryonic and Adult Tissues
Defrance, Florence Ajchenbaum-Cymbalista, Pierre-Yves Mol. Cell. Biol., Jan 2003; 23: 150 - 162.
Simonin, Sophie Feldblum, and Jean-Pierre Kolb
Involvement of BAFF and APRIL in the resistance to apoptosis
of B-CLL through an autocrine pathway
Blood, Jan 2004; 103: 679 - 688.
Appendix 131
References
Ai Q. Truong-Tran, Richard E. Ruffin, Paul S. Foster, Aulikki M. Rainer Riesenberg, Alexander Buchner, Heike Pohla, and Horst
Koskinen, Peter Coyle, Jeffrey C. Philcox, Allan M Rofe, and Lindhofer
Peter D. Zalewski Lysis of Prostate Carcinoma Cells by Trifunctional Bispecific
Altered Zinc Homeostasis and Caspase-3 Activity in Murine Antibodies (EpCAM x CD3)
Allergic Airway Inflammation J. Histochem. Cytochem., Jul 2001; 49: 911 - 918.
Am. J. Respir. Cell Mol. Biol., Sep 2002; 27: 286 - 296.
Peter Birner, Monika Schindl, Andreas Obermair, Gerhard
Kristin R. Coulter, Andrea Doseff, Patricia Sweeney, Yijie Wang, Breitenecker, and Georg Oberhuber
Clay B. Marsh, Mark D. Wewers, and Daren L. Knoell Expression of Hypoxia-inducible Factor 1 in Epithelial Ovarian
Opposing Effect by Cytokines on Fas-Mediated Apoptosis in Tumors: Its Impact on Prognosis and on Response to Chemo-
A549 Lung Epithelial Cells therapy
Am. J. Respir. Cell Mol. Biol., Jan 2002; 26: 58 - 66. Clin. Cancer Res., Jun 2001; 7: 1661 - 1668.
Michael Kasper, Cora Roehlecke, Martin Witt, Heinz Fehren- Declan F. McCole, Lars Eckmann, Fabrice Laurent, and Martin F.
bach, Andreas Hofer, Toshio Miyata, Cora Weigert, Richard H. Kagnoff
W. Funk, and Erwin D. Schleicher Intestinal Epithelial Cell Apoptosis following Cryptosporidium
Induction of Apoptosis by Glyoxal in Human Embryonic Lung parvum Infection
Epithelial Cell Line L132 Infect. Immun., Mar 2000; 68: 1710 - 1713.
Am. J. Respir. Cell Mol. Biol., Oct 2000; 23: 485 - 491.
Stacy M. Stabler, Lisa L. Ostrowski, Susan M. Janicki, and

Caspase 3 Activity Assay, Cat.-No. 12 012 952 001
Mervyn J. Monteiro
A Myristoylated Calcium-binding Protein that Preferentially Yossi Gottfried, Asaf Rotem, Rona Lotan, Hermann Steller, and
Interacts with the Alzheimer's Disease Presenilin 2 Protein Sarit Larisch
J. Cell Biol., Jun 1999; 145: 1277 - 1292. The mitochondrial ARTS protein promotes apoptosis through
targeting XIAP
Christian Bojarski, Jrg Weiske, Torsten Schneberg, Werner
Schrder, Joachim Mankertz, Jrg-Dieter Schulzke, Peter EMBO J., Mar 2004; 10.1038/sj.emboj.7600155.
Florian, Michael Fromm, Rudolf Tauber, and Otmar Huber Kyeong Cheon Jung, Weon Seo Park, Hae Jung Kim, Eun Young
The specific fates of tight junction proteins in apoptotic Choi, Myeong-Cherl Kook, Han-Woong Lee, and Youngmee Bae
epithelial cells TCR-Independent and Caspase-Independent Apoptosis of
J. Cell Sci., Mar 2004; 10.1242/jcs.01071. Murine Thymocytes by CD24 Cross-Linking
J. Immunol., Jan 2004; 172: 795 - 802.
Matthias Bruewer, Andreas Luegering, Torsten Kucharzik,
Charles A. Parkos, James L. Madara, Ann M. Hopkins, and Debabrata Mandal, Veronique Baudin-Creuza, Asima Bhatta-
Asma Nusrat charyya, Shresh Pathak, Jean Delaunay, Manikuntala Kundu,
Proinflammatory Cytokines Disrupt Epithelial Barrier Function and Joyoti Basu
by Apoptosis-Independent Mechanisms Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic
J. Immunol., Dec 2003; 171: 6164 - 6172. Domain of the Human Erythroid Anion Exchanger 1 (Band 3)
J. Biol. Chem., Dec 2003; 278: 52551 - 52558.
Carolyn L. Cannon, Michael P. Kowalski, Kimberly S. Stopak,
and Gerald B. Pier Niels W. C. J. van de Donk, Diana Schotte, Marloes M. J.
Pseudomonas aeruginosaInduced Apoptosis Is Defective in Kamphuis, Arinne M. W. van Marion, Berris van Kessel,
Respiratory Epithelial Cells Expressing Mutant Cystic Fibrosis Andries C. Bloem, and Henk M. Lokhorst
Transmembrane Conductance Regulator Protein Geranylgeranylation Is Critical for the Regulation of
Am. J. Respir. Cell Mol. Biol., Aug 2003; 29: 188 - 197. Survival and Proliferation of Lymphoma Tumor Cells
Clin. Cancer Res., Nov 2003; 9: 5735 - 5748.
Alison J. Faragher and Andrew M. Fry
Nek2A kinase stimulates centrosome disjunction and is Toshiki Itoh, Cristin O'Shea, and Stuart Linn
required for formation of bipolar mitotic spindles Impaired Regulation of Tumor Suppressor p53 Caused by Muta-
Mol. Biol. Cell, Jul 2003; 14: 2876 - 2889. tions in the Xeroderma Pigmentosum DDB2 Gene: Mutual Reg-
Julie Guillermet, Nathalie Saint-Laurent, Philippe Rochaix, Oliv- ulatory Interactions between p48DDB2 and p53
Mol. Cell. Biol., Nov 2003; 23: 7540 - 7553.
ier Cuvillier, Thierry Levade, Andrew V. Schally, Lucien Praday-
rol, Louis Buscail, Christiane Susini, and Corinne Bousquet P. Kelk, A. Johansson, R. Claesson, L. Hnstrm, and S. Kalfas
Somatostatin receptor subtype 2 sensitizes human pancreatic Caspase 1 Involvement in Human Monocyte Lysis Induced by
cancer cells to death ligand-induced apoptosis Actinobacillus actinomycetemcomitans Leukotoxin
PNAS, Jan 2003; 100: 155 - 160. Infect. Immun., Aug 2003; 71: 4448 - 4455.
Guo-Zhong Tao, Lusijah S. Rott, Anson W. Lowe, and M. Bishr Lin-Tao Jia, Li-Hong Zhang, Cui-Juan Yu, Jing Zhao, Yan-Ming
Omary Xu, Jun-Hao Gui, Ming Jin, Zong-Ling Ji, Wei-Hong Wen,
C
Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Cheng-Ji Wang, Si-Yi Chen, and An-Gang Yang
Activation and Cyclin/Cyclin-dependent Kinase Degradation Specific Tumoricidal Activity of a Secreted Proapoptotic Protein
J. Biol. Chem., May 2002; 277: 19295 - 19303. Consisting of HER2 Antibody and Constitutively Active
Caspase-3
Naoko Yagishita, Yoko Yamamoto, Tatsuya Yoshizawa, Keisuke
Sekine, Yoshikatsu Uematsu, Hisashi Murayama, Yumiko Nagai, Cancer Res., Jun 2003; 63: 3257 - 3262.
Wojciech Krezel, Pierre Chambon, Toshio Matsumoto, and Tommer Ravid, Adili Tsaba, Peter Gee, Reuven Rasooly, Edward
Shigeaki Kato A. Medina, and Tzipora Goldkorn
Aberrant Growth Plate Development in VDR/RXR Double Null Ceramide accumulation precedes caspase-3 activation during
Mutant Mice apoptosis of A549 human lung adenocarcinoma cells
Endocrinology, Dec 2001; 142: 5332 - 5341. Am J Physiol Lung Cell Mol Physiol, Jun 2003; 284: 1082 - 1092.

References
Mizue Nakajoh, Takeyasu Fukushima, Tomoko Suzuki, Anti-Poly (ADP-Ribose) Polymerase,

Mutsuo Yamaya, Katsutoshi Nakayama, Kiyohisa Sekizawa, Cat.-No. 11 835 238 001
and Hidetada Sasaki
Christian Bojarski, Jrg Weiske, Torsten Schneberg, Werner
Retinoic Acid Inhibits Elastase-Induced Injury in Human Lung
Epithelial Cell Lines Schrder, Joachim Mankertz, Jrg-Dieter Schulzke, Peter
Florian, Michael Fromm, Rudolf Tauber, and Otmar Huber
Am. J. Respir. Cell Mol. Biol., Mar 2003; 28: 296 - 304.
The specific fates of tight junction proteins in apoptotic
Thomas Mund, Andreas Gewies, Nicole Schoenfeld, Manuel epithelial cells
K.A. Bauer, and Stefan Grimm J. Cell Sci., Mar 2004; 10.1242/jcs.01071.
Spike, a novel BH3-only protein, regulates apoptosis at the
Ju-Hyung Woo, Young-Ho Kim, Yun-Jung Choi, Dae-Gon Kim,
endoplasmic reticulum
FASEB J, Feb 2003; 10.1096/fj.02-0657fje. Kyung-Seop Lee, Jae Hoon Bae, Do Sik Min, Jong-Soo Chang,
Yong-Jin Jeong, Young Han Lee, Jong-Wook Park, and Taeg Kyu
Andreas Gewies and Stefan Grimm Kwon
UBP41 Is a Proapoptotic Ubiquitin-specific Protease Molecular mechanisms of curcumin-induced cytotoxicity:
Cancer Res., Feb 2003; 63: 682 - 688. induction of apoptosis through generation of reactive oxygen
species, down-regulation of Bcl-XL and IAP, the release of
Christelle Forcet, Xin Ye, Laure Granger, Vronique Corset,
cytochrome c and inhibition of Akt
Hwain Shin, Dale E. Bredesen, and Patrick Mehlen
Carcinogenesis, Jul 2003; 24: 1199 - 1208.
The dependence receptor DCC (deleted in colorectal cancer)
defines an alternative mechanism for caspase activation Michael D. Gober, Cynthia C. Smith, Kaori Ueda, Jeffrey A.
PNAS, Mar 2001; 98: 3416 - 3421. Toretsky, and Laure Aurelian
Forced Expression of the H11 Heat Shock Protein Can Be Regu-
Christelle Forcet, Xin Ye, Laure Granger, Vronique Corset, lated by DNA Methylation and Trigger Apoptosis in Human
Hwain Shin, Dale E. Bredesen, and Patrick Mehlen
Cells
The dependence receptor DCC (deleted in colorectal cancer)
J. Biol. Chem., Sep 2003; 278: 37600 - 37609.
defines an alternative mechanism for caspase activation
PNAS, Mar 2001; 98: 3416 - 3421. Kyung-Hee Chun, Jerome W. Kosmeder, II, Shihua Sun, John M.
Pezzuto, Reuben Lotan, Waun Ki Hong, and Ho-Young Lee
Michael Gekle, Gerald Schwerdt, Ruth Freudinger, Sigrid
Effects of Deguelin on the Phosphatidylinositol 3-Kinase/Akt
Mildenberger, Doris Wilflingseder, Verena Pollack, Martina Pathway and Apoptosis in Premalignant Human Bronchial Epi-
Dander, and Herbert Schramek
thelial Cells
Ochratoxin A Induces JNK Activation and Apoptosis in MDCK-
J Natl Cancer Inst, Feb 2003; 95: 291 - 302.
C7 Cells at Nanomolar Concentrations
J. Pharmacol. Exp. Ther., Jun 2000; 293: 837 - 844. Olivier Sordet, Cdric Rb, Stphanie Plenchette, Yal Zermati,
Olivier Hermine, William Vainchenker, Carmen Garrido, Eric
Quy N. Diep, Hope D. Intengan, and Ernesto L. Schiffrin
Solary, and Laurence Dubrez-Daloz
Endothelin-1 Attenuates 3 Fatty AcidInduced Apoptosis by Specific involvement of caspases in the differentiation of mono-
Inhibition of Caspase 3
cytes into macrophages
Hypertension, Jan 2000; 35: 287 - 291.
Blood, Dec 2002; 100: 4446 - 4453.
Victor T. Solovyan, Zinayida A. Bezvenyuk, Antero Salminen,
Homogeneous Caspases Assay, fluorimetric, Caroline A. Austin, and Michael J. Courtney
Cat.-No. 03 005 372 001 The Role of Topoisomerase II in the Excision of DNA Loop
Domains during Apoptosis
Leng Wen, Li Zhuang, Xia Luo, and Ping Wei J. Biol. Chem., Jun 2002; 277: 21458 - 21467.
TL1A-induced NF-B Activation and c-IAP2 Production Prevent
DR3-mediated Apoptosis in TF-1 Cells George Zachos, Margy Koffa, Chris M. Preston, J. Barklie
J. Biol. Chem., Oct 2003; 278: 39251 - 39258. Clements, and Joe Conner
Herpes Simplex Virus Type 1 Blocks the Apoptotic Host Cell
Anke Niedernberg, Andree Blaukat, Torsten Schneberg, and Defense Mechanisms That Target Bcl-2 and Manipulates Acti-
Evi Kostenis vation of p38 Mitogen-Activated Protein Kinase To Improve Viral
Regulated and constitutive activation of specific signalling Replication
pathways by the human S1P5 receptor J. Virol., Mar 2001; 75: 2710 - 2728.
Gareth J. Inman, Ulrich K. Binn, Gillian A. Parker, Paul J. Farrell,
Lucas C. Armstrong, Benny Bjrkblom, Kurt D. Hankenson, and Martin J. Allday
Anthony W. Siadak, Charlotte E. Stiles, and Paul Bornstein Activators of the Epstein-Barr Virus Lytic Program Concomi-
Thrombospondin 2 Inhibits Microvascular Endothelial Cell Pro- tantly Induce Apoptosis, but Lytic Gene Expression Protects
liferation by a Caspase-independent Mechanism from Cell Death
Mol. Biol. Cell, Jun 2002; 13: 1893 - 1905. J. Virol., Mar 2001; 75: 2400 - 2410.
Douglas D. Bannerman, Joan C. Tupper, Ryan D. Erwert,

Robert K. Winn, and John M. Harlan
Divergence of Bacterial Lipopolysaccharide Pro-apoptotic
Signaling Downstream of IRAK-1
J. Biol. Chem., Mar 2002; 277: 8048 - 8053.
Ricky W. Johnstone, Mark Gerber, Theresa Landewe, Anne
Tollefson, William S. Wold, and Ali Shilatifard
Functional Analysis of the Leukemia Protein ELL: Evidence for a
Role in the Regulation of Cell Growth and Survival
Mol. Cell. Biol., Mar 2001; 21: 1672 - 1681. C
Appendix 133
References
Michael Schmidt, Norbert Lgering, Andreas Lgering, Hans- Christian Kannemeier, Nadia Al-Fakhri, Klaus T. Preissner, and
Gerd Pauels, Klaus Schulze-Osthoff, Wolfram Domschke, and Sandip M. Kanse
Torsten Kucharzik Factor VII activating protease (FSAP) inhibits growth factor-
Role of the CD95/CD95 Ligand System in Glucocorticoid- mediated cell proliferation and migration of vascular smooth
Induced Monocyte Apoptosis muscle cells.
J. Immunol., Jan 2001; 166: 1344 - 1351. FASEB J, Feb 2004; 10.1096/fj.03-0898fje.
Gareth J. Inman and Martin J. Allday Antonio Parrado, Macarena Robledo, M. Rosa Moya-Quiles,
Apoptosis Induced by TGF-1 in Burkitts Lymphoma Cells Is Luis A. Marn, Christine Chomienne, Rose Ann Padua, and M.
Caspase 8 Dependent But Is Death Receptor Independent Roco lvarez-Lpez
J. Immunol., Sep 2000; 165: 2500 - 2510. The promyelocytic leukemia zinc finger protein down-regulates
apoptosis and expression of the proapoptotic BID protein in
Gareth J. Inman and Martin J. Allday
lymphocytes
Resistance to TGF-1 correlates with a reduction of TGF- type II
PNAS, Feb 2004; 101: 1898 - 1903.
receptor expression in Burkitts lymphoma and EpsteinBarr
virus-transformed B lymphoblastoid cell lines Herv Le Stunff, Rodolphe ;Auger, Jean Kanellopoulos, and
J. Gen. Virol., Jun 2000; 81: 1567 - 1578. Marie-Nolle Raymond
The Pro-451 to Leu polymorphism within the C-terminal tail of
M. Maddalena Di Somma, Francesca Somma, Maria Saveria
P2X7 receptor impairs cell death but not phospholipase D acti-
Gilardini Montani, Rosamaria Mangiacasale, Enrico Cundari, vation in murine thymocytes
and Enza Piccolella
J. Biol. Chem., Feb 2004; 10.1074/jbc.M313064200.
TCR Engagement Regulates Differential Responsiveness of
Human Memory T Cells to Fas (CD95)-Mediated Apoptosis Erica D. Smith, Yanfei Xu, Brett N. Tomson, Cindy G. Leung,
J. Immunol., Apr 1999; 162: 3851 - 3858. Yuko Fujiwara, Stuart H. Orkin, and John D. Crispino
More than blood, a Novel Gene Required for Mammalian
Postimplantation Development
In Situ Cell Death Detection Kit, Fluorescein, Mol. Cell. Biol., Feb 2004; 24: 1168 - 1173.
Cat.-No. 11 684 795 001
Maryam Diarra-Mehrpour, Samuel Arrabal, Abdelali Jalil, Xavier
Chi-Huang Chen, Shang-Sen Lee, Da-Chang Chen, Hsin-Hsuan Pinson, Catherine Gaudin, Genevive Pitu, Amandine Pitaval,
Chien, I-Ching Chen, Yung-Ning Chu, Jah-Yao Liu, Wei-Hwa Hugues Ripoche, Marc Eloit, Dominique Dormont, and Salem
Chen, and Gwo-Jang Wu Chouaib
Apoptosis and Kinematics of Ejaculated Spermatozoa in Prion Protein Prevents Human Breast Carcinoma Cell Line from
Patients With Varicocele Tumor Necrosis Factor -Induced Cell Death
J Androl, May 2004; 25: 348 - 353. Cancer Res., Jan 2004; 64: 719 - 727.
Gerhard Schratt, Ulrike Philippar, Dirk Hockemeyer, Heinz Takashi Maeda, Caroline M. Alexander, and Andreas Friedl
Schwarz, Siegfried Alberti, and Alfred Nordheim Induction of Syndecan-1 Expression in Stromal Fibroblasts Pro-
SRF regulates Bcl-2 expression and promotes cell survival dur- motes Proliferation of Human Breast Cancer Cells
ing murine embryonic development Cancer Res., Jan 2004; 64: 612 - 621.
EMBO J., Apr 2004; 10.1038/sj.emboj.7600188. Rodrigo Cuervo and Luis Covarrubias
Pascal Froment, Christophe Staub, Stphanie Hembert, Death is the major fate of medial edge epithelial cells and the
Claudine Pisselet, Michle Magistrini, Bernadette Delaleu, cause of basal lamina degradation during palatogenesis
Danielle Seurin, Jon E. Levine, Larry Johnson, Michel Binoux, Development, Jan 2004; 131: 15 - 24.
and Philippe Monget
T. T. T. Nguyen, E. Tran, T. H. Nguyen, P. T. Do, T. H. Huynh, and
Reproductive Abnormalities in Human Insulin-Like Growth Fac-
H. Huynh
tor-Binding Protein-1 Transgenic Male Mice The role of activated MEK-ERK pathway in quercetin-induced
Endocrinology, Apr 2004; 145: 2080 - 2091.
growth inhibition and apoptosis in A549 lung cancer cells
Nasrin Mesaeli and Clark Phillipson Carcinogenesis, Dec 2003; 10.1093/carcin/bgh052.
Impaired p53 Expression, Function, and Nuclear Localization in Benjamin D. Yu, Michelle Becker-Hapak, Eric L. Snyder, Marc
Calreticulin-deficient Cells
Vooijs, Catherine Denicourt, and Steven F. Dowdy
Mol. Biol. Cell, Apr 2004; 15: 1862 - 1870.
Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-
Deepak Raina, Surender Kharbanda, and Donald Kufe dependent kinases 4/6 activity in melanocyte survival
The MUC1 oncoprotein activates the anti-apoptotic P13K/Akt PNAS, Dec 2003; 100: 14881 - 14886.
and Bcl-xL pathways in rat 3Y1 fibroblasts
Bernhard Stockmeyer, Thomas Beyer, Winfried Neuhuber,
J. Biol. Chem., Mar 2004; 10.1074/jbc.M310538200. Roland Repp, Joachim R. Kalden, Thomas Valerius, and Martin
Stefania Croci, Lorena Landuzzi, Annalisa Astolfi, Giordano Herrmann
C
Nicoletti, Angelo Rosolen, Francesca Sartori, Matilde Y. Follo, Polymorphonuclear Granulocytes Induce Antibody-Dependent
Noelynn Oliver, Carla De Giovanni, Patrizia Nanni, and Pier- Apoptosis in Human Breast Cancer Cells
Luigi Lollini J. Immunol., Nov 2003; 171: 5124 - 5129.
Inhibition of Connective Tissue Growth Factor (CTGF/CCN2)
Kevin H. Mayo, Ruud P. M. Dings, Carolee Flader, Irina Nes-
Expression Decreases the Survival and Myogenic Differentia- melova, Balasz Hargittai, Daisy W. J. van der Schaft, Loes I. van
tion of Human Rhabdomyosarcoma Cells
Eijk, Dinesha Walek, Judy Haseman, Thomas R. Hoye, and Arjan
Cancer Res., Mar 2004; 64: 1730 - 1736.
W. Griffioen
Design of a Partial Peptide Mimetic of Anginex with Antiangio-
genic and Anticancer Activity
J. Biol. Chem., Nov 2003; 278: 45746 - 45752.

References
John W. Calvert, Changman Zhou, Anil Nanda, and John H. Shane W. Rau, Dena B. Dubal, Martina Bttner, Lynnette M.
Zhang Gerhold, and Phyllis M. Wise
Effect of hyperbaric oxygen on apoptosis in neonatal hypoxia- Estradiol Attenuates Programmed Cell Death after Stroke-Like
ischemia rat model Injury
J Appl Physiol, Nov 2003; 95: 2072 - 2080. J. Neurosci., Dec 2003; 23: 11420 - 11426.
Marjorie A. Robbins, Lola Maksumova, Emma Pocock, and Gregory Kennedy, Jun Komano, and Bill Sugden
Janet K. Chantler Epstein-Barr virus provides a survival factor to Burkitt's lympho-
Nuclear Factor-B Translocation Mediates Double-Stranded mas
Ribonucleic Acid-Induced NIT-1 -Cell Apoptosis and Up-Reg- PNAS, Nov 2003; 100: 14269 - 14274.
ulates Caspase-12 and Tumor Necrosis Factor Receptor-Asso-
ciated Ligand (TRAIL) Jiu Jiang, Lisa L. Lau, and Hao Shen
Selective Depletion of Nonspecific T Cells During the Early
Endocrinology, Oct 2003; 144: 4616 - 4625.
Stage of Immune Responses to Infection
XINGZHI XU and DAVID F. STERN1 J. Immunol., Oct 2003; 171: 4352 - 4358.
NFBD1/MDC1 regulates ionizing radiation-induced focus for-
Jiu Jiang, Farvardin Anaraki, Kenneth J. Blank, and Donna M.
mation by DNA checkpoint signaling and repair factors
Murasko
FASEB J, Oct 2003; 17: 1842 - 1848.
Cutting Edge: T Cells from Aged Mice Are Resistant to Deple-
Xianmin Meng, John F. Klement, Dominic A. Leperi, David E. tion Early During Virus Infection
Birk, Takako Sasaki, Rupert Timpl, Jouni Uitto, and Leena
J. Immunol., Oct 2003; 171: 3353 - 3357.
Pulkkinen
Targeted Inactivation of Murine Laminin 2-Chain Gene Recapit- May Nour, Alexander B. Quiambao, Ward M. Peterson, Muayyad
ulates Human Junctional Epidermolysis Bullosa R. Al-Ubaidi, and Muna I. Naash
J. Invest. Dermatol., Oct 2003; 121: 720 - 731. P2Y2 Receptor Agonist INS37217 Enhances Functional Recov-
ery after Detachment Caused by Subretinal Injection in Normal
Andrew Zloza, Yvonne B. Sullivan, Elizabeth Connick, Alan L.
Landay, and Lena Al-Harthi and rds Mice
Invest. Ophthalmol. Vis. Sci., Oct 2003; 44: 4505 - 4514.
CD8+ T cells that express CD4 on their surface
(CD4dimCD8bright T cells) recognize an antigen-specific Timur O. Yarovinsky, Linda S. Powers, Noah S. Butler, Mary A.
target, are detected in vivo, and can be productively infected by Bradford, Martha M. Monick, and Gary W. Hunninghake
T-tropic HIV Adenoviral Infection Decreases Mortality from Lipopolysaccha-
Blood, Sep 2003; 102: 2156 - 2164. ride-Induced Liver Failure Via Induction of TNF- Tolerance
Annette Lasham, Stephanie Moloney, Tracy Hale, Craig Homer, J. Immunol., Sep 2003; 171: 2453 - 2460.
You Fang Zhang, J. Greg Murison, Antony W. Braithwaite, and Elisabeth Larsen, Christine Gran, Barbro Elisabet Sther, Erling
James Watson Seeberg, and Arne Klungland
The Y-box-binding Protein, YB1, Is a Potential Negative Regula- Proliferation Failure and Gamma Radiation Sensitivity of Fen1
tor of the p53 Tumor Suppressor Null Mutant Mice at the Blastocyst Stage
J. Biol. Chem., Sep 2003; 278: 35516 - 35523. Mol. Cell. Biol., Aug 2003; 23: 5346 - 5353.
Jinn-Yang Chen, Chin-Wen Chi, Hui-Ling Chen, Chiung-Pei Yanhong Hao, Liangxue Lai, Jiude Mao, Gi-Sun Im, Aaron Bonk,
Wan, Wu-Chang Yang, and An-Hang Yang and Randall S. Prather
TNF- renders human peritoneal mesothelial cells sensitive to Apoptosis and In Vitro Development of Preimplantation Porcine
anti-Fas antibody-induced apoptosis Embryos Derived In Vitro or by Nuclear Transfer
Nephrol. Dial. Transplant., Sep 2003; 18: 1741 - 1747. Biol. Reprod., Aug 2003; 69: 501 - 507.
Man-Seong Park, Adolfo Garca-Sastre, Jerome F. Cros, Christo- Nicole Maas-Szabowski, Anja Strker, and Norbert E. Fusenig
pher F. Basler, and Peter Palese Epidermal tissue regeneration and stromal interaction in HaCaT
Newcastle Disease Virus V Protein Is a Determinant of Host cells is initiated by TGF-
Range Restriction J. Cell Sci., Jul 2003; 116: 2937 - 2948.
J. Virol., Sep 2003; 77: 9522 - 9532.
D. Magnus Eklund and Johan Edqvist
Localization of Nonspecific Lipid Transfer Proteins Correlate
with Programmed Cell Death Responses during Endosperm
In Situ Cell Death Detection Kit, TMR red,
Cat.-No. 12 156 792 001 Degradation in Euphorbia lagascae Seedlings
Plant Physiology, Jul 2003; 132: 1249 - 1259.
Gerry Melino, Francesca Bernassola, Marco Ranalli, Karen Yee,
E. A. Eugenin, T. G. D'Aversa, L. Lopez, T. M. Calderon, and J. W.
Wei Xing Zong, Marco Corazzari, Richard A. Knight, Doug R.
Green, Craig Thompson, and Karen H. Vousden Berman
MCP-1 (CCL2) protects human neurons and astrocytes from
C
p73 Induces Apoptosis via PUMA Transactivation and Bax
NMDA or HIV-tat-induced apoptosis
Mitochondrial Translocation
J. Biol. Chem., Feb 2004; 279: 8076 - 8083. J. NeuRochem., Jun 2003; 85: 1299 - 1311.
Adi Inbal, Daniel Levanon, and Adi Salzberg
Wulin Aerbajinai, Y. Terry Lee, Urszula Wojda, Valarie A. Barr,
Multiple roles for u-turn/ventral veinless in the development of
and Jeffery L. Miller
Cloning and Characterization of a Gene Expressed during Ter- Drosophila PNS
Development, Jun 2003; 130: 2467 - 2478.
minal Differentiation That Encodes a Novel Inhibitor of Growth
J. Biol. Chem., Jan 2004; 279: 1916 - 1921.
Appendix 135
References
Wolfgang Fierlbeck, Ailian Liu, Raluca Coyle, and Barbara J. Kathrin Maedler, Jos Oberholzer, Pascal Bucher, Giatgen A.
Ballermann Spinas, and Marc Y. Donath
Endothelial Cell Apoptosis during Glomerular Capillary Lumen Monounsaturated Fatty Acids Prevent the Deleterious Effects of
Formation In Vivo Palmitate and High Glucose on Human Pancreatic -Cell Turn-
J. Am. Soc. Nephrol., May 2003; 14: 1349 - 1354. over and Function
Diabetes, Mar 2003; 52: 726 - 733.
Charvi A. Patel, Muhammad Mukhtar, and Roger J. Pomerantz
Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis C. Kitamura, Y. Ogawa, T. Nishihara, T. Morotomi, and M.
in Human Neuronal Cells Terashita
J. Virol., Oct 2000; 74: 9717 - 9726. Transient Co-localization of c-Jun N-terminal Kinase and c-Jun
with Heat Shock Protein 70 in Pulp Cells during Apoptosis
J. Dent. Res., Feb 2003; 82: 91 - 95.
In Situ Cell Death Detection Kit, AP,
Birgitte Viuff, Kirsten Tjrnehj, Lars E. Larsen, Christine M.
Cat.-No. 11 684 809 001
Rntved, se Uttenthal, Leif Rnsholt, and Soren Alexandersen
Xiao-Nan Li, Suhag Parikh, Qin Shu, Hye-Lim Jung, Chi-Wan Replication and Clearance of Respiratory Syncytial Virus: Apop-
Chow, Laszlo Perlaky, Hon-Chiu Eastwood Leung, Jack Su, tosis Is an Important Pathway of Virus Clearance after Experi-
Susan Blaney, and Ching C. Lau mental Infection with Bovine Respiratory Syncytial Virus
Phenylbutyrate and Phenylacetate Induce Differentiation and Am. J. Pathol., Dec 2002; 161: 2195 - 2207.
Inhibit Proliferation of Human Medulloblastoma Cells Koji Shinozaki, Toshihiko Miyagi, Michio Yoshida, Takaki Miyata,
Clin. Cancer Res., Feb 2004; 10: 1150 - 1159.
Masaharu Ogawa, Shinichi Aizawa, and Yoko Suda
Estbaliz L. Fernndez, Anne-Lee Gustafson, Maria Andersson, Absence of Cajal-Retzius cells and subplate neurons associated
Bjorn Hellman, and Lennart Dencker with defects of tangential cell migration from ganglionic emi-
Cadmium-Induced Changes in Apoptotic Gene Expression Lev- nence in Emx1/2 double mutant cerebral cortex
els and DNA Damage in Mouse Embryos Are Blocked by Zinc Development, Jul 2002; 129: 3479 - 3492.
Toxicol. Sci., Nov 2003; 76: 162 - 170. Lori M. Roberts, Jenny A. Visser, and Holly A. Ingraham
Wolfgang Zink, Christoph Seif, Jrgen R. E. Bohl, Nicola Hacke, Involvement of a matrix metalloproteinase in MIS-induced cell
Peter M. Braun, Barbara Sinner, Eike Martin, Rainer H. A. Fink, death during urogenital development
and Bernhard M. Graf Development, Mar 2002; 129: 1487 - 1496.
The Acute Myotoxic Effects of Bupivacaine and Ropivacaine
Hitoshi Akiba, Jeanne Kehren, Marie-Thrse Ducluzeau, Maya
After Continuous Peripheral Nerve Blockades
Krasteva, Franoise Horand, Dominique Kaiserlian, Fumio
Anesth. Analg., Oct 2003; 97: 1173 - 1179. Kaneko, and Jean-Franois Nicolas
Christophe Van de Wiele, Christophe Lahorte, Hubert Skin Inflammation During Contact Hypersensitivity Is Mediated
Vermeersch, D. Loose, Kris Mervillie, Neil D. Steinmetz, by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing
Jean-Luc Vanderheyden, Claude A. Cuvelier, Guido Slegers, and Keratinocyte Apoptosis
Rudi A. Dierck J. Immunol., Mar 2002; 168: 3079 - 3087.
Quantitative Tumor Apoptosis Imaging Using Technetium-99m
Lisa Wise-Faberowski, Mohan K. Raizada, and Colin Sumners
HYNIC Annexin V Single Photon Emission Computed Tomogra- Oxygen and Glucose Deprivation-Induced Neuronal Apoptosis
phy
is Attenuated by Halothane and Isoflurane
J. Clin. Oncol., Sep 2003; 21: 3483 - 3487.
Anesth. Analg., Nov 2001; 93: 1281 - 1287.
Robert A. Ritzel and Peter C. Butler Toshimasa Yamauchi, Junji Kamon, Hironori Waki, Koji
Replication Increases -Cell Vulnerability to Human Islet Amy-
Murakami, Kiyoto Motojima, Kajuro Komeda, Tomohiro Ide,
loid Polypeptide-Induced Apoptosis
Naoto Kubota, Yasuo Terauchi, Kazuyuki Tobe, Hiroshi Miki,
Diabetes, Jul 2003; 52: 1701 - 1708. Atsuko Tsuchida, Yasuo Akanuma, Ryozo Nagai, Satoshi
Francesca Mascia, Valentina Mariani, Giampiero Girolomoni, Kimura, and Takashi Kadowaki
and Saveria Pastore The Mechanisms by Which Both Heterozygous Peroxisome
Blockade of the EGF Receptor Induces a Deranged Chemokine Proliferator-activated Receptor (PPAR) Deficiency and PPAR
Expression in Keratinocytes Leading to Enhanced Skin Inflam- Agonist Improve Insulin Resistance
mation J. Biol. Chem., Oct 2001; 276: 41245 - 41254.
Am. J. Pathol., Jul 2003; 163: 303 - 312. Annemiek Beverdam, Antje Brouwer, Mark Reijnen, Jeroen Kor-
Xian-Yong Ma, Hong Wang, Bo Ding, Haihong Zhong, Sankar ving, and Frits Meijlink
Ghosh, and Peter Lengyel Severe nasal clefting and abnormal embryonic apoptosis in
The Interferon-inducible p202a Protein Modulates NF-B Activity Alx3/Alx4 double mutant mice
by Inhibiting the Binding to DNA of p50/p65 Heterodimers and Development, Oct 2001; 128: 3975 - 3986.
C
p65 Homodimers While Enhancing the Binding of p50
Dana G. Mordue, Fernando Monroy, Marie La Regina, Charles
Homodimers A. Dinarello, and L. David Sibley
J. Biol. Chem., Jun 2003; 278: 23008 - 23019.
Acute Toxoplasmosis Leads to Lethal Overproduction of Th1
Michael Abdo, Susan Hisheh, and Arun Dharmarajan Cytokines
Role of Tumor Necrosis Factor-Alpha and the Modulating Effect J. Immunol., Oct 2001; 167: 4574 - 4584.
of the Caspases in Rat Corpus Luteum Apoptosis
Biol. Reprod., Apr 2003; 68: 1241 - 1248.

References
Daniela Dyntar, Monika Eppenberger-Eberhardt, Kathrin Shuji Takiguchi, Norihiro Sugino, Kikue Esato, Ayako Karube-
Maedler, Martin Pruschy, Hans M. Eppenberger, Giatgen A. Harada, Aki Sakata, Yasuhiko Nakamura, Hitoshi Ishikawa, and
Spinas, and Marc Y. Donath Hiroshi Kato
Glucose and Palmitic Acid Induce Degeneration of Myofibrils Differential Regulation of Apoptosis in the Corpus Luteum of
and Modulate Apoptosis in Rat Adult Cardiomyocytes Pregnancy and Newly Formed Corpus Luteum after Parturition
Diabetes, Sep 2001; 50: 2105 - 2113. in Rats
Biol. Reprod., Feb 2004; 70: 313 - 318.
Kathrin Maedler, Giatgen A. Spinas, Roger Lehmann, Pavel
Sergeev, Markus Weber, Adriano Fontana, Nurit Kaiser, and Hidenori Ozaki, Kazuaki Nakamura, Jun-ichi Funahashi, Keiko
Marc Y. Donath Ikeda, Gen Yamada, Hisashi Tokano, Hiro-oki Okamura, Ken
Glucose Induces -Cell Apoptosis Via Upregulation of the Fas Kitamura, Shigeaki Muto, Hayato Kotaki, Katsuko Sudo, Reiko
Receptor in Human Islets Horai, Yoichiro Iwakura, and Kiyoshi Kawakami
Diabetes, Aug 2001; 50: 1683 - 1690. Six1 controls patterning of the mouse otic vesicle
Development, Feb 2004; 131: 551 - 562.
Annett Jungmann, Hermann Nieper, and Hermann Mller
Apoptosis is induced by infectious bursal disease virus replica- OV Anichtchik, J Kaslin, N Peitsaro, M Scheinin, and P Panula
tion in productively infected cells as well as in antigen-negative Neurochemical and behavioural changes in zebrafish Danio
cells in their vicinity rerio after systemic administration of 6-hydroxydopamine and
J. Gen. Virol., May 2001; 82: 1107 - 1115. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
J Neurochem, Jan 2004; 88(2): 443-53.
J. ALLEN D. COOPER, Jr., A. LYNN RIDGEWAY, JOHN
PEARSON, and RACHEL R. CULBRETH Luciano Dalla Libera, Barbara Ravara, Maurizio Volterrani,
Attenuation of Interleukin 8-induced Nasal Inflammation by an Valerio Gobbo, Mila Della Barbera, Annalisa Angelini, Daniela
Inhibitor Peptide Danieli Betto, Elena Germinario, and Giorgio Vescovo
Am. J. Respir. Crit. Care Med., Apr 2001; 163: 1198 - 1205. Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and
function in experimental heart failure
Batya Cohen, Dalit Barkan, Yinon Levy, Iris Goldberg, Eduard Am J Physiol Cell Physiol, Jan 2004; 286: 138 - 144.
Fridman, Juri Kopolovic, and Menachem Rubinstein
Leptin Induces Angiopoietin-2 Expression in Adipose Tissues Juan F. Medina, Sergio Recalde, Jess Prieto, Jon Lecanda,
J. Biol. Chem., Mar 2001; 276: 7697 - 7700. Elena Sez, Colin D. Funk, Paola Vecino, Marian A. van Roon,
Roelof Ottenhoff, Piter J. Bosma, Conny T. Bakker, and Ronald P.
Franoise Trousse, Pilar Esteve, and Paola Bovolenta
J. Oude Elferink
BMP4 Mediates Apoptotic Cell Death in the Developing Chick
Anion exchanger 2 is essential for spermiogenesis in mice
Eye PNAS, Dec 2003; 100: 15847 - 15852.
J. Neurosci., Feb 2001; 21: 1292 - 1301.
Arturo Chavez-Reyes, John M. Parant, Lisa L. Amelse, Roberto
Antony W. Wood and Glen J. Van Der Kraak
Montes de Oca Luna, Stanley J. Korsmeyer, and Guillermina
Apoptosis and Ovarian Function: Novel Perspectives from the Lozano
Teleosts
Switching Mechanisms of Cell Death in mdm2- and mdm4-null
Biol. Reprod., Jan 2001; 64: 264 - 271.
Mice by Deletion of p53 Downstream Targets
Cancer Res., Dec 2003; 63: 8664 - 8669.
In Situ Cell Death Detection Kit, POD, HE Paczoska-Eliasiewicz, A Gertler, M Proszkowiec, J Proud-
Cat.-No. 11 684 817 001 man, A Hrabia, A Sechman, M Mika, T Jacek, S Cassy, N
Ravner, and J Rzasa
Ken-ichi Kimura, Akitoshi Kodama, Yosihiro Hayasaka, and Attenuation by leptin of the effects of fasting on ovarian func-
Takumi Ohta tion in hens (Gallus domesticus)
Activation of the cAMP/PKA signaling pathway is required for Reproduction, Dec 2003; 126: 739 - 751.
post-ecdysial cell death in wing epidermal cells of Drosophila
melanogaster Robert D. Knight, Sreelaja Nair, Sarah S. Nelson, Ali Afshar,
Development, Apr 2004; 131: 1597 - 1606. Yashar Javidan, Robert Geisler, Gerd-Joerg Rauch, and Thomas
F. Schilling
Ivan Orlandi, Maurizio Bettiga, Lilia Alberghina, and Marina Vai lockjaw encodes a zebrafish tfap2a required for early neural
Transcriptional Profiling of ubp10 Null Mutant Reveals Altered crest development
Subtelomeric Gene Expression and Insurgence of Oxidative Development, Dec 2003; 130: 5755 - 5768.
Stress Response
J. Biol. Chem., Feb 2004; 279: 6414 - 6425. Ilka B. Schiffer, Susanne Gebhard, Carolin K. Heimerdinger,
Annette Heling, Jochem Hast, Ursula Wollscheid, Barbara
Satomi Kuramochi-Miyagawa, Tohru Kimura, Takashi W. Ijiri, Seliger, Berno Tanner, Sandra Gilbert, Thomas Beckers, Silke
Taku Isobe, Noriko Asada, Yukiko Fujita, Masahito Ikawa, Baasner, Walburgis Brenner, Christian Spangenberg, Dirk
Naomi Iwai, Masaru Okabe, Wei Deng, Haifan Lin, Yoichi Prawitt, Tatjana Trost, Wolfgang G. Schreiber, Bernhard Zabel,
C
Matsuda, and Toru Nakano Manfred Thelen, Hans-Anton Lehr, Franz Oesch, and Jan G.
Mili, a mammalian member of piwi family gene, is essential for Hengstler
spermatogenesis Switching Off HER-2/neu in a Tetracycline-Controlled Mouse
Development, Feb 2004; 131: 839 - 849. Tumor Model Leads to Apoptosis and Tumor-Size-Dependent
Remission
Claudia Orelio, Kirsty N Harvey, Colin Miles, Robert A Oosten-
dorp, Karin van der Horn, and Elaine Dzierzak Cancer Res., Nov 2003; 63: 7221 - 7231.
The role of apoptosis in the development of AGM hematopoie-
tic stem cells revealed by Bcl-2 overexpression
Blood, Feb 2004; 10.1182/blood-2003-06-1827.
Appendix 137
References
Cristiane G. Lin, Shr-Jeng Leu, Ningyu Chen, Christopher M. Alana M. Thackray and Raymond Bujdoso
Tebeau, Shao-Xia Lin, Cho-Yau Yeung, and Lester F. Lau PrPc Expression Influences the Establishment of Herpes Sim-
CCN3 (NOV) Is a Novel Angiogenic Regulator of the CCN Pro- plex Virus Type 1 Latency
tein Family J. Virol., Mar 2002; 76: 2498 - 2509.
J. Biol. Chem., Jun 2003; 278: 24200 - 24208.
Hiroyuki Koizumi, Noritaka Yamaguchi, Mitsuharu Hattori,

Annexin-V-FLUOS, Cat.-No. 11 828 681 001
Tomo-o Ishikawa, Junken Aoki, Makoto M. Taketo, Keizo Inoue,
and Hiroyuki Arai Gero Kramer, Hamdiye Erdal, Helena J. M. M. Mertens, Marius
Targeted Disruption of Intracellular Type I Platelet Activating Nap, Julian Mauermann, Georg Steiner, Michael Marberger,
Factor-acetylhydrolase Catalytic Subunits Causes Severe Kenneth Bivn, Maria C. Shoshan, and Stig Linder
Impairment in Spermatogenesis Differentiation between Cell Death Modes Using Measure-
J. Biol. Chem., Mar 2003; 278: 12489 - 12494. ments of Different Soluble Forms of Extracellular Cytokeratin 18
Cancer Res., Mar 2004; 64: 1751 - 1756.
Torsten Wuestefeld, Christian Klein, Konrad L. Streetz, Ulrich
Betz, Jrg Lauber, Jan Buer, Michael P. Manns, Werner Mller, Rung-chi Li, Tereza Cindrova-Davies, Jeremy N. Skepper, and
and Christian Trautwein Lynda A. Sellers
Interleukin-6/Glycoprotein 130-dependent Pathways Are Pro- Prostacyclin Induces Apoptosis of Vascular Smooth Muscle
tective during Liver Regeneration Cells by a cAMP-Mediated Inhibition of Extracellular Signal-
J. Biol. Chem., Mar 2003; 278: 11281 - 11288. Regulated Kinase Activity and Can Counteract the Mitogenic
Activity of Endothelin-1 or Basic Fibroblast Growth Factor
Geert Hamer, Hermien L. Roepers-Gajadien, Annemarie van
Circ. Res., Feb 2004; 10.1161/01.RES.0000121568.40692.97.
Duyn-Goedhart, Iris S. Gademan, Henk B. Kal, Paul P.W. van
Buul, Terry Ashley, and Dirk G. de Rooij Gong Yang, Kathy Qi Cai, Jennifer A. Thompson-Lanza, Robert
Function of DNA-Protein Kinase Catalytic Subunit During the C. Bast, Jr., and Jinsong Liu
Early Meiotic Prophase Without Ku70 and Ku86 Inhibition of Breast and Ovarian Tumor Growth through Multiple
Biol. Reprod., Mar 2003; 68: 717 - 721. Signaling Pathways by Using Retrovirus-mediated Small Inter-
fering RNA against Her-2/neu Gene Expression
Ulrike Wille, Eric N. Villegas, Linden Craig, Robert Peach, and
J. Biol. Chem., Feb 2004; 279: 4339 - 4345.
Christopher A. Hunter
Contribution of Interleukin-12 (IL-12) and the CD28/B7 and Olena Yermolaieva, Rong Xu, Carrie Schinstock, Nathan Brot,
CD40/CD40 Ligand Pathways to the Development of a Patho- Herbert Weissbach, Stefan H. Heinemann, and Toshinori Hoshi
logical T-Cell Response in IL-10-Deficient Mice Methionine sulfoxide reductase A protects neuronal cells
Infect. Immun., Dec 2002; 70: 6940 - 6947. against brief hypoxia/reoxygenation
PNAS, Feb 2004; 101: 1159 - 1164.
Hairong Peng, Vonda Wright, Arvydas Usas, Brian Gearhart,
Hsain-Chung Shen, James Cummins, and Johnny Huard Shanhong Ling, Aozhi Dai, Rodney J. Dilley, Margaret Jones,
Synergistic enhancement of bone formation and healing by Evan Simpson, Paul A. Komesaroff, and Krishnankutty Sudhir
stem cellexpressed VEGF and bone morphogenetic protein-4 Endogenous Estrogen Deficiency Reduces Proliferation and
J. Clin. Invest., Sep 2002; 110: 751 - 759. Enhances Apoptosis-Related Death in Vascular Smooth Muscle
Heung Tae Kim, Byung Chul Kim, Isaac Yi Kim, Mizuko Mamura, Cells: Insights From the Aromatase-Knockout Mouse
Circulation, Feb 2004; 109: 537 - 543.
Do Hwan Seong, Ja-June Jang, and Seong-Jin Kim
Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apo- Mark A. Guthridge, Emma F. Barry, Fernando A. Felquer, Bar-
ptosis through Cleavage of BAD in TSU-PR1 Human Cancer bara J. McClure, Frank C. Stomski, Hayley Ramshaw, and Angel
Cells F. Lopez
J. Biol. Chem., Aug 2002; 277: 32510 - 32515. The phosphoserine-585dependent pathway of the GM-CSF/
Byung-Chul Kim, Heung-Tae Kim, Mizuko Mamura, Indu S. IL-3/IL-5 receptors mediates hematopoietic cell survival
through activation of NF-B and induction of bcl-2
Ambudkar, Kyeong-Sook Choi, and Seong-Jin Kim
Blood, Feb 2004; 103: 820 - 827.
Tumor Necrosis Factor Induces Apoptosis in Hepatoma Cells by
Increasing Ca2+ Release from the Endoplasmic Reticulum and Petranel T. Ferrao, Michelle J. Frost, Shoo-Peng Siah, and Leonie
Suppressing Bcl-2 Expression K. Ashman
J. Biol. Chem., Aug 2002; 277: 31381 - 31389. Overexpression of P-glycoprotein in K562 cells does not confer
Li Zeng, Herv Kempf, L. Charles Murtaugh, Mie Elissa Sato, resistance to the growth inhibitory effects of imatinib (STI571)
in vitro
and Andrew B. Lassar
Blood, Dec 2003; 102: 4499 - 4503.
Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is
maintained by BMP signals to induce somitic chondrogenesis Yuan Lin, Angela C. Bright, Terri A. Rothermel, and Biao He
Genes & Dev., Aug 2002; 16: 1990 - 2005. Induction of Apoptosis by Paramyxovirus Simian Virus 5 Lack-
C
ing a Small Hydrophobic Gene
Ken-ichi Sunayama, Hiroyuki Konno, Toshio Nakamura, Hide-
humi Kashiwabara, Tsuyoshi Shoji, Toshihiro Tsuneyoshi, and J. Virol., Mar 2003; 77: 3371 - 3383.
Satoshi Nakamura E. Lpez, S. Figueroa, M. J. Oset-Gasque, and M. P. Gonzlez
The role of cyclooxygenase-2 (COX-2) in two different morpho- Apoptosis and necrosis: two distinct events induced by cad-
logical stages of intestinal polyps in APC474 knockout mice mium in cortical neurons in culture
Carcinogenesis, Aug 2002; 23: 1351 - 1359. Br. J. Pharmacol., Mar 2003; 138: 901 - 911.
Mary L. Alpaugh, James S. Tomlinson, Yin Ye, and Sanford H. Hui Xu, Dengping Yin, Bashoo Naziruddin, Libing Chen, Aileen
Barsky Stark, Yuanyuan Wei, Ying Lei, JiKun Shen, John S. Logan,
Relationship of Sialyl-Lewisx/a Underexpression and E-Cad- Guerard W. Byrne, and Anita S.-F. Chong
herin Overexpression in the Lymphovascular Embolus of Inflam- The In Vitro and In Vivo Effects of Anti-Galactose Antibodies on
matory Breast Carcinoma Endothelial Cell Activation and Xenograft Rejection
Am. J. Pathol., Aug 2002; 161: 619 - 628. J. Immunol., Feb 2003; 170: 1531 - 1539.

References
Pattabhiraman Shankaranarayanan and Santosh Nigam Veronica A. Tovar Sepulveda, Xiaoli Shen, and Miriam Falzon
IL-4 Induces Apoptosis in A549 Lung Adenocarcinoma Cells: Intracrine PTHrP Protects against Serum Starvation-Induced
Evidence for the Pivotal Role of 15-Hydroxyeicosatetraenoic Apoptosis and Regulates the Cell Cycle in MCF-7 Breast
Acid Binding to Activated Peroxisome Proliferator-Activated Cancer Cells
Receptor Transcription Factor Endocrinology, Feb 2002; 143: 596 - 606.
J. Immunol., Jan 2003; 170: 887 - 894.
Lee Carpenter, Damien Cordery, and Trevor J. Biden
Katja Issbrcker, Hugo H. Marti, Stefan Hippenstiel, Georg Inhibition of Protein Kinase C Protects Rat INS-1 Cells Against
Springmann, Robert Voswinckel, Andreas Gaumann, Georg Interleukin-1 and Streptozotocin-Induced Apoptosis
Breier, Hannes C. A. Drexler, Norbert Suttorp, and Matthias Diabetes, Feb 2002; 51: 317 - 324.
Clauss
p38 MAP Kinase - a molecular switch between VEGF-induced Yoshiki Yanagawa, Norifumi Iijima, Kazuya Iwabuchi, and
Kazunori Ono
angiogenesis and vascular hyperpermeability
Activation of extracellular signal-related kinase by TNF- con-
FASEB J, Dec 2002; 10.1096/fj.02-0329fje.
trols the maturation and function of murine dendritic cells
Margaret Park, Amanda Helip-Wooley, and Jess Thoene J. Leukoc. Biol., Jan 2002; 71: 125 - 132.
Lysosomal Cystine Storage Augments Apoptosis in Cultured
Thomas D. Batiuk, Carol Schnizlein-Bick, Zoya Plotkin, and
Human Fibroblasts and Renal Tubular Epithelial Cells
Pierre C. Dagher
J. Am. Soc. Nephrol., Dec 2002; 13: 2878 - 2887.
Guanine nucleosides and Jurkat cell death: roles of ATP deple-
Theocharis Panaretakis, Katja Pokrovskaja, Maria C. Shoshan, tion and accumulation of deoxyribonucleotides
and Dan Grandr Am J Physiol Cell Physiol, Dec 2001; 281: 1776 - 1784.
Activation of Bak, Bax, and BH3-only Proteins in the Apoptotic
Response to Doxorubicin
J. Biol. Chem., Nov 2002; 277: 44317 - 44326. Annexin-V-FLUOS Staining Kit, Cat.-No. 11 858 777 001
Michelle J. Frost, Petranel T. Ferrao, Timothy P. Hughes, and Caroline Paris, Philippe M. Loiseau, Christian Bories, and Jaque-
Leonie K. Ashman line Brard
Juxtamembrane Mutant V560GKit Is More Sensitive to Imatinib Miltefosine Induces Apoptosis-Like Death in Leishmania dono-
(STI571) Compared with Wild-Type c-Kit Whereas the Kinase vani Promastigotes
Domain Mutant D816VKit Is Resistant Antimicrob. Agents Chemother., Mar 2004; 48: 852 - 859.
Mol. Cancer Ther., Oct 2002; 1: 1115 - 1124.
Yayoi Kaneko, Kenji Kitazato, and Yuji Basaki
Satoshi Murasawa, Joan Llevadot, Marcy Silver, Jeffrey M. Isner, Integrin-linked kinase regulates vascular morphogenesis
Douglas W. Losordo, and Takayuki Asahara induced by vascular endothelial growth factor
Constitutive Human Telomerase Reverse Transcriptase Expres- J. Cell Sci., Jan 2004; 117: 407 - 415.
sion Enhances Regenerative Properties of Endothelial Progeni-
tor Cells Silke Appel, Andreas M. Boehmler, Frank Grnebach, Martin R.
Circulation, Aug 2002; 106: 1133 - 1139. Mller, Anette Rupf, Markus M. Weck, Ulrike Hartmann, Volker
L. Reichardt, Lothar Kanz, Tim H. Brmmendorf, and Peter Bros-
Ilse Decordier, Lubina Dillen, Enrico Cundari, and Micheline sart
Kirsch-Volders Imatinib mesylate affects the development and function of den-
Elimination of micronucleated cells by apoptosis after treatment dritic cells generated from CD34+ peripheral blood progenitor
with inhibitors of microtubules cells
Mutagenesis, Jul 2002; 17: 337 - 344. Blood, Jan 2004; 103: 538 - 544.
Juan J. Muoz, Luis M. Alonso-C., Rosa Sacedn, Tessa Cromp- Jianwu Pei and Thomas A. Ficht
ton, Angeles Vicente, Eva Jimnez, Alberto Varas, and Agustn Brucella abortus Rough Mutants Are Cytopathic for Macroph-
G. Zapata ages in Culture
Expression and Function of the Eph A Receptors and Their Infect. Immun., Jan 2004; 72: 440 - 450.
Ligands Ephrins A in the Rat Thymus
J. Immunol., Jul 2002; 169: 177 - 184. Rena Balzan, Karen Sapienza, Dolores R. Galea, Neville Vas-
sallo, Hank Frey, and William H. Bannister
Chin-Ju Tsi, Yee Chao, Ching-Wen Chen, and Wan Wan Lin Aspirin commits yeast cells to apoptosis depending on carbon
Aurintricarboxylic Acid Protects against Cell Death Caused by source
Lipopolysaccharide in Macrophages by Decreasing Inducible Microbiology, Jan 2004; 150: 109 - 115.
Nitric-Oxide Synthase Induction via IB Kinase, Extracellular Sig-
nal-Regulated Kinase, and p38 Mitogen-Activated Protein Bing Z. Carter, Steven M. Kornblau, Twee Tsao, Rui-Yu Wang,
Kinase Inhibition Wendy D. Schober, Michele Milella, Hsi-Guang Sung, John C.
Mol. Pharmacol., Jul 2002; 62: 90 - 101. Reed, and Michael Andreeff
Caspase-independent cell death in AML: caspase inhibition in
C
PART VIII. NERVOUS SYSTEM AND ARTHRITIS: vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin
DAVID S. JESSOP, LOUISE J. RICHARDS, and MICHAEL S. does not affect cell survival or prognosis
HARBUZ Blood, Dec 2003; 102: 4179 - 4186.
Opioid Peptides Endomorphin-1 and Endomorphin-2 in the
Immune System in Humans and in a Rodent Model of Inflam- Nicola A. Johnson, Shiladitya Sengupta, Samir A. Saidi, Khasha-
mation yar Lessan, Stephen D. Charnock-Jones, Laurie Scott, Richard
Ann. N.Y. Acad. Sci., Jun 2002; 966: 456 - 463. Stephens, Tom C. Freeman, Brian D. M. Tom, Michael Harris,
Gareth Denyer, Mallik Sundaram, Ram Sasisekharan, Stephen
Lingge Lu, Kristina Holmqvist, Michael Cross, and Michael K. Smith, and Cristin G. Print
Welsh Endothelial cells preparing to die by apoptosis initiate a pro-
Role of the Src Homology 2 Domain-containing Protein Shb in gram of transcriptome and glycome regulation
Murine Brain Endothelial Cell Proliferation and Differentiation FASEB J, Nov 2003; 10.1096/fj.03-0097fje.
Cell Growth Differ., Mar 2002; 13: 141 - 148.
Appendix 139
References
Alessio Nencioni, Kirsten Lauber, Frank Grnebach, Luk Van Fabrice Bureau, Alain Vanderplasschen, Fabrice Jaspar, Frdric
Parijs, Claudio Denzlinger, Sebastian Wesselborg, and Peter Minner, Paul-Pierre Pastoret, Marie-Paule Merville, Vincent
Brossart Bours, and Pierre Lekeux
Cyclopentenone Prostaglandins Induce Lymphocyte Apoptosis Constitutive nuclear factor-B activity preserves homeostasis of
by Activating the Mitochondrial Apoptosis Pathway Indepen- quiescent mature lymphocytes and granulocytes by controlling
dent of External Death Receptor Signaling the expression of distinct Bcl-2 family proteins
J. Immunol., Nov 2003; 171: 5148 - 5156. Blood, May 2002; 99: 3683 - 3691.
Francesco D'Alo', Lisa M. Johansen, Erik A. Nelson, Hanna S. S. Radulovic, P. W. Price, M. S. Beier, J. Gaywee, J. A. Macaluso,
Radomska, Erica K. Evans, Pu Zhang, Claus Nerlov, and Daniel and A. Azad
G. Tenen Rickettsia-Macrophage Interactions: Host Cell Responses to
The amino terminal and E2F interaction domains are critical for Rickettsia akari and Rickettsia typhi
C/EBP-mediated induction of granulopoietic development of Infect. Immun., May 2002; 70: 2576 - 2582.
hematopoietic cells
Blood, Nov 2003; 102: 3163 - 3171. Han-Ming Shen, Jun Dai, Sin-Eng Chia, Alvin Lim, and Choon-
Nam Ong
Martin Sattler, Yuri B. Pride, Patrick Ma, Jessica L. Gramlich, Detection of apoptotic alterations in sperm in subfertile patients
Stephanie C. Chu, Laura A. Quinnan, Sheri Shirazian, Congxin and their correlations with sperm quality
Liang, Klaus Podar, James G. Christensen, and Ravi Salgia Hum. Reprod., May 2002; 17: 1266 - 1273.
A Novel Small Molecule Met Inhibitor Induces Apoptosis in
Claudio Hetz, Mara Rosa Bono, Luis Felipe Barros, and Rosalba
Cells Transformed by the Oncogenic TPR-MET Tyrosine Kinase
Lagos
Cancer Res., Sep 2003; 63: 5462 - 5469.
Microcin E492, a channel-forming bacteriocin from Klebsiella
Toshiaki Hayashi, Teru Hideshima, Masaharu Akiyama, Noopur pneumoniae, induces apoptosis in some human cell lines
Raje, Paul Richardson, Dharminder Chauhan, and Kenneth C. PNAS, Mar 2002; 99: 2696 - 2701.
Anderson
Ex vivo induction of multiple myelomaspecific cytotoxic T Min Huang, Piotr Kozlowski, Matthew Collins, Yanhong Wang,
Timothy A. Haystead, and Lee M. Graves
lymphocytes
Caspase-Dependent Cleavage of Carbamoyl Phosphate
Blood, Aug 2003; 102: 1435 - 1442.
Synthetase II during Apoptosis
Guido Janeke, Wilfried Siefken, Stefanie Carstensen, Gunja Mol. Pharmacol., Mar 2002; 61: 569 - 577.
Springmann, Oliver Bleck, Hans Steinhart, Peter Hger, Klaus-
Kaoruko Tada Iida, Hiroaki Suzuki, Hirohito Sone, Hitoshi
Peter Wittern, Horst Wenck, Franz Stb, Gerhard Sauermann,
Volker Schreiner, and Thomas Doering Shimano, Hideo Toyoshima, Shigeru Yatoh, Tomoichiro Asano,
Yukichi Okuda, and Nobuhiro Yamada
Role of Taurine Accumulation in Keratinocyte Hydration
Insulin Inhibits Apoptosis of Macrophage Cell Line, THP-1 Cells,
J. Invest. Dermatol., Aug 2003; 121: 354 - 361.
via Phosphatidylinositol-3-KinaseDependent Pathway
Howard Clark, Nades Palaniyar, Peter Strong, Jess Edmondson, Arterioscler. Thromb. Vasc. Biol., Mar 2002; 22: 380 - 386.
Samuel Hawgood, and Kenneth B. M. Reid
Robert A. Mitchell, Hong Liao, Jason Chesney, Gunter Fingerle-
Surfactant Protein D Reduces Alveolar Macrophage Apoptosis
In Vivo Rowson, John Baugh, John David, and Richard Bucala
Macrophage migration inhibitory factor (MIF) sustains mac-
J. Immunol., Sep 2002; 169: 2892 - 2899.
rophage proinflammatory function by inhibiting p53: Regulatory
Yoichiro Kakugawa, Tadashi Wada, Kazunori Yamaguchi, Hide- role in the innate immune response
aki Yamanami, Kiyoaki Ouchi, Ikuro Sato, and Taeko Miyagi PNAS, Jan 2002; 99: 345 - 350.
Up-regulation of plasma membrane-associated ganglioside
Taro Matsumoto, Ingela Turesson, Majlis Book, Pr Gerwins,
sialidase (Neu3) in human colon cancer and its involvement in
apoptosis suppression and Lena Claesson-Welsh
p38 MAP kinase negatively regulates endothelial cell survival,
PNAS, Aug 2002; 99: 10718 - 10723.
proliferation, and differentiation in FGF-2stimulated angiogen-
Toshiaki Hayashi, Teru Hideshima, Masaharu Akiyama, Paul esis
Richardson, Robert L. Schlossman, Dharminder Chauhan, J. Cell Biol., Jan 2002; 156: 149 - 160.
Nikhil C. Munshi, Samuel Waxman, and Kenneth C. Anderson
Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma
Cells in the Bone Marrow Microenvironment Annexin-V-Alexa 568, Cat.-No. 11 985 485 001
Mol. Cancer Ther., Aug 2002; 1: 851 - 860. (new Cat.-No. 03 703 126 001)
Volker Brinkmann, Michael D. Davis, Christopher E. Heise, Rauf Latif, Nicole Kerlero de Rosbo, Tany Amarant, Rino Rap-
Rainer Albert, Sylvain Cottens, Robert Hof, Christian Bruns, Eva puoli, Gregor Sappler, and Avraham Ben-Nun
Prieschl, Thomas Baumruker, Peter Hiestand, Carolyn A. Foster, Reversal of the CD4+/CD8+ T-Cell Ratio in Lymph Node Cells
Markus Zollinger, and Kevin R. Lynch
C
upon In Vitro Mitogenic Stimulation by Highly Purified, Water-
The Immune Modulator FTY720 Targets Sphingosine 1-Phos- Soluble S3-S4 Dimer of Pertussis Toxin
phate Receptors Infect. Immun., May 2001; 69: 3073 - 3081.
J. Biol. Chem., Jun 2002; 277: 21453 - 21457.
Felipe Diaz-Griffero, Steven A. Hoschander, and Jrgen Bro-
Srikanth Yellayi, Afia Naaz, Melissa A. Szewczykowski, Tomomi jatsch
Sato, Jeffrey A. Woods, Jongsoo Chang, Mariangela Segre, Clint Bystander Killing during Avian Leukosis Virus Subgroup B
D. Allred, William G. Helferich, and Paul S. Cooke Infection Requires TVBS3 Signaling
The phytoestrogen genistein induces thymic and immune J. Virol., Dec 2003; 77: 12552 - 12561.
changes: A human health concern?
PNAS, May 2002; 99: 7616 - 7621.

References
Hong-Lin Su, Ching-Len Liao, and Yi-Ling Lin Jaleh Doostzadeh-Cizeron, Nicholas H. A. Terry, and David W.
Japanese Encephalitis Virus Infection Initiates Endoplasmic Goodrich
Reticulum Stress and an Unfolded Protein Response The Nuclear Death Domain Protein p84N5 Activates a G2/M
J. Virol., May 2002; 76: 4162 - 4171. Cell Cycle Checkpoint Prior to the Onset of Apoptosis
J. Biol. Chem., Jan 2001; 276: 1127 - 1132.
Amir Abbas Samani, Eric Chevet, Lucia Fallavollita, Jacques
Galipeau, and Pnina Brodt Helena Helmby, Gun Jnsson, and Marita Troye-Blomberg
Loss of Tumorigenicity and Metastatic Potential in Carcinoma Cellular Changes and Apoptosis in the Spleens and Peripheral
Cells Expressing the Extracellular Domain of the Type 1 Insulin- Blood of Mice Infected with Blood-Stage Plasmodium chabaudi
Like Growth Factor Receptor chabaudi AS
Cancer Res., May 2004; 64: 3380 - 3385. Infect. Immun., Mar 2000; 68: 1485 - 1490.
Yu-Hauh Wu, Sheue-Fang Shih, and Jung-Yaw Lin Frank Thvenod, Jenny M. Friedmann, Alice D. Katsen, and
Ricin Triggers Apoptotic Morphological Changes through Ingeborg A. Hauser
Caspase-3 Cleavage of BAT3 Up-regulation of Multidrug Resistance P-glycoprotein via
J. Biol. Chem., Apr 2004; 279: 19264 - 19275. Nuclear Factor-B Activation Protects Kidney Proximal Tubule
Cells from Cadmium- and Reactive Oxygen Species-induced
Simona Caporali, Manami Imai, Lucia Altucci, Massimo
Apoptosis
Cancemi, Silvana Caristi, Luigi Cicatiello, Filomena Matarese,
J. Biol. Chem., Jan 2000; 275: 1887 - 1896.
Roberta Penta, Dipak K. Sarkar, Francesco Bresciani, and
Alessandro Weisz
Distinct Signaling Pathways Mediate Stimulation of Cell Cycle
Annexin-V-Biotin, Cat.-No. 11 828 690 001
Progression and Prevention of Apoptotic Cell Death by Estrogen
in Rat Pituitary Tumor PR1 Cells Mounira Djerbi, Khairul-Bariah Abdul-Majid, Manuchehr
Mol. Biol. Cell, Dec 2003; 14: 5051 - 5059. Abedi-Valugerdi, Tomas Olsson, Robert A. Harris, and Alf
Grandien
Alessio Cardinale, Ilaria Filesi, Sonia Mattei, and Silvia Biocca
Evidence for proteasome dysfunction in cytotoxicity mediated Expression of the Long Form of Human FLIP by Retroviral Gene
Transfer of Hemopoietic Stem Cells Exacerbates Experimental
by anti-Ras intracellular antibodies
Autoimmune Encephalomyelitis
Eur. J. Biochem., Aug 2003; 270: 3389 - 3397.
J. Immunol., Feb 2003; 170: 2064 - 2073.
Kamel Izeradjene, Jean-Pierre Revillard, and Laurent Genestier
Tom Taghon, Magda De Smedt, Frank Stolz, Maggy Cnockaert,
Inhibition of thymidine synthesis by folate analogues induces a
Jean Plum, and Georges Leclercq
FasFas ligand-independent deletion of superantigen-reactive
peripheral T cells Enforced Expression of GATA-3 Severely Reduces Human Thy-
mic Cellularity
Int. Immunol., Jan 2001; 13: 85 - 93.
J. Immunol., Oct 2001; 167: 4468 - 4475.
Jaleh Doostzadeh-Cizeron, Shenmin Yin, and David W. Goo-
drich Elad Katz, Maureen R. Deehan, Sandra Seatter, Caroline Lord,
Roger D. Sturrock, and Margaret M. Harnett
Apoptosis Induced by the Nuclear Death Domain Protein
B Cell Receptor-Stimulated Mitochondrial Phospholipase A2
p84N5 Is Associated with Caspase-6 and NF-B Activation
J. Biol. Chem., Aug 2000; 275: 25336 - 25341. Activation and Resultant Disruption of Mitochondrial Mem-
brane Potential Correlate with the Induction of Apoptosis in
Gautam Bandyopadhyay, Tanusree Biswas, Keshab C Roy, WEHI-231 B Cells
Swapan Mandal, Chhabinath Mandal, Bikas C Pal, Samir J. Immunol., Jan 2001; 166: 137 - 147.
Bhattacharya, Srabanti Rakshit, Dilip K Bhattacharya, Utpal
Masako Nishizawa, Masakazu Kamata, Ryoichi Katsumata, and
Chaudhuri, Aditya Konar, and Santu Bandyopadhyay
Yoko Aida
Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers
p38 mitogen-activated protein kinase-dependent apoptosis in A Carboxy-Terminally Truncated Form of the Human Immunode-
ficiency Virus Type 1 Vpr Protein Induces Apoptosis via G1 Cell
chronic myelogenous leukemic cells
Cycle Arrest
Blood, Jun 2004; 10.1182/blood-2003-11-4065.
J. Virol., Jul 2000; 74: 6058 - 6067.
Hyungshin Yim, Ying Hua Jin, Byoung Duck Park, Hye Jin Choi,
Francesco Puppo, Paola Contini, Massimo Ghio, Sabrina Brenci,
and Seung Ki Lee
Marco Scudeletti, Gilberto Filaci, Soldano Ferrone, and
Caspase-3-mediated Cleavage of Cdc6 Induces Nuclear Local-
ization of p49-truncated Cdc6 and Apoptosis Francesco Indiveri
Soluble human MHC class I molecules induce soluble Fas
Mol. Biol. Cell, Oct 2003; 14: 4250 - 4259.
ligand secretion and trigger apoptosis in activated CD8+ Fas
Dominique Garcin, Joseph Curran, Masae Itoh, and Daniel (CD95)+ T lymphocytes
Kolakofsky Int. Immunol., Feb 2000; 12: 195 - 203.
Longer and Shorter Forms of Sendai Virus C Proteins Play Dif-
C
Josefa Blanco-Rodrguez and Carmen Martnez-Garca
ferent Roles in Modulating the Cellular Antiviral Response
J. Virol., Aug 2001; 75: 6800 - 6807. Apoptosis Is Physiologically Restricted to a Specialized Cyto-
plasmic Compartment in Rat Spermatids
Yaron Shav-Tal, Michal Cohen, Smadar Lapter, Billy Dye, James Biol. Reprod., Dec 1999; 61: 1541 - 1547.
G. Patton, Joel Vandekerckhove, and Dov Zipori
Nuclear Relocalization of the Pre-mRNA Splicing Factor PSF Bruno Verhasselt, Evelien Naessens, Chris Verhofstede, Magda
De Smedt, Sigrid Schollen, Tessa Kerre, Dominique Vanhecke,
during Apoptosis Involves Hyperphosphorylation, Masking of
and Jean Plum
Antigenic Epitopes, and Changes in Protein Interactions
Mol. Biol. Cell, Aug 2001; 12: 2328 - 2340. Human Immunodeficiency Virus nef Gene Expression Affects
Generation and Function of Human T Cells, But Not Dendritic
Cells
Blood, Oct 1999; 94: 2809 - 2818.
Appendix 141
References
M. Ghio, P. Contini, C. Mazzei, S. Brenci, G. Barberis, G. Filaci, Alberto Chiarugi, Giovanni Mario Pitari, Rosa Costa, Margherita
F. Indiveri, and F. Puppo Ferrante, Loredana Villari, Matilde Amico-Roxas, Thophile
Soluble HLA Class I, HLA Class II, and Fas Ligand in Blood Godfraind, Alfredo Bianchi, and Salvatore Salomone
Components: A Possible Key to Explain the Immunomodulatory Effect of prolonged incubation with copper on endothelium-
Effects of Allogeneic Blood Transfusions dependent relaxation in rat isolated aorta
Blood, Mar 1999; 93: 1770 - 1777. Br. J. Pharmacol., Aug 2002; 136: 1185 - 1193.
Eduardo Correa-Meyer, Liuska Pesce, Carmen Guerrero, and

Jacob I. Sznajder
Cytotoxicity Detection Kit (LDH), Cat.-No. 11 644 793 001
Mechanotransduction in the Lung: Cyclic stretch activates
Markus Meissner, Monika Stein, Carmen Urbich, Kerstin Reis- ERK1/2 via G proteins and EGFR in alveolar epithelial cells
inger, Guntram Suske, Bart Staels, Roland Kaufmann, and Jens Am J Physiol Lung Cell Mol Physiol, May 2002; 282: 883 - 891.
Gille
RUI F.M. SILVA, CECLIA M.P. RODRIGUES, and DORA BRITES
PPAR Activators Inhibit Vascular Endothelial Growth Factor
Rat Cultured Neuronal and Glial Cells Respond Differently to
Receptor-2 Expression by Repressing Sp1-Dependent DNA Toxicity of Unconjugated Bilirubin
Binding and Transactivation
Pediatr. Res., Apr 2002; 51: 535 - 541.
Circ. Res., Feb 2004; 94: 324 - 332.
Jonathan G. Crowston, Lydia H. Chang, Peter H. Constable, Julie
Sabine Morand, Valrie Buchillier, Fabienne Maurer, Christophe
T. Daniels, Arne N. Akbar, and Peng T. Khaw
Bonny, Yvan Arsenijevic, Francis L. Munier, and Daniel F. Schor- Apoptosis Gene Expression and Death Receptor Signaling in
deret
Mitomycin-CTreated Human Tenon Capsule Fibroblasts
Induction of Apoptosis in Human Corneal and HeLa Cells by
Invest. Ophthalmol. Vis. Sci., Mar 2002; 43: 692 - 699.
Mutated BIGH3
Invest. Ophthalmol. Vis. Sci., Jul 2003; 44: 2973 - 2979. M Nakajima, M Miura, T Aosaki, and T Shirasawa
Deficiency of presenilin-1 increases calcium-dependent vulner-
Alberto Chiarugi, Elena Meli, Maura Calvani, Roberta Picca,
ability of neurons to oxidative stress in vitro.
Roberto Baronti, Emidio Camaioni, Gabriele Costantino, Maura J Neurochem, Aug 2001; 78(4): 807-14.
Marinozzi, Domenico E. Pellegrini-Giampietro, Roberto Pellic-
ciari, and Flavio Moroni Monika Raisova, Amir M. Hossini, Jrgen Eberle, Christian
Novel Isoquinolinone-Derived Inhibitors of Poly(ADP-ribose) Riebeling, Thomas Wieder, Isrid Sturm, Peter T. Daniel,
Polymerase-1: Pharmacological Characterization and Neuro- Constantin E. Orfanos, and Christoph C. Geilen
protective Effects in an in Vitro Model of Cerebral Ischemia The Bax/Bcl-2 Ratio Determines the Susceptibility of Human
J. Pharmacol. Exp. Ther., Jun 2003; 305: 943 - 949. Melanoma Cells to CD95/Fas-Mediated Apoptosis
J. Invest. Dermatol., Aug 2001; 117: 333 - 340.
Nicholas Mitsiades, Constantine S. Mitsiades, Paul G. Richard-
son, Ciaran McMullan, Vassiliki Poulaki, Galinos Fanourakis, Stig O.P. Jacobsson and Christopher J. Fowler
Robert Schlossman, Dharminder Chauhan, Nikhil C. Munshi, Characterization of palmitoylethanolamide transport in mouse
Teru Hideshima, Victoria M. Richon, Paul A. Marks, and Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leu-
Kenneth C. Anderson kaemia cells: comparison with anandamide
Molecular sequelae of histone deacetylase inhibition in human Br. J. Pharmacol., Apr 2001; 132: 1743 - 1754.
malignant B cells
HANS-PETER DEIGNER, RALF CLAUS, GABRIEL A.
Blood, May 2003; 101: 4055 - 4062.
BONATERRA, CHRISTOF GEHRKE, NILOFAR BIBAK, MARKUS
K Wosik, J Antel, T Kuhlmann, W Bruck, B Massie, and J Nal- BLAESS, MICHAEL CANTZ, JRGEN METZ, and RALF
bantoglu KINSCHERF
Oligodendrocyte injury in multiple sclerosis: a role for p53. Ceramide induces aSMase expression: implications for
J Neurochem, May 2003; 85(3): 635-44. oxLDL-induced apoptosis
FASEB J, Mar 2001; 15: 807 - 814.
Alexandra Aicher, Winfried Brenner, Maaz Zuhayra, Cornel
Badorff, Schirin Massoudi, Birgit Assmus, Thomas Eckey, Akihiko Hirata, Masahiko Igarashi, Hiroshi Yamaguchi, Akira
Eberhard Henze, Andreas M. Zeiher, and Stefanie Dimmeler Suwabe, Makoto Daimon, Takeo Kato, and Makoto Tominaga
Assessment of the Tissue Distribution of Transplanted Human Nifedipine suppresses neointimal thickening by its inhibitory
Endothelial Progenitor Cells by Radioactive Labeling effect on vascular smooth muscle cell growth via a MEK-ERK
Circulation, Apr 2003; 107: 2134 - 2139. pathway coupling with Pyk2
Br. J. Pharmacol., Dec 2000; 131: 1521 - 1530.
Raquel Blanco, Luis Carrasco, and Ivn Ventoso
Cell Killing by HIV-1 Protease Nicolas Ancellin, Chantal Colmont, Joseph Su, Qin Li, Nanette
J. Biol. Chem., Jan 2003; 278: 1086 - 1093. Mittereder, Sung-Suk Chae, Steingrimur Stefansson, Gene Liau,
and Timothy Hla
Nicholas Mitsiades, Constantine S. Mitsiades, Vassiliki Poulaki,
Extracellular Export of Sphingosine Kinase-1 Enzyme. SPHIN-
C
Dharminder Chauhan, Galinos Fanourakis, Xuesong Gu,
GOSINE 1-PHOSPHATE GENERATION AND THE INDUCTION
Charles Bailey, Marie Joseph, Towia A. Libermann, Steven P. OF ANGIOGENIC VASCULAR MATURATION
Treon, Nikhil C. Munshi, Paul G. Richardson, Teru Hideshima,
J. Biol. Chem., Feb 2002; 277: 6667 - 6675.
and Kenneth C. Anderson
Molecular sequelae of proteasome inhibition in human multiple Cristina Cebrin, Cristina Arest, Antoni Nicols, Pere Oliv,
myeloma cells Ana Carceller, Jaume Piulats, and Anna Meseguer
PNAS, Oct 2002; 99: 14374 - 14379. Kidney Androgen-regulated Protein Interacts with Cyclophilin B
and Reduces Cyclosporine A-mediated Toxicity in Proximal
Tubule Cells
J. Biol. Chem., Jul 2001; 276: 29410 - 29419.

References
Silke Reinartz, Siegmund Khler, Harald Schlebusch, Karl Krista, Patrizia Russo, Dario Arzani, Sonya Trombino, and Carla Falugi
Patrick Giffels, Kirsten Renke, Jens Huober, Volker Mbus, Rolf c-myc Down-Regulation Induces Apoptosis in Human Cancer
Kreienberg, Andreas duBois, Paul Sabbatini, and Uwe Wagner Cell Lines Exposed to RPR-115135 (C31H29NO4), a Non-Pepti-
Vaccination of Patients with Advanced Ovarian Carcinoma with domimetic Farnesyltransferase Inhibitor
the Anti-Idiotype ACA125: Immunological Response and Sur- J. Pharmacol. Exp. Ther., Jan 2003; 304: 37 - 47.
vival (Phase Ib/II)
Antonia M. Joussen, Vassiliki Poulaki, Nicholas Mitsiades,
Clin. Cancer Res., Mar 2004; 10: 1580 - 1587.
Wen-yi Cai, Izumi Suzuma, John Pak, Shyr-Te Ju, Susan L. Rook,
G. Zhang, P. H. H. Lopez, C. Y. Li, N. R. Mehta, J. W. Griffin, R. L. Peter Esser, Constantin Mitsiades, Bernd Kirchhof, Anthony P.
Schnaar, and K. A. Sheikh Adamis, and Lloyd Paul Aiello
Anti-ganglioside antibody-mediated neuronal cytotoxicity and Suppression of Fas-FasL-induced endothelial cell apoptosis
its protection by intravenous immunoglobulin: implications for prevents diabetic blood-retinal barrier breakdown in a model of
immune neuropathies streptozotocin-induced diabetes
Brain, Feb 2004; 10.1093/brain/awh127. FASEB J, Nov 2002; 10.1096/fj.02-0157fje.
Rebecca R. Foster, Rachel Hole, Karen Anderson, Simon C. M Toyoda, H Takagi, N Horiguchi, S Kakizaki, K Sato, H
Satchell, Richard J. Coward, Peter W. Mathieson, David A. Takayama, and M Mori
Gillatt, Moin A. Saleem, David O. Bates, and Steven J. Harper A ligand for peroxisome proliferator activated receptor inhibits
Functional evidence that vascular endothelial growth factor may cell growth and induces apoptosis in human liver cancer cells
act as an autocrine factor on human podocytes Gut, Apr 2002; 50: 563 - 567.
Am J Physiol Renal Physiol, Jun 2003; 284: 1263 - 1273.
Nicoletta Di Simone, Roberta Castellani, Dario Caliandro, and
E C Clark, S D Patel, P R Chadwick, G Warhurst, A Curry, and G Alessandro Caruso
L Carlson Monoclonal Anti-Annexin V Antibody Inhibits Trophoblast
Glutamine deprivation facilitates tumour necrosis factor Gonadotropin Secretion and Induces Syncytiotrophoblast Apo-
induced bacterial translocation in Caco-2 cells by depletion of ptosis
enterocyte fuel substrate Biol. Reprod., Dec 2001; 65: 1766 - 1770.
Gut, Feb 2003; 52: 224 - 230.
Jingxuan Pan, Guangpu Xu, and Sai-Ching Jim Yeung
Andreas C. Tomac, Alan D. Agulnick, Norman Haughey, Cytochrome c Release Is Upstream to Activation of Caspase-9,
Chen-Fu Chang, Yajun Zhang, Cristina Bckman, Marisela Caspase-8, and Caspase-3 in the Enhanced Apoptosis of Ana-
Morales, Mark P. Mattson, Yun Wang, Heiner Westphal, and plastic Thyroid Cancer Cells Induced by Manumycin and Pacli-
Barry J. Hoffer taxel
Effects of cerebral ischemia in mice deficient in Persephin J. Clin. Endocrinol. Metab., Oct 2001; 86: 4731 - 4740.
PNAS, Jul 2002; 99: 9521 - 9526.
Ching-Len Liao, Yi-Ling Lin, Bi-Ching Wu, Chang-Huei Tsao,
Jill C. Todt, Bin Hu, Antonello Punturieri, Joanne Sonstein, Timo- Mei-Chuan Wang, Chiu-I Liu, Yue-Ling Huang, Jui-Hui Chen,
thy Polak, and Jeffrey L. Curtis Jia-Pey Wang, and Li-Kuang Chen
Activation of Protein Kinase C II by the Stereo-specific Phos- Salicylates Inhibit Flavivirus Replication Independently of Block-
phatidylserine Receptor Is Required for Phagocytosis of Apop- ing Nuclear Factor Kappa B Activation
totic Thymocytes by Resident Murine Tissue Macrophages J. Virol., Sep 2001; 75: 7828 - 7839.
J. Biol. Chem., Sep 2002; 277: 35906 - 35914.
Kei Yamamoto, Ryuichi Morishita, Shin-ichiro Hayashi, Hidet-
sugu Matsushita, Hidenori Nakagami, Atsushi Moriguchi, Kunio
Matsumoto, Toshikazu Nakamura, Yasufumi Kaneda, and
Cellular DNA Fragmentation ELISA,
Toshio Ogihara
Cat.- No. 11 585 045 001
Contribution of Bcl-2, but Not Bcl-xL and Bax, to Antiapoptotic
Makio Mogi and Akifumi Togari Actions of Hepatocyte Growth Factor in Hypoxia-Conditioned
Activation of Caspases Is Required for Osteoblastic Differentia- Human Endothelial Cells
tion Hypertension, May 2001; 37: 1341 - 1348.
J. Biol. Chem., Nov 2003; 278: 47477 - 47482. Q. Chang and B. L. Tepperman
Luis Miguel Blanco-Colio, Begoa Muoz-Garca, Jose Luis The role of protein kinase C isozymes in TNF--induced cytotox-
Martn-Ventura, Corina Lorz, Cristina Daz, Gonzalo Hernndez, icity to a rat intestinal epithelial cell line
and Jess Egido Am J Physiol Gastrointest Liver Physiol, Apr 2001; 280: 572 -
3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors 583.
Decrease Fas Ligand Expression and Cytotoxicity in Activated
XiaoYing Li, Michela Marani, Roberta Mannucci, Berma Kinsey,
Human T Lymphocytes Francesca Andriani, Ildo Nicoletti, Larry Denner, and Marco
Circulation, Sep 2003; 108: 1506 - 1513.
Marcelli
Overexpression of BCL-XL Underlies the Molecular Basis for
C
Yong-Taek Jun, Hee-Jung Kim, Min-Jin Song, Ji-Hyang Lim,
Dong-Gun Lee, Kyung-Ja Han, Su-Mi Choi, Jin-Hong Yoo, Wan- Resistance to Staurosporine-induced Apoptosis in PC-3 Cells
Shik Shin, and Jung-Hyun Choi Cancer Res., Feb 2001; 61: 1699 - 1706.
In Vitro Effects of Ciprofloxacin and Roxithromycin on Apoptosis
Hidetsugu Matsushita, Ryuichi Morishita, Toshie Nata,
of Jurkat T Lymphocytes Motokuni Aoki, Hironori Nakagami, Yoshiaki Taniyama, Kei
Antimicrob. Agents Chemother., Mar 2003; 47: 1161 - 1164.
Yamamoto, Jitsuo Higaki, Kaneda Yasufumi, and Toshio Ogihara
Satoshi Hirakawa, Young-Kwon Hong, Natasha Harvey, Vivien Hypoxia-Induced Endothelial Apoptosis Through Nuclear Fac-
Schacht, Kant Matsuda, Towia Libermann, and Michael Detmar tor-B (NF-B)Mediated bcl-2 Suppression : In Vivo Evidence of
Identification of Vascular Lineage-Specific Genes by Transcrip- the Importance of NF-B in Endothelial Cell Regulation
tional Profiling of Isolated Blood Vascular and Lymphatic Endot- Circ. Res., May 2000; 86: 974 - 981.
helial Cells
Am. J. Pathol., Feb 2003; 162: 575 - 586.
Appendix 143
References
Cell Proliferation Jennifer J. Schlezinger, Brenda A. Jensen, Koren K. Mann,

Heui-Young Ryu, and David H. Sherr
Cell Proliferation Kit I (MTT), Cat.-No. 11 465 007 001 Peroxisome Proliferator-Activated Receptor -Mediated NF-B
Activation and Apoptosis in Pre-B Cells
Alexey V. Gordadze, Chisaroka W. Onunwor, RongSheng Peng, J. Immunol., Dec 2002; 169: 6831 - 6841.
David Poston, Elisabeth Kremmer, and Paul D. Ling
EBNA2 Amino Acids 3 to 30 Are Required for Induction of Louise McHugh, Stella Hu, B. K. Lee, Kenneth Santora, Paul E.
LMP-1 and Immortalization Maintenance Kennedy, Edward A. Berger, Ira Pastan, and Dean H. Hamer
J. Virol., Apr 2004; 78: 3919 - 3929. Increased Affinity and Stability of an Anti-HIV-1 Envelope
Immunotoxin by Structure-based Mutagenesis
K. Mimori, Y. Tanaka, K. Yoshinaga, T. Masuda, K. Yamashita,
J. Biol. Chem., Sep 2002; 277: 34383 - 34390.
M. Okamoto, H. Inoue, and M. Mori
Clinical significance of the overexpression of the candidate Guoxiong Xu, Marie-Jose Guimond, Chandan Chakraborty,
oncogene CYP24 in esophageal cancer and Peeyush K. Lala
Ann. Onc., Feb 2004; 15: 236 - 241. Control of Proliferation, Migration, and Invasiveness of Human
Extravillous Trophoblast by Decorin, a Decidual Product
Liang-Nian Song, Meghan Coghlan, and Edward P. Gelmann
Biol. Reprod., Aug 2002; 67: 681 - 689.
Antiandrogen Effects of Mifepristone on Coactivator and Core-
pressor Interactions with the Androgen Receptor Seckho Ha, Sulra Lee, Myungil Chung, and Yunjaie Choi
Mol. Endocrinol., Jan 2004; 18: 70 - 85. Mouse ING1 Homologue, a Protein Interacting with A1,
Enhances Cell Death and Is Inhibited by A1 in Mammary Epi-
Christopher S. Stipp, Tatiana V. Kolesnikova, and Martin E.
thelial Cells
Hemler
Cancer Res., Mar 2002; 62: 1275 - 1278.
EWI-2 regulates 31 integrindependent cell functions on
laminin-5 Zhong Ma, Donna J. Webb, Minji Jo, and Steven L. Gonias
J. Cell Biol., Dec 2003; 163: 1167 - 1177. Endogenously produced urokinase-type plasminogen activator
is a major determinant of the basal level of activated ERK/MAP
Minji Jo, Keena S. Thomas, Lihua Wu, and Steven L. Gonias kinase and prevents apoptosis in MDA-MB-231 breast cancer
Soluble Urokinase-type Plasminogen Activator Receptor Inhib-
cells
its Cancer Cell Growth and Invasion by Direct Urokinase-inde-
J. Cell Sci., Sep 2001; 114: 3387 - 3396.
pendent Effects on Cell Signaling
J. Biol. Chem., Nov 2003; 278: 46692 - 46698. Alexey V. Gordadze, RongSheng Peng, Jie Tan, GuoZhen Liu,
Richard Sutton, Bettina Kempkes, George W. Bornkamm, and
Oliver Planz, Stephan Pleschka, Katja Oesterle, Friederike
Paul D. Ling
Berberich-Siebelt, Christina Ehrhardt, Lothar Stitz, and Stephan Notch1IC Partially Replaces EBNA2 Function in B Cells Immor-
Ludwig
talized by Epstein-Barr Virus
Borna Disease Virus Nucleoprotein Interacts with the Cdc2-
J. Virol., Jul 2001; 75: 5899 - 5912.
Cyclin B1 Complex
J. Virol., Oct 2003; 77: 11186 - 11192. Wojciech Rzeski, Lechoslaw Turski, and Chrysanthy Ikonomidou
From the Cover: Glutamate antagonists limit tumor growth
Dean H. Hamer, Sven Bocklandt, Louise McHugh, Tae-Wook
PNAS, May 2001; 98: 6372 - 6377.
Chun, Peter M. Blumberg, Dina M. Sigano, and Victor E.
Marquez Marc Leng, Daniel Locker, Marie-Josphe Giraud-Panis, Annie
Rational Design of Drugs That Induce Human Immunodefi- Schwartz, Francesco P. Intini, Giovanni Natile, Claudio Pisano,
ciency Virus Replication Angelina Boccarelli, Domenico Giordano, and Mauro Coluccia
J. Virol., Oct 2003; 77: 10227 - 10236. Replacement of an NH3 by an Iminoether in Transplatin Makes
an Antitumor Drug from an Inactive Compound
Santiago Canals, Maria Jos Casarejos, Sonsoles de Bernardo,
Mol. Pharmacol., Dec 2000; 58: 1525 - 1535.
Eulalia Rodrguez-Martn, and Maria Angeles Mena
Nitric Oxide Triggers the Toxicity due to Glutathione Depletion Chaoyong Zhu, Magnus Johansson, and Anna Karlsson
in Midbrain Cultures through 12-Lipoxygenase Incorporation of Nucleoside Analogs into Nuclear or Mitochon-
J. Biol. Chem., Jun 2003; 278: 21542 - 21549. drial DNA Is Determined by the Intracellular Phosphorylation
Site
Yin Li, Anthony J. Raffo, Lisa Drew, Yuehua Mao, Andy Tran,
J. Biol. Chem., Aug 2000; 275: 26727 - 26731.
Daniel P. Petrylak, and Robert L. Fine
Fas-Mediated Apoptosis Is Dependent on Wild-Type p53 Status Toshihiro Nanki and Peter E. Lipsky
in Human Cancer Cells Expressing a Temperature-Sensitive p53 Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator
Mutant Alanine-143 for CD4+ T Cell Activation
Cancer Res., Apr 2003; 63: 1527 - 1533. J. Immunol., May 2000; 164: 5010 - 5014.
Ulrike Krummrei, Etienne-Emile Baulieu, and Batrice
C
Chambraud
Cell Proliferation Kit II (XTT), Cat.-No. 11 465 015 001
The FKBP-associated protein FAP48 is an antiproliferative
molecule and a player in T cell activation that increases IL2 Evguenia S. Svarovskaia, Rebekah Barr, Xuechun Zhang,
synthesis Godwin C. G. Pais, Christophe Marchand, Yves Pommier,
PNAS, Mar 2003; 100: 2444 - 2449. Terrence R. Burke, Jr., and Vinay K. Pathak
Karsten Spiekermann, Ralf J. Dirschinger, Ruth Schwab, Ksenia Azido-Containing Diketo Acid Derivatives Inhibit Human Immu-
nodeficiency Virus Type 1 Integrase In Vivo and Influence the
Bagrintseva, Florian Faber, Christian Buske, Susanne Schnittger,
Frequency of Deletions at Two-Long-Terminal-Repeat-Circle
Louise M. Kelly, D. Gary Gilliland, and Wolfgang Hiddemann
The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and Junctions
J. Virol., Apr 2004; 78: 3210 - 3222.
induces growth arrest and apoptosis in AML-derived cell lines
expressing a constitutively activated FLT3
Blood, Feb 2003; 101: 1494 - 1504.

References
Y Tsunemitsu, S Kagawa, N Tokunaga, S Otani, T Umeoka, J A Masaru Nawa, Tomohiko Takasaki, Ken-Ichiro Yamada, Ichiro
Roth, B Fang, N Tanaka, and T Fujiwara Kurane, and Toshitaka Akatsuka
Molecular therapy for peritoneal dissemination of xenotrans- Interference in Japanese encephalitis virus infection of Vero
planted human MKN-45 gastric cancer cells with adenovirus cells by a cationic amphiphilic drug, chlorpromazine
mediated Bax gene transfer J. Gen. Virol., Jul 2003; 84: 1737 - 1741.
Gut, Apr 2004; 53: 554 - 560.
Shivanand P. Lad, Daniel A. Peterson, Ralph A. Bradshaw, and
Harutaka Katano, Lesley Pesnicak, and Jeffrey I. Cohen Kenneth E. Neet
Simvastatin induces apoptosis of Epstein-Barr virus (EBV)- Individual and Combined Effects of TrkA and p75NTR Nerve
transformed lymphoblastoid cell lines and delays development Growth Factor Receptors: A ROLE FOR THE HIGH AFFINITY
of EBV lymphomas RECEPTOR SITE
PNAS, Mar 2004; 10.1073/pnas.0305149101. J. Biol. Chem., Jun 2003; 278: 24808 - 24817.
Federica Piccioni, Benjamin R. Roman, Kenneth H. Fischbeck, Silke Meiners, Dirk Heyken, Andrea Weller, Antje Ludwig, Karl
and J. Paul Taylor Stangl, Peter-M. Kloetzel, and Elke Krger
A screen for drugs that protect against the cytotoxicity of poly- Inhibition of Proteasome Activity Induces Concerted Expression
glutamine-expanded androgen receptor of Proteasome Genes and de Novo Formation of Mammalian
Hum. Mol. Genet., Feb 2004; 13: 437 - 446. Proteasomes
J. Biol. Chem., Jun 2003; 278: 21517 - 21525.
Shuhong Wu, Hongbo Zhu, Jian Gu, Lidong Zhang, Fuminori
Teraishi, John J. Davis, Dietmar A. Jacob, and Bingliang Fang Congxiao Zhang, Judit Baffi, Scott W. Cousins, and Karl G.
Induction of Apoptosis and Down-Regulation of Bcl-XL in Csaky
Cancer Cells by a Novel Small Molecule, 2[ [3-(2,3-Dichlo- Oxidant-induced cell death in retinal pigment epithelium cells
rophenoxy)propyl]amino]ethanol mediated through the release of apoptosis-inducing factor
Cancer Res., Feb 2004; 64: 1110 - 1113. J. Cell Sci., May 2003; 116: 1915 - 1923.
Takeshi Kawashima, Shunsuke Kagawa, Naoya Kobayashi, Chengchen Lufei, Jing Ma, Guochang Huang, Tong Zhang,
Yoshiko Shirakiya, Tatsuo Umeoka, Fuminori Teraishi, Masaki Veronica Novotny-Diermayr, Chin Thing Ong, and Xinmin Cao
Taki, Satoru Kyo, Noriaki Tanaka, and Toshiyoshi Fujiwara GRIM-19, a death-regulatory gene product, suppresses Stat3
Telomerase-Specific Replication-Selective Virotherapy for activity via functional interaction
Human Cancer EMBO J., Mar 2003; 22: 1325 - 1335.
Clin. Cancer Res., Jan 2004; 10: 285 - 292.
J. Blake Marriott, Ian A. Clarke, Anna Czajka, Keith Dredge, Kay
Ada Girnita, Leonard Girnita, Fabrizio del Prete, Armando Childs, Hon-Wah Man, Peter Schafer, Sowmya Govinda, George
Bartolazzi, Olle Larsson, and Magnus Axelson W. Muller, David I. Stirling, and Angus G. Dalgleish
Cyclolignans as Inhibitors of the Insulin-Like Growth Factor-1 A Novel Subclass of Thalidomide Analogue with Anti-Solid
Receptor and Malignant Cell Growth Tumor Activity in Which Caspase-dependent Apoptosis is Asso-
Cancer Res., Jan 2004; 64: 236 - 242. ciated with Altered Expression of bcl-2 Family Proteins
Cancer Res., Feb 2003; 63: 593 - 599.
Stefania Fontana, Daniele Moratto, Surinder Mangal, Maria De
Francesco, William Vermi, Simona Ferrari, Fabio Facchetti, Necil Hiroaki Kawasaki and Kazunari Taira
Kutukculer, Claudia Fiorini, Marzia Duse, Pranab K. Das, Luigi Short hairpin type of dsRNAs that are controlled by tRNAVal
D. Notarangelo, Alessandro Plebani, and Raffaele Badolato promoter significantly induce RNAi-mediated gene silencing in
Functional defects of dendritic cells in patients with CD40 defi- the cytoplasm of human cells
ciency Nucleic Acids Res., Jan 2003; 31: 700 - 707.
Blood, Dec 2003; 102: 4099 - 4106.
Peter Y. Hahn, Scott E. Evans, Theodore J. Kottom, Joseph E.
Weibo Zhou, P. Jeanette Simpson, Jill M. McFadden, Craig A. Standing, Richard E. Pagano, and Andrew H. Limper
Townsend, Susan M. Medghalchi, Aravinda Vadlamudi, Michael Pneumocystis carinii Cell Wall -Glucan Induces Release of
L. Pinn, Gabriele V. Ronnett, and Francis P. Kuhajda Macrophage Inflammatory Protein-2 from Alveolar Epithelial
Fatty Acid Synthase Inhibition Triggers Apoptosis during S Cells via a Lactosylceramide-mediated Mechanism
Phase in Human Cancer Cells J. Biol. Chem., Jan 2003; 278: 2043 - 2050.
Cancer Res., Nov 2003; 63: 7330 - 7337.
Akihito Ishigami, Toshiko Fujita, Setsuko Handa, Takuji
Ming-Yu Cao, Yoon Lee, Ning-Ping Feng, Keyong Xiong, Hong- Shirasawa, Haruhiko Koseki, Tsuneo Kitamura, Nobuyuki
nan Jin, Ming Wang, Aikaterini Vassilakos, Stphane Viau, Jim Enomoto, Nobuhiro Sato, Tatsuo Shimosawa, and Naoki
A. Wright, and Aiping H. Young Maruyama
Adenovirus-Mediated Ribonucleotide Reductase R1 Gene Senescence Marker Protein-30 Knockout Mouse Liver Is Highly
Therapy of Human Colon Adenocarcinoma Susceptible to Tumor Necrosis Factor-- and Fas-Mediated Apo-
Clin. Cancer Res., Oct 2003; 9: 4553 - 4561. ptosis
Am. J. Pathol., Oct 2002; 161: 1273 - 1281.
C
Akira Asai, Yuko Oshima, Yoshihiro Yamamoto, Taka-aki Uochi,
Hideaki Kusaka, Shiro Akinaga, Yoshinori Yamashita, Krisztina NELSON ARISPE and MICHAEL DOH
Pongracz, Ronald Pruzan, Ellen Wunder, Mieczyslaw Piatyszek, Plasma membrane cholesterol controls the cytotoxicity of
Shihong Li, Allison C. Chin, Calvin B. Harley, and Sergei Alzheimers disease AP (140) and (142) peptides
Gryaznov FASEB J, Oct 2002; 16: 1526 - 1536.
A Novel Telomerase Template Antagonist (GRN163) as a Poten-
Yuntao Xie, Bjrn Skytting, Gunnar Nilsson, Alessandra Gas-
tial Anticancer Agent
Cancer Res., Jul 2003; 63: 3931 - 3939. barri, Karl Haslam, Armando Bartolazzi, Bertha Brodin, Nils
Mandahl, and Olle Larsson
Leonard Girnita, Ada Girnita, and Olle Larsson SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sar-
Mdm2-dependent ubiquitination and degradation of the insu- coma Cells: A Gain of Function of the t(X;18)(p11.2;q11.2)
lin-like growth factor 1 receptor Translocation
PNAS, Jul 2003; 100: 8247 - 8252. Cancer Res., Jul 2002; 62: 3861 - 3867.
Appendix 145
References
Tongyu Lin, Jian Gu, Lidong Zhang, Xuefeng Huang, L. Clifton Kai Kraemer, Susanne Fuessel, Uta Schmidt, Matthias Kotzsch,
Stephens, Steven A. Curley, and Bingliang Fang Bernd Schwenzer, Manfred P. Wirth, and Axel Meye
Targeted Expression of Green Fluorescent Protein/Tumor Antisense-mediated hTERT Inhibition Specifically Reduces the
Necrosis Factor-related Apoptosis-inducing Ligand Fusion Growth of Human Bladder Cancer Cells
Protein from Human Telomerase Reverse Transcriptase Clin. Cancer Res., Sep 2003; 9: 3794 - 3800.
Promoter Elicits Antitumor Activity without Toxic Effects on
Dinja Oosterhoff, M. Adhiambo Witlox, Victor W. van
Primary Human Hepatocytes
Cancer Res., Jul 2002; 62: 3620 - 3625. Beusechem, Hidde J. Haisma, Gerard R. Schaap, Johannes
Bras, Frank A. Kruyt, Bonnie Molenaar, Epie Boven, Paul I. J. M.
Shu-Fen Wu, Chyan-Jang Lee, Ching-Len Liao, Raymond A. Wuisman, Herbert M. Pinedo, and Winald R. Gerritsen
Dwek, Nicole Zitzmann, and Yi-Ling Lin Gene-directed Enzyme Prodrug Therapy for Osteosarcoma:
Antiviral Effects of an Iminosugar Derivative on Flavivirus Sensitization to CPT-11 in Vitro and in Vivo by Adenoviral Deliv-
Infections ery of a Gene Encoding Secreted Carboxylesterase-2
J. Virol., Apr 2002; 76: 3596 - 3604. Mol. Cancer Ther., Aug 2003; 2: 765 - 771.
Natalia I. Dmitrieva, Dmitry V. Bulavin, and Maurice B. Burg

High NaCl causes Mre11 to leave the nucleus, disrupting DNA
Cell Proliferation Reagent WST-1,
damage signaling and repair
Cat.-No. 11 644 807 001
Am J Physiol Renal Physiol, Aug 2003; 285: 266 - 274.
Dong Cho Han, Mi-Young Lee, Ki Deok Shin, Sun Bok Jeon, Shunli Ding, Tatyana Merkulova-Rainon, Zhong Chao Han, and
Jung Min Kim, Kwang-Hee Son, Hyoung-Chin Kim, Hwan-Mook
Grard Tobelem
Kim, and Byoung-Mog Kwon
HGF receptor up-regulation contributes to the angiogenic phe-
2'-Benzoyloxycinnamaldehyde Induces Apoptosis in Human notype of human endothelial cells and promotes angiogenesis
Carcinoma via Reactive Oxygen Species
in vitro
J. Biol. Chem., Feb 2004; 279: 6911 - 6920.
Blood, Jun 2003; 101: 4816 - 4822.
Chang Han, Jing Leng, A. Jake Demetris, and Tong Wu Vania De Arcangelis, Dario Coletti, Marco Conti, Michel
Cyclooxygenase-2 Promotes Human Cholangiocarcinoma
Lagarde, Mario Molinaro, Sergio Adamo, Georges Nemoz, and
Growth: Evidence for Cyclooxygenase-2-Independent Mecha-
Fabio Naro
nism in Celecoxib-Mediated Induction of p21waf1/cip1 and IGF-I-induced Differentiation of L6 Myogenic Cells Requires the
p27kip1 and Cell Cycle Arrest
Activity of cAMP-Phosphodiesterase
Cancer Res., Feb 2004; 64: 1369 - 1376.
Mol. Biol. Cell, Apr 2003; 14: 1392 - 1404.
Lei Li, Zhiwei Feng, and Alan G. Porter Jaime R. Merchan, Barden Chan, Sujata Kale, Lowell E. Schnip-
JNK-dependent Phosphorylation of c-Jun on Serine 63 Medi-
per, and Vikas P. Sukhatme
ates Nitric Oxide-induced Apoptosis of Neuroblastoma Cells
In Vitro and In Vivo Induction of Antiangiogenic Activity by Plas-
J. Biol. Chem., Feb 2004; 279: 4058 - 4065. minogen Activators and Captopril
Alexias Safi, Marie Vandromme, Sabine Caussanel, Laure J Natl Cancer Inst, Mar 2003; 95: 388 - 399.
Valdacci, Dominique Baas, Marc Vidal, Gilbert Brun, Laurent
Seongsoo Sohn, Wonhee Hur, Myung Kuk Joe, Ji-Hyun Kim,
Schaeffer, and Evelyne Goillot Zee-Won Lee, Kwon-Soo Ha, and Changwon Kee
Role for the Pleckstrin Homology Domain-Containing Protein
Expression of Wild-Type and Truncated Myocilins in Trabecular
CKIP-1 in Phosphatidylinositol 3-Kinase-Regulated Muscle
Meshwork Cells: Their Subcellular Localizations and
Differentiation Cytotoxicities
Mol. Cell. Biol., Feb 2004; 24: 1245 - 1255.
Invest. Ophthalmol. Vis. Sci., Dec 2002; 43: 3680 - 3685.
Elisa Tedeschi, Marta Menegazzi, Ying Yao, Hisanori Suzuki,
Yves Langelier, Stphane Bergeron, Stphane Chabaud, Julie
Ulrich Frstermann, and Hartmut Kleinert Lippens, Claire Guilbault, A. Marie-Jose Sasseville, Stphan
Green Tea Inhibits Human Inducible Nitric-Oxide Synthase
Denis, Dick D. Mosser, and Bernard Massie
Expression by Down-Regulating Signal Transducer and Activa-
The R1 subunit of herpes simplex virus ribonucleotide reduc-
tor of Transcription-1 Activation tase protects cells against apoptosis at, or upstream of,
Mol. Pharmacol., Jan 2004; 65: 111 - 120.
caspase-8 activation
Darshan S. Sappal, A. Kathleen McClendon, James A. Fleming, J. Gen. Virol., Nov 2002; 83: 2779 - 2789.
Vala Thoroddsen, Kelly Connolly, Corinne Reimer, Ronald K. Olga N. Ilinskaya, Florian Dreyer, Vladimir A. Mitkevich, Kevin L.
Blackman, Christine E. Bulawa, Neil Osheroff, Peter Charlton,
Shaw, C. Nick Pace, and Alexander A. Makarov
and Laura A. Rudolph-Owen
Changing the net charge from negative to positive makes ribo-
Biological characterization of MLN944: A potent DNA binding nuclease Sa cytotoxic
agent
Protein Sci., Oct 2002; 11: 2522 - 2525.
Mol. Cancer Ther., Jan 2004; 3: 47 - 58.
C
Andrea Elsner, Bernd Kreikemeyer, Andrea Braun-Kiewnick,
R.J. Aitken, A.L. Ryan, B.J. Curry, and M.A. Baker Barbara Spellerberg, Bettina A. Buttaro, and Andreas Podbielski
Multiple forms of redox activity in populations of human sper-
Involvement of Lsp, a Member of the LraI-Lipoprotein Family in
matozoa
Streptococcus pyogenes, in Eukaryotic Cell Adhesion and Inter-
Mol. Hum. Reprod., Nov 2003; 9: 645 - 661. nalization
Friederike Herr, Olin D. Liang, Julio Herrero, Uwe Lang, Klaus T. Infect. Immun., Sep 2002; 70: 4859 - 4869.
Preissner, Victor K. M. Han, and Marek Zygmunt
Possible Angiogenic Roles of Insulin-Like Growth Factor II and
Its Receptors in Uterine Vascular Adaptation to Pregnancy
J. Clin. Endocrinol. Metab., Oct 2003; 88: 4811 - 4817.

References
Zhiwei Feng, Lei Li, Poh Yong Ng, and Alan G. Porter Yasutoshi Tatsumi, Satoshi Ohta, Hiroshi Kimura, Toshiki
Neuronal Differentiation and Protection from Nitric Oxide- Tsurimoto, and Chikashi Obuse
Induced Apoptosis Require c-Jun-Dependent Expression of The ORC1 Cycle in Human Cells: I. CELL CYCLE-REGULATED
NCAM140 OSCILLATION OF HUMAN ORC1
Mol. Cell. Biol., Aug 2002; 22: 5357 - 5366. J. Biol. Chem., Oct 2003; 278: 41528 - 41534.
ROK HUMAR, FABRICE N. KIEFER, HARTMUT BERNS, Yi Cao, Zhefeng Zhao, Joanna Gruszczynska-Biegala, and Anna
THRSE J. RESINK, and EDOUARD J. BATTEGAY Zolkiewska
Hypoxia enhances vascular cell proliferation and angiogenesis Role of Metalloprotease Disintegrin ADAM12 in Determination
in vitro via rapamycin (mTOR) -dependent signaling of Quiescent Reserve Cells during Myogenic Differentiation In
FASEB J, Jun 2002; 16: 771 - 780. Vitro
Mol. Cell. Biol., Oct 2003; 23: 6725 - 6738.
Fabienne Brilot, Wassim Chehadeh, Chantal Charlet-Renard,
Henri Martens, Vincent Geenen, and Didier Hober Jan-Willem Taanman, John R. Muddle, and Ania C. Muntau
Persistent Infection of Human Thymic Epithelial Cells by Cox- Mitochondrial DNA depletion can be prevented by dGMP and
sackievirus B4 dAMP supplementation in a resting culture of deoxyguanosine
J. Virol., May 2002; 76: 5260 - 5265. kinase-deficient fibroblasts
Hum. Mol. Genet., Aug 2003; 12: 1839 - 1845.
Haisong Ju, Sandhya Nerurkar, Charles F. Sauermelch, Alan R.
Olzinski, Rosanna Mirabile, Dawn Zimmerman, John C. Lee, Heather J. Evans, Janea K. Sweet, Robert L. Price, Michael Yost,
Jerry Adams, Joseph Sisko, Marinella Berova, and Robert N. and Richard L. Goodwin
Willette Novel 3D culture system for study of cardiac myocyte
Sustained Activation of p38 Mitogen-Activated Protein Kinase development
Contributes to the Vascular Response to Injury Am J Physiol Heart Circ Physiol, Jul 2003; 285: 570 - 578.
J. Pharmacol. Exp. Ther., Apr 2002; 301: 15 - 20.
Sanna Pasonen-Seppnen, Susanna Karvinen, Kari Trrnen,
MOLECULAR BASIS OF CELL AND DEVELOPMENTAL Juha M. T. Hyttinen, Tiina Jokela, Mikko J. Lammi, Markku I.
BIOLOGY: Tammi, and Raija Tammi
Long-Yuan Li, Hsiu-Ming Shih, Mei-Ying Liu, and Jen-Yang EGF Upregulates, Whereas TGF- Downregulates, the Hyaluro-
Chen nan Synthases Has2 and Has3 in Organotypic Keratinocyte Cul-
The Cellular Protein PRA1 Modulates the Anti-apoptotic Activity tures: Correlations with Epidermal Proliferation and
of Epstein-Barr Virus BHRF1, a Homologue of Bcl-2, through Differentiation
Direct Interaction J. Invest. Dermatol., Jun 2003; 120: 1038 - 1044.
J. Biol. Chem., Jul 2001; 276: 27354 - 27362.
Eric Julien and Winship Herr
Chuanhai Guo, Shihui Yu, Alan T. Davis, Huamin Wang, Jeffrey Proteolytic processing is necessary to separate and ensure
E. Green, and Khalil Ahmed proper cell growth and cytokinesis functions of HCF-1
A Potential Role of Nuclear Matrix-associated Protein Kinase EMBO J., May 2003; 22: 2360 - 2369.
CK2 in Protection against Drug-induced Apoptosis in Cancer
Kaiwen Ma, Keigo Araki, Solachuddin J.A. Ichwan, Tamaki
Cells
J. Biol. Chem., Feb 2001; 276: 5992 - 5999. Suganuma, Mimi Tamamori-Adachi, and Masa-Aki Ikeda
E2FBP1/DRIL1, an AT-Rich Interaction Domain-Family Tran-
Takako Akiyama, Shin Takasawa, Koji Nata, Seiichi Kobayashi, scription Factor, Is Regulated by p53
Michiaki Abe, Nausheen J. Shervani, Takayuki Ikeda, Kei Naka- Mol. Cancer Res., Apr 2003; 1: 438 - 444.
gawa, Michiaki Unno, Seiki Matsuno, and Hiroshi Okamoto
Yoshiyuki Konishi and Azad Bonni
Activation of Reg gene, a gene for insulin-producing -cell
The E2F-Cdc2 Cell-Cycle Pathway Specifically Mediates Activity
regeneration: Poly(ADP-ribose) polymerase binds Reg pro-
moter and regulates the transcription by autopoly(ADP- Deprivation-Induced Apoptosis of Postmitotic Neurons
J. Neurosci., Mar 2003; 23: 1649 - 1658.
ribosyl)ation
PNAS, Jan 2001; 98: 48 - 53. Jean Buteau, Sylvain Foisy, Erik Joly, and Marc Prentki
Glucagon-Like Peptide 1 Induces Pancreatic -Cell proliferation
Via Transactivation of the Epidermal Growth Factor Receptor
5-Bromo-2-deoxy-uridine Labeling and Detection Kit I, Diabetes, Jan 2003; 52: 124 - 132.
Cat.-No. 11 296 736 001
Mary Ann Stepp, Heather E. Gibson, Purvi H. Gala, Drina D. Sta.
Sandra Bauer, Jrgen Pollheimer, Johannes Hartmann, Peter Iglesia, Ahdeah Pajoohesh-Ganji, Sonali Pal-Ghosh, Marcus
Husslein, John D. Aplin, and Martin Knfler Brown, Christopher Aquino, Arnold M. Schwartz, Olga Gold-
Tumor Necrosis Factor- Inhibits Trophoblast Migration through berger, Michael T. Hinkes, and Merton Bernfield
Elevation of Plasminogen Activator Inhibitor-1 in First-Trimester Defects in keratinocyte activation during wound healing in the
Villous Explant Cultures syndecan-1-deficient mouse
C
J. Clin. Endocrinol. Metab., Feb 2004; 89: 812 - 822. J. Cell Sci., Dec 2002; 115: 4517 - 4531.
Igor Oruetxebarria, Francesca Venturini, Tuija Kekarainen, Ada Xiaodong Shu, Weicheng Wu, Raymond D. Mosteller, and
Houweling, Lobke M. P. Zuijderduijn, Adone Mohd-Sarip, Rob- Daniel Broek
ert G. J. Vries, Rob C. Hoeben, and C. Peter Verrijzer Sphingosine Kinase Mediates Vascular Endothelial Growth Fac-
p16INK4a Is Required for hSNF5 Chromatin Remodeler- tor-Induced Activation of Ras and Mitogen-Activated Protein
induced Cellular Senescence in Malignant Rhabdoid Tumor Kinases
Cells Mol. Cell. Biol., Nov 2002; 22: 7758 - 7768.
J. Biol. Chem., Jan 2004; 279: 3807 - 3816.
Appendix 147
References
Tamaki Suganuma, Masahiro Kawabata, Tomoko Ohshima, and R. Montgomery Gill and Paul A. Hamel
Masa-Aki Ikeda Subcellular Compartmentalization of E2F Family Members Is
Growth suppression of human carcinoma cells by reintroduc- Required for Maintenance of the Postmitotic State in Terminally
tion of the p300 coactivator Differentiated Muscle
PNAS, Oct 2002; 99: 13073 - 13078. J. Cell Biol., Mar 2000; 148: 1187 - 1202.
Kirsi Rilla, Mikko J. Lammi, Reijo Sironen, Kari Trrnen, Merja John Lynch, Eun-Ran Suh, Debra G. Silberg, Steven Rulyak,
Luukkonen, Vincent C. Hascall, Ronald J. Midura, Mika Nadine Blanchard, and Peter G. Traber
Hyttinen, Jukka Pelkonen, Markku Tammi, and Raija Tammi The Caudal-related Homeodomain Protein Cdx1 Inhibits Prolif-
Changed lamellipodial extension, adhesion plaques and migra- eration of Intestinal Epithelial Cells by Down-regulation of D-
tion in epidermal keratinocytes containing constitutively type Cyclins
expressed sense and antisense hyaluronan synthase 2 (Has2) J. Biol. Chem., Feb 2000; 275: 4499 - 4506.
genes
J. Cell Sci., Sep 2002; 115: 3633 - 3643.
Satoshi Kurosaka, Yasumitsu Nagao, Naojiro Minami, 5-Bromo-2-deoxy-uridine Labeling and Detection Kit II,
Cat.-No. 11 299 964 001
Masayasu Yamada, and Hiroshi Imai
Dependence of DNA Synthesis and In Vitro Development of Kenneth H. H. Wong, Heather D. Wintch, and Mario R. Capecchi
Bovine Nuclear Transfer Embryos on the Stage of the Cell Cycle Hoxa11 Regulates Stromal Cell Death and Proliferation during
of Donor Cells and Recipient Cytoplasts Neonatal Uterine Development
Biol. Reprod., Aug 2002; 67: 643 - 647. Mol. Endocrinol., Jan 2004; 18: 184 - 193.
Michael L. Whitfield, Gavin Sherlock, Alok J. Saldanha, John I. MARIE-THERESE SIMON, ALAIN PAULOIN, GUILLAUME
Murray, Catherine A. Ball, Karen E. Alexander, John C. Matese, NORMAND, HANH-TU LIEU, HELENE MOULY, GERARD PIV-
Charles M. Perou, Myra M. Hurt, Patrick O. Brown, and David ERT, FRANOISE CARNOT, J. GUILHERME TRALHAO, CHRIS-
Botstein TIAN BRECHOT, and LAURENCE CHRISTA
Identification of Genes Periodically Expressed in the Human HIP/PAP stimulates liver regeneration after partial hepatectomy
Cell Cycle and Their Expression in Tumors and combines mitogenic and anti-apoptotic functions through
Mol. Biol. Cell, Jun 2002; 13: 1977 - 2000. the PKA signaling pathway
Fang Liao, Jacqueline F. Doody, Jay Overholser, Bridget Finnerty, FASEB J, Aug 2003; 17: 1441 - 1450.
Rajiv Bassi, Yan Wu, Elisabetta Dejana, Paul Kussie, Peter Andrs F. Muro, Anil K. Chauhan, Srecko Gajovic, Alessandra
Bohlen, and Daniel J. Hicklin Iaconcig, Fabiola Porro, Giorgio Stanta, and Francisco E. Baralle
Selective Targeting of Angiogenic Tumor Vasculature by Vascu- Regulated splicing of the fibronectin EDA exon is essential for
lar Endothelial-cadherin Antibody Inhibits Tumor Growth with- proper skin wound healing and normal lifespan
out Affecting Vascular Permeability J. Cell Biol., Jul 2003; 162: 149 - 160.
Cancer Res., May 2002; 62: 2567 - 2575.
Yoshikazu Nakamura, Kiyoko Fukami, Haiyan Yu, Kei Takenaka,
Christopher J. McGann, Shannon J. Odelberg, and Mark T. Yuki Kataoka, Yuji Shirakata, Shin-Ichi Nishikawa, Koji Hashim-
Keating oto, Nobuaki Yoshida, and Tadaomi Takenawa
Mammalian myotube dedifferentiation induced by newt regen- Phospholipase C1 is required for skin stem cell lineage commit-
eration extract ment
PNAS, Nov 2001; 98: 13699 - 13704. EMBO J., Jun 2003; 22: 2981 - 2991.
Salvador Soriano, David E. Kang, Maofu Fu, Richard Pestell, Jijun Chen, Yajun Tu, Cheil Moon, Eiichiro Nagata, and Gabriele
Nathalie Chevallier, Hui Zheng, and Edward H. Koo V. Ronnett
Presenilin 1 Negatively Regulates -Catenin/T Cell Factor/Lym- Heme oxygenase-1 and heme oxygenase-2 have distinct roles
phoid Enhancer Factor-1 Signaling Independently of -Amyloid in the proliferation and survival of olfactory receptor neurons
Precursor Protein and Notch Processing mediated by cGMP and bilirubin, respectively
J. Cell Biol., Feb 2001; 152: 785 - 794. J. Neurochem., Jun 2003; 85: 1247 - 1261.
Jacob B. Hansen, Hongbin Zhang, Thomas H. Rasmussen, Ras- Ryuichi Yamada, Yoko Mizutani-Koseki, Takanori Hasegawa,
mus K. Petersen, Esben N. Flindt, and Karsten Kristiansen Noriko Osumi, Haruhiko Koseki, and Naoki Takahashi
Peroxisome Proliferator-activated Receptor (PPAR)-mediated Cell-autonomous involvement of Mab21l1 is essential for lens
Regulation of Preadipocyte Proliferation and Gene Expression Is placode development
Dependent on cAMP Signaling Development, May 2003; 130: 1759 - 1770.
J. Biol. Chem., Jan 2001; 276: 3175 - 3182.
Maarit Rossi, Hiroyuki Morita, Raija Sormunen, Sari Airenne,
Hitoshi Komuro, Ellada Yacubova, Elina Yacubova, and Pasko Marjut Kreivi, Ling Wang, Naomi Fukai, Bjorn R. Olsen, Karl
Rakic Tryggvason, and Raija Soininen
Mode and Tempo of Tangential Cell Migration in the Cerebellar
C
Heparan sulfate chains of perlecan are indispensable in the
External Granular Layer lens capsule but not in the kidney
J. Neurosci., Jan 2001; 21: 527 - 540. EMBO J., Jan 2003; 22: 236 - 245.
STEPHAN J. RESHKIN, ANTONIA BELLIZZI, SANDRA CAL- B. R. Wamhoff, D. K. Bowles, N. J. Dietz, Q. Hu, and M. Sturek
DEIRA, VALENTINA ALBARANI, ILARIA MALANCHI, MAN- Exercise training attenuates coronary smooth muscle pheno-
UELA POIGNEE, MARIANNA ALUNNI-FABBRONI, VALERIA typic modulation and nuclear Ca2+ signaling
CASAVOLA, and MASSIMO TOMMASINO Am J Physiol Heart Circ Physiol, Dec 2002; 283: 2397 - 2410.
Na+/H+ exchanger-dependent intracellular alkalinization is an
early event in malignant transformation and plays an essential
role in the development of subsequent transformation-associ-
ated phenotypes
FASEB J, Nov 2000; 14: 2185 - 2197.

References
James J. A. Contos, Isao Ishii, Nobuyuki Fukushima, Marcy A. Emmanuelle Charpentier, Robert M. Lavker, Elizabeth Acquista,
Kingsbury, Xiaoqin Ye, Shuji Kawamura, Joan Heller Brown, and and Pamela Cowin
Jerold Chun Plakoglobin Suppresses Epithelial Proliferation and Hair Growth
Characterization of lpa2 (Edg4) and lpa1/lpa2 (Edg2/Edg4) In Vivo
Lysophosphatidic Acid Receptor Knockout Mice: Signaling J. Cell Biol., Apr 2000; 149: 503 - 520.
Deficits without Obvious Phenotypic Abnormality Attributable
Mette Christiansen, Marie Kveiborg, Moustapha Kassem, Brian
to lpa2
Mol. Cell. Biol., Oct 2002; 22: 6921 - 6929. F. C. Clark, and Suresh I. S. Rattan
CBFA1 and Topoisomerase I mRNA Levels Decline During Cel-
Stacey Rentschler, Jennifer Zander, Kathleen Meyers, David lular Aging of Human Trabecular Osteoblasts
France, Rebecca Levine, George Porter, Scott A. Rivkees, Gre- J. Gerontol. A Biol. Sci. Med. Sci., Apr 2000; 55: 194 - 200.
gory E. Morley, and Glenn I. Fishman
Birgit Stallmeyer, Heiko Kmpfer, Nicole Kolb, Josef
Neuregulin-1 promotes formation of the murine cardiac con-
Pfeilschifter, and Stefan Frank
duction system
PNAS, Aug 2002; 99: 10464 - 10469. The Function of Nitric Oxide in Wound Repair: Inhibition of
Inducible Nitric Oxide-Synthase Severely Impairs Wound Reepi-
Laura P. Stabile, Autumn L. Gaither Davis, Christopher T. thelialization
Gubish, Toni M. Hopkins, James D. Luketich, Neil Christie, Syd- J. Invest. Dermatol., Dec 1999; 113: 1090 - 1098.
ney Finkelstein, and Jill M. Siegfried
Human Non-Small Cell Lung Tumors and Cells Derived from Jacob George, Dominique Roulot, Victor E. Koteliansky, and D.
Montgomery Bissell
Normal Lung Express Both Estrogen Receptor and and Show
In vivo inhibition of rat stellate cell activation by soluble trans-
Biological Responses to Estrogen
Cancer Res., Apr 2002; 62: 2141 - 2150. forming growth factor type II receptor: A potential new therapy
for hepatic fibrosis
Eleonora Minina, Hans Markus Wenzel, Conny Kreschel, Seth PNAS, Oct 1999; 96: 12719 - 12724.
Karp, William Gaffield, Andrew P. McMahon, and Andrea
Vortkamp ALESSANDRO FATATIS and RICHARD J. MILLER
Cell cycle control of PDGF-induced Ca2+ signaling through
BMP and Ihh/PTHrP signaling interact to coordinate chondro-
modulation of sphingolipid metabolism
cyte proliferation and differentiation
Development, Nov 2001; 128: 4523 - 4534. FASEB J, Aug 1999; 13: 1291 - 1301.
Masahiro Hitomi and Dennis W. Stacey
Kamilah Alexander and Philip W. Hinds
Cellular Ras and Cyclin D1 Are Required during Different Cell
Requirement for p27KIP1 in Retinoblastoma Protein-Mediated
Senescence Cycle Periods in Cycling NIH 3T3 Cells
Mol. Cell. Biol., Jul 1999; 19: 4623 - 4632.
Mol. Cell. Biol., Jun 2001; 21: 3616 - 3631.
Shu Wakino, Ulrich Kintscher, Sarah Kim, Simon Jackson, Fen

Yin, Sunil Nagpal, Roshantha A. S. Chandraratna, Willa A. 5-Bromo-2-deoxy-uridine Labeling and Detection Kit III,
Hsueh, and Ronald E. Law Cat.-No. 11 444 611 001
Retinoids Inhibit Proliferation of Human Coronary Smooth Mus-
cle Cells by Modulating Cell Cycle Regulators Hideyuki Sakoda, Yukiko Gotoh, Hideki Katagiri, Mineo
Arterioscler. Thromb. Vasc. Biol., May 2001; 21: 746 - 751. Kurokawa, Hiraku Ono, Yukiko Onishi, Motonobu Anai,
Takehide Ogihara, Midori Fujishiro, Yasushi Fukushima, Miho
A. Cevik Tufan and Rocky S. Tuan Abe, Nobuhiro Shojima, Masatoshi Kikuchi, Yoshitomo Oka,
Wnt regulation of limb mesenchymal chondrogenesis is accom- Hisamaru Hirai, and Tomoichiro Asano
panied by altered N-cadherin-related functions Differing Roles of Akt and Serum- and Glucocorticoid-
FASEB J, Apr 2001; 10.1096/fj.00-0784fje. regulated Kinase in Glucose Metabolism, DNA Synthesis, and
Cor F. Calkhoven, Christine Mller, and Achim Leutz Oncogenic Activity
J. Biol. Chem., Jul 2003; 278: 25802 - 25807.
Translational control of C/EBP and C/EBP isoform expression
Genes & Dev., Aug 2000; 14: 1920 - 1932. Jeremy K. Wong, Hoa H. Le, Attila Zsarnovszky, and Scott M.
Maria Luiza C. Albuquerque, Christopher M. Waters, Ushma Belcher
Estrogens and ICI182,780 (Faslodex) Modulate Mitosis and Cell
Savla, H. William Schnaper, and Annette S. Flozak
Death in Immature Cerebellar Neurons via Rapid Activation of
Shear stress enhances human endothelial cell wound closure in
vitro p44/p42 Mitogen-Activated Protein Kinase
J. Neurosci., Jun 2003; 23: 4984 - 4995.
Am J Physiol Heart Circ Physiol, Jul 2000; 279: 293 - 302.
D. Sun, Y. Gong, H. Kojima, G. Wang, E. Ravinsky, M. Zhang, and
Stephan Goetze, Ulrich Kintscher, Hiroaki Kawano, Yasuko
Kawano, Shu Wakino, Eckart Fleck, Willa A. Hsueh, and Ronald G. Y. Minuk
Increasing cell membrane potential and GABAergic activity
E. Law
C
inhibits malignant hepatocyte growth
Tumor Necrosis Factor Inhibits Insulin-induced Mitogenic
Signaling in Vascular Smooth Muscle Cells Am J Physiol Gastrointest Liver Physiol, Jun 2003; 285: 12 - 19.
J. Biol. Chem., Jun 2000; 275: 18279 - 18283. Senji Kasahara, Kazuki Ando, Kuniaki Saito, Kenji Sekikawa,
Hiroyasu Ito, Tetsuya Ishikawa, Hiroo Ohnishi, Mitsuru Seishima,
Charlotte M. Boney, Philip A. Gruppuso, Ronald A. Faris, and A.
Raymond Frackelton, Jr. Shinichi Kakumu, and Hisataka Moriwaki
Lack of Tumor Necrosis Factor Alpha Induces Impaired Prolifer-
The Critical Role of Shc in Insulin-Like Growth Factor-I-Medi-
ation of Hepatitis B Virus-Specific Cytotoxic T Lymphocytes
ated Mitogenesis and Differentiation in 3T3-L1 Preadipocytes
Mol. Endocrinol., Jun 2000; 14: 805 - 813. J. Virol., Feb 2003; 77: 2469 - 2476.
Appendix 149
References
Oren Z. Lerman, Robert D. Galiano, Mary Armour, Jamie P. Michael D. Island, Xaioling Cui, and John W. Warren
Levine, and Geoffrey C. Gurtner Effect of Escherichia coli Cytotoxic Necrotizing Factor 1 on
Cellular Dysfunction in the Diabetic Fibroblast: Impairment in Repair of Human Bladder Cell Monolayers In Vitro
Migration, Vascular Endothelial Growth Factor Production, and Infect. Immun., Jul 1999; 67: 3657 - 3661.
Response to Hypoxia
R. JESNOWSKI, P. MLLER, W. SCHARECK, S. LIEBE, and M.
Am. J. Pathol., Jan 2003; 162: 303 - 312.
LHR
Lasse Riemann and Farooq Azam Immortalized Pancreatic Duct Cells in vitro and in vivo
Widespread N-Acetyl-D-Glucosamine Uptake among Pelagic Ann. N.Y. Acad. Sci., Jun 1999; 880: 50 - 65.
Marine Bacteria and Its Ecological Implications
Appl. Envir. Microbiol., Nov 2002; 68: 5554 - 5562.
Ren Gysin, Angelo Azzi, and Theresa Visarius Cell Proliferation ELISA, BrdU (colorimetric),
Cat.-No. 11 647 229 001
g-Tocopherol inhibits human cancer cell cycle progression and
cell proliferation by down-regulation of cyclins Cell Proliferation ELISA, BrdU (chemiluminescent),
FASEB J, Oct 2002; 10.1096/fj.02-0362fje. Cat.-No. 11 669 915 001
Eva Jimnez, Rosa Sacedn, Angeles Vicente, Carmen Hernn- Kana Mizuno, Hiroyuki Okamoto, and Takeshi Horio
dez-Lpez, Agustn G. Zapata, and Alberto Varas Ultraviolet B Radiation Suppresses Endocytosis, Subsequent
Rat Peripheral CD4+CD8+ T Lymphocytes Are Partially Immu- Maturation, and Migration Activity of Langerhans Cell-Like
nocompetent Thymus-Derived Cells That Undergo Post-Thymic Dendritic Cells
Maturation to Become Functionally Mature CD4+ T Lympho- J. Invest. Dermatol., Feb 2004; 122: 300 - 306.
cytes
J. Immunol., May 2002; 168: 5005 - 5013. Marc-Andr Raymond, Anik Dsormeaux, Patrick Laplante,
Normand Vigneault, Janos G. Filep, Karine Landry, Alexey V.
Carmen Hernndez-Lpez, Alberto Varas, Rosa Sacedn, Eva Pshezhetsky, and Marie-Jose Hbert
Jimnez, Juan Jos Muoz, Agustn G. Zapata, and Angeles Apoptosis of endothelial cells triggers a caspase-dependent
Vicente anti-apoptotic paracrine loop active on vascular smooth muscle
Stromal cell-derived factor 1/CXCR4 signaling is critical for cells
early human T-cell development FASEB J, Feb 2004; 10.1096/fj.03-0573fje.
Blood, Jan 2002; 99: 546 - 554.
Souichi Yoshida, Tasuku Harada, Masahiro Mitsunari, Tomio
Li-Fen Lee, Junlin Guan, Yun Qiu, and Hsing-Jien Kung Iwabe, Yasuko Sakamoto, Satoru Tsukihara, Yumiko Iba, Sayako
Neuropeptide-Induced Androgen Independence in Prostate Horie, and Naoki Terakawa
Cancer Cells: Roles of Nonreceptor Tyrosine Kinases Etk/Bmx, Hepatocyte Growth Factor/Met System Promotes Endometrial
Src, and Focal Adhesion Kinase and Endometriotic Stromal Cell Invasion via Autocrine and
Mol. Cell. Biol., Dec 2001; 21: 8385 - 8397. Paracrine Pathways
Wojciech Rzeski, Lechoslaw Turski, and Chrysanthy Ikonomidou J. Clin. Endocrinol. Metab., Feb 2004; 89: 823 - 832.
From the Cover: Glutamate antagonists limit tumor growth Alexias Safi, Marie Vandromme, Sabine Caussanel, Laure Val-
PNAS, May 2001; 98: 6372 - 6377. dacci, Dominique Baas, Marc Vidal, Gilbert Brun, Laurent
Satomichi Yoshimura, Jan Bondeson, Brian M. J. Foxwell, Fion- Schaeffer, and Evelyne Goillot
Role for the Pleckstrin Homology Domain-Containing Protein
ula M. Brennan, and Marc Feldmann
CKIP-1 in Phosphatidylinositol 3-Kinase-Regulated Muscle Dif-
Effective antigen presentation by dendritic cells is NF-B depen-
dent: coordinate regulation of MHC, co-stimulatory molecules ferentiation
Mol. Cell. Biol., Feb 2004; 24: 1245 - 1255.
and cytokines
Int. Immunol., May 2001; 13: 675 - 683. ENMEI LIU, HELEN K.W. LAW, and YU-LUNG LAU
Alberto Varas, Eva Jimnez, Rosa Sacedn, Margarita Insulin-Like Growth Factor I Promotes Maturation and Inhibits
Apoptosis of Immature Cord Blood MonocyteDerived Den-
Rodrguez-Mahou, Enrique Maroto, Agustn G. Zapata, and
dritic Cells through MEK and PI 3-Kinase Pathways
Angeles Vicente
Analysis of the Human Neonatal Thymus: Evidence for a Tran- Pediatr. Res., Dec 2003; 54: 919 - 925.
sient Thymic Involution Yun-Hee Noh, Kant Matsuda, Young-Kwon Hong, Rainer Kunst-
J. Immunol., Jun 2000; 164: 6260 - 6267. feld, Lucia Riccardi, Manuel Koch, Hajimu Oura, Soheil S.
Israel Rubinstein Dadras, Michael Streit, and Michael Detmar
An N-Terminal 80 kDa Recombinant Fragment of Human
Smokeless tobacco potentiates VIP-induced DNA synthesis
Thrombospondin-2 Inhibits Vascular Endothelial Growth Factor
and inactivates NEP 24.11 in oral keratinocytes
Am J Physiol Cell Physiol, Feb 2000; 278: 391 - 396. Induced Endothelial Cell Migration In Vitro and Tumor Growth
and Angiogenesis In Vivo
C
Michael J. Kennedy, Robert J. Yancey, Jr., Margaret S. Sanchez, J. Invest. Dermatol., Dec 2003; 121: 1536 - 1543.
Robert A. Rzepkowski, Sandra M. Kelly, and Roy Curtiss, III
Attenuation and Immunogenicity of cya crp Derivatives of Sal- K Yoshida, A Tsutsumi, Y Ohnishi, T Akimoto, H Murata, and T
Sumida
monella choleraesuis in Pigs
T cell epitopes of prothrombin in patients with antiphospholipid
Infect. Immun., Sep 1999; 67: 4628 - 4636.
syndrome
Rosa Sacedn, Angeles Vicente, Alberto Varas, Eva Jimnez, Ann. Rheum. Dis, Sep 2003; 62: 905 - 906.
Juan Jos Muoz, and Agustn G. Zapata
Glucocorticoid-mediated regulation of thymic dendritic cell
function
Int. Immunol., Aug 1999; 11: 1217 - 1224.

References
ENMEI LIU, HELEN K.W. LAW, and YU-LUNG LAU Kelly L. Davenpeck, Cezary Marcinkiewicz, Dian Wang, Rodica
BCG Promotes Cord Blood Monocyte-Derived Dendritic Cell Niculescu, Yi Shi, Jack L. Martin, and Andrew Zalewski
Maturation with Nuclear Rel-B Up-regulation and Cytosolic IB Regional Differences in Integrin Expression : Role of 51 in
and Degradation Regulating Smooth Muscle Cell Functions
Pediatr. Res., Jul 2003; 54: 105 - 112. Circ. Res., Feb 2001; 88: 352 - 358.
Mitsuhiro Matsuo, Hiroaki Sakurai, and Ikuo Saiki Masahide Tokunou, Toshiro Niki, Keisuke Eguchi, Sanae Iba,
ZD1839, a Selective Epidermal Growth Factor Receptor Tyrosine Hitoshi Tsuda, Tesshi Yamada, Yoshihiro Matsuno, Haruhiko
Kinase Inhibitor, Shows Antimetastatic Activity Using a Hepato- Kondo, Yukihito Saitoh, Hiroji Imamura, and Setsuo Hirohashi
cellular Carcinoma Model c-MET Expression in Myofibroblasts : Role in Autocrine Activa-
Mol. Cancer Ther., Jun 2003; 2: 557 - 561. tion and Prognostic Significance in Lung Adenocarcinoma
Am. J. Pathol., Apr 2001; 158: 1451 - 1463.
Jason S. Taub, Rishu Guo, L. M. Fredrik Leeb-Lundberg, John F.
Madden, and Yehia Daaka Marie-Claude Amoureux, Bruce A. Cunningham, Gerald M.
Bradykinin Receptor Subtype 1 Expression and Function in Edelman, and Kathryn L. Crossin
Prostate Cancer N-CAM Binding Inhibits the Proliferation of Hippocampal Pro-
Cancer Res., May 2003; 63: 2037 - 2041. genitor Cells and Promotes Their Differentiation to a Neuronal
Phenotype
Takao Sakai, Shaohua Li, Denitsa Docheva, Carsten Grashoff,
J. Neurosci., May 2000; 20: 3631 - 3640.
Keiko Sakai, Gnter Kostka, Attila Braun, Alexander Pfeifer,
Peter D. Yurchenco, and Reinhard Fssler KD Coutant, AB de Fraissinette, A Cordier, and P Ulrich
Integrin-linked kinase (ILK) is required for polarizing the epi- Modulation of the activity of human monocyte-derived dendritic
blast, cell adhesion, and controlling actin accumulation cells by chemical haptens, a metal allergen, and a staphylococ-
Genes & Dev., Apr 2003; 17: 926 - 940. cal superantigen
Toxicol. Sci., Dec 1999; 52: 189 - 198.
Yvonne E.M. Dommels, Merel M.G. Haring, Nynke G.M. Keestra,
Gerrit M. Alink, Peter J. van Bladeren, and Ben van Ommen
The role of cyclooxygenase in n-6 and n-3 polyunsaturated fatty
In Situ Cell Proliferation Kit, FLUOS,
acid mediated effects on cell proliferation, PGE2 synthesis and
Cat.-No. 11 810 740 001
cytotoxicity in human colorectal carcinoma cell lines
Carcinogenesis, Mar 2003; 24: 385 - 392. Juan Carlos Casar, Claudio Cabello-Verrugio, Hugo Olguin,
Rebeca Aldunate, Nibaldo C. Inestrosa, and Enrique Brandan
Rainer Wessely, Ludger Hengst, Birgit Jaschke, Franziska
Heparan sulfate proteoglycans are increased during skeletal
Wegener, Thomas Richter, Raffaella Lupetti, Makarios Paschali-
dis, Albert Schmig, Richard Brandl, and Franz-Josef Neumann muscle regeneration: requirement of syndecan-3 for successful
fiber formation
A central role of interferon regulatory factor-1 for the limitation
J. Cell Sci., Jan 2004; 117: 73 - 84.
of neointimal hyperplasia
Hum. Mol. Genet., Jan 2003; 12: 177 - 187. Iwan Walev, Sebastian Chakrit Bhakdi, Fred Hofmann, Nabil
Djonder, Angela Valeva, Klaus Aktories, and Sucharit Bhakdi
Marc-Andr Raymond, Luigina Mollica, Normand Vigneault,
Delivery of proteins into living cells by reversible membrane
Anik Dsormeaux, John S.D. Chan, Janos G. Filep, and Marie-
Jose Hbert permeabilization with streptolysin-O
PNAS, Mar 2001; 98: 3185 - 3190.
Blockade of the apoptotic machinery by cyclosporin A redirects
cell death toward necrosis in arterial endothelial cells: regula- Amrit Mann, Kai Breuhahn, Peter Schirmacher, Arnd Wilhelmi,
tion by reactive oxygen species and cathepsin D Carsten Beyer, Andrea Rosenau, Suat zbek, Stefan Rose-John,
FASEB J, Jan 2003; 10.1096/fj.02-0500fje. and Manfred Blessing
Up- and Down-Regulation of Granulocyte/Macrophage-Colony
Craig H. Selzman, Stephanie A. Miller, Michael A. Zimmerman,
Fabia Gamboni-Robertson, Alden H. Harken, and Anirban Stimulating Factor Activity in Murine Skin Increase Susceptibil-
ity to Skin Carcinogenesis by Independent Mechanisms
Banerjee
Cancer Res., Mar 2001; 61: 2311 - 2319.
Monocyte chemotactic protein-1 directly induces human
vascular smooth muscle proliferation Mario Encinas, Montse Iglesias, Yuhui Liu, Hongyin Wang,
Am J Physiol Heart Circ Physiol, Oct 2002; 283: 1455 - 1461. Ashraf Muhaisen, Valentin Cea, Carme Gallego, and Joan X.
Comella
Kahoru Nishina, Katsuya Mikawa, Osamu Morikawa, Hidefumi
Obara, and R. J. Mason Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and
Brain-Derived Neurotrophic Factor Gives Rise to Fully Differen-
The Effects of Intravenous Anesthetics and Lidocaine on Prolif-
tiated, Neurotrophic Factor-Dependent, Human Neuron-Like
eration of Cultured Type II Pneumocytes and Lung Fibroblasts
Anesth. Analg., Feb 2002; 94: 385 - 388. Cells
J. Neurochem., Sep 2000; 75: 991 - 1003.
Torsten R. Dunkern and Bernd Kaina
C
Satomi S. Tanaka, Yayoi Toyooka, Ryuko Akasu, Yuko Katoh-
Cell proliferation and DNA Breaks Are Involved in Ultraviolet
Light-induced Apoptosis in Nucleotide Excision Repair-defi- Fukui, Yoko Nakahara, Rika Suzuki, Minesuke Yokoyama, and
Toshiaki Noce
cient Chinese Hamster Cells
The mouse homolog of Drosophila Vasa is required for the
Mol. Biol. Cell, Jan 2002; 13: 348 - 361.
development of male germ cells
Ferruccio Galbiati, Daniela Volonte', Jun Liu, Franco Capozza, Genes & Dev., Apr 2000; 14: 841 - 853.
Philippe G. Frank, Liang Zhu, Richard G. Pestell, and Michael P.
Qianghua Hu and Sankar N. Maity
Lisanti
Caveolin-1 Expression Negatively Regulates Cell Cycle Progres- Stable Expression of a Dominant Negative Mutant of CCAAT
Binding Factor/NF-Y in Mouse Fibroblast Cells Resulting in
sion by Inducing G0/G1 Arrest via a p53/p21WAF1/Cip1-depen-
Retardation of Cell Growth and Inhibition of Transcription of
dent Mechanism
Mol. Biol. Cell, Aug 2001; 12: 2229 - 2244. Various Cellular Genes
J. Biol. Chem., Feb 2000; 275: 4435 - 4444.
Appendix 151
References
Toshihiro Yoshizawa, Yasuaki Yamagishi, Naoteru Koseki, Jun Anti-Bromodeoxyuridine-Fluorescein formalin grade,
Goto, Hideaki Yoshida, Futoshi Shibasaki, Shinichi Shoji, and Cat.-No. 11 202 693 001
Ichiro Kanazawa
James A. Irving, Sain S. Shushanov, Robert N. Pike, Evgenya Y.
Cell cycle arrest enhances the in vitro cellular toxicity of the
truncated MachadoJoseph disease gene product with an Popova, Dieter Brmme, Theresa H. T. Coetzer, Stephen P. Bot-
tomley, Iaroslava A. Boulynko, Sergei A. Grigoryev, and James C.
expanded polyglutamine stretch
Whisstock
Hum. Mol. Genet., Jan 2000; 9: 69 - 78.
Inhibitory Activity of a Heterochromatin-associated Serpin
(MENT) against Papain-like Cysteine Proteinases Affects Chro-
matin Structure and Blocks Cell Proliferation
Anti-Bromodeoxyuridine formalin grade,
J. Biol. Chem., Apr 2002; 277: 13192 - 13201.
Cat.-No. 11 170 376 001
Masaaki Hayashi, Richard I. Tapping, Ta-Hsiang Chao, Jeng-Fan
J. Norrman, C. W. David, S. N. Sauter, H. M. Hammon, and J. W.
Lo, Charles C. King, Young Yang, and Jiing-Dwan Lee
Blum
BMK1 Mediates Growth Factor-induced Cell Proliferation
Effects of dexamethasone on lymphoid tissue in the gut and through Direct Cellular Activation of Serum and Glucocorticoid-
thymus of neonatal calves fed with colostrum or milk replacer
inducible Kinase
J Anim Sci, Sep 2003; 81: 2322 - 2332.
J. Biol. Chem., Mar 2001; 276: 8631 - 8634.
Dong-Woon Kim, Jin-Sook Kwon, Young-Gyu Kim, Maeng Sup
Thomas F. Westbrook, Don X. Nguyen, Barry R. Thrash, and
Kim, Gwan-Sun Lee, Tae-Jin Youn, and Myeong-Chan Cho Dennis J. McCance
Novel Oral Formulation of Paclitaxel Inhibits Neointimal Hyper-
E7 Abolishes Raf-Induced Arrest via Mislocalization of p21Cip1
plasia in a Rat Carotid Artery Injury Model
Mol. Cell. Biol., Oct 2002; 22: 7041 - 7052.
Circulation, Mar 2004; 109: 1558 - 1563.
Paola Secchiero, Arianna Gonelli, Claudio Celeghini, Prisco
Allen D. Everett, Jill V. Narron, Tamara Stoops, Hideji Nakamura,
Mirandola, Lia Guidotti, Giuseppe Visani, Silvano Capitani, and
and Amy Tucker
Giorgio Zauli
Hepatoma Derived Growth Factor is a Pulmonary Endothelial Activation of the nitric oxide synthase pathway represents a key
Cell Expressed Angiogenic Factor
component of tumor necrosis factor-related apoptosis-inducing
Am J Physiol Lung Cell Mol Physiol, Jan 2004; 10.1152/
ligand-mediated cytotoxicity on hematologic malignancies
ajplung.00427.2003. Blood, Oct 2001; 98: 2220 - 2228.
J. Norrman, C. W. David, S. N. Sauter, H. M. Hammon, and J. W.
Hanna E. Kleczkowska, Giancarlo Marra, Teresa Lettieri, and
Blum
Josef Jiricny
Effects of dexamethasone on lymphoid tissue in the gut and hMSH3 and hMSH6 interact with PCNA and colocalize with it
thymus of neonatal calves fed with colostrum or milk replacer
to replication foci
J Anim Sci, Sep 2003; 81: 2322 - 2332.
Genes & Dev., Mar 2001; 15: 724 - 736.
Adarsh V. Mudgil, Nadav Segal, Frank Andriani, Youai Wang, Bing Jiang, Amrita Kamat, and Carole R. Mendelson
Norbert E. Fusenig, and Jonathan A. Garlick
Hypoxia Prevents Induction of Aromatase Expression in Human
Ultraviolet B Irradiation Induces Expansion of Intraepithelial
Trophoblast Cells in Culture: Potential Inhibitory Role of the
Tumor Cells in a Tissue Model of Early Cancer Progression Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian
J. Invest. Dermatol., Jul 2003; 121: 191 - 197.
Achaete-Scute Homologous Protein-2)
Jo-Ellen Murphy, Romeo E. Morales, Jordan Scott, and Thomas Mol. Endocrinol., Oct 2000; 14: 1661 - 1673.
S. Kupper ZE Smith and DR Higgs
IL-1, Innate Immunity, and Skin Carcinogenesis: The Effect of
The pattern of replication at a human telomeric region
Constitutive Expression of IL-1 in Epidermis on Chemical Car-
(16p13.3): its relationship to chromosome structure and gene
cinogenesis expression
J. Immunol., Jun 2003; 170: 5697 - 5703.
Hum. Mol. Genet., Aug 1999; 8: 1373 - 1386.
Rebecca M. Wolf, Nicole Draghi, Xiquan Liang, Chengkai Dai,
Teresa Lettieri, Giancarlo Marra, Gabriele Aquilina, Margherita
Lene Uhrbom, Charlotta Eklf, Bengt Westermark, Eric C. Hol- Bignami, Nigel E.A. Crompton, Fabio Palombo, and Josef Jiricny
land, and Marilyn D. Resh
Effect of hMSH6 cDNA expression on the phenotype of mis-
p190RhoGAP can act to inhibit PDGF-induced gliomas in mice:
match repair-deficient colon cancer cell line HCT15
a putative tumor suppressor encoded on human Chromosome Carcinogenesis, Mar 1999; 20: 373 - 382.
19q13.3
Genes & Dev., Feb 2003; 17: 476 - 487. Manho Kim, H-S Lee, Genevieve LaForet, Charmian McIntyre,
Eileen J. Martin, Patrick Chang, Tae Wan Kim, M. Williams, P. H.
Julie M. Hastings, Diana R. Licence, Graham J. Burton, D. Reddy, Dan Tagle, Frederick M. Boyce, Lisa Won, Alfred Heller,
Stephen Charnock-Jones, and Stephen K. Smith
Neil Aronin, and Marian DiFiglia
C
Soluble Vascular Endothelial Growth Factor Receptor 1 Inhibits
Mutant Huntingtin Expression in Clonal Striatal Cells: Dissocia-
Edema and Epithelial Proliferation Induced by 17-Estradiol in tion of Inclusion Formation and Neuronal Survival by Caspase
the Mouse Uterus
Inhibition
Endocrinology, Jan 2003; 144: 326 - 334.
J. Neurosci., Feb 1999; 19: 964 - 973.

References
Anti-Bromodeoxyuridine-Peroxidase, Fab fragments

formalin grade, Cat.-No. 11 585 860 001
Atsushi Terunuma, Kimberly L. Jackson, Veena Kapoor, William

G. Telford, and Jonathan C. Vogel
Side Population Keratinocytes Resembling Bone Marrow Side
Population Stem Cells Are Distinct From Label-Retaining Kerat-
inocyte Stem Cells
J. Invest. Dermatol., Nov 2003; 121: 1095 - 1103.
Neela Patel, Li Sun, Deborah Moshinsky, Hui Chen, Kathleen M.

Leahy, Phuong Le, Katherine G. Moss, Xueyan Wang, Audie
Rice, Danny Tam, A. Douglas Laird, Xiaoming Yu, Qingling
Zhang, Cho Tang, Gerald McMahon, and Anthony Howlett
A Selective and Oral Small Molecule Inhibitor of Vascular Epi-
thelial Growth Factor Receptor (VEGFR)-2 and VEGFR-1 Inhib-
its Neovascularization and Vascular Permeability
J. Pharmacol. Exp. Ther., Sep 2003; 306: 838 - 845.
Liang Ma, Jian Liu, Tobey Wu, Maksim Plikus, Ting-Xin Jiang,
Qun Bi, Yi-Hsin Liu, Sven Mller-Rver, Heiko Peters, John P.
Sundberg, Rob Maxson, Richard L. Maas, and Cheng-Ming
Chuong
`Cyclic alopecia' in Msx2 mutants: defects in hair cycling and
hair shaft differentiation
Development, Jan 2003; 130: 379 - 389.
Michael R. Albert, Ruth-Ann Foster, and Jonathan C. Vogel

Murine Epidermal Label-Retaining Cells Isolated by Flow
Cytometry do not Express the Stem Cell Markers CD34, Sca-1,
or Flk-1
J. Invest. Dermatol., Oct 2001; 117: 943 - 948.
Tino D. Piscione, Tien Phan, and Norman D. Rosenblum

BMP7 controls collecting tubule cell proliferation and apoptosis
via Smad1-dependent and -independent pathways
Am J Physiol Renal Physiol, Jan 2001; 280: 19 - 33.
Martha Campbell-Thompson, Gregory Y. Lauwers, Kristen K.

Reyher, Josh Cromwell, and Kathleen T. Shiverick
17-Estradiol Modulates Gastroduodenal Preneoplastic Alter-
ations in Rats Exposed to the Carcinogen N-Methyl-N'-Nitro-
Nitrosoguanidine
Endocrinology, Oct 1999; 140: 4886 - 4894.
C
Appendix 153
References
General references
3.3 General references

1. Schwartzman, R. A. and Cidlowski, J. A. (1993) Apoptosis: the biochemistry and molecular biology of
programmed cell death. Endocrine Rev. 14, 133.
2. Vermes, I. and Haanan, C. (1994) Apoptosis and programmed cell death in health and disease.
Adv. Clin. Chem. 31, 177.
3. Berke, G. (1991) Debate: the mechanism of lymphocyte-mediated killing. Lymphocyte-triggered

internal target disintegration. Immunol. Today 12, 396.
4. Krhenbhl, O. and Tschopp, J. (1991) Debate: the mechanism of lymphocyte-mediated killing.

Perforin-induced pore formation. Immunol. Today 12, 399.
5. Van Furth, R. and Van Zwet, T. L. (1988) Immunocytochemical detection of 5-bromo-2-deoxyuridine

incorporation in individual cells. J. Immunol. Methods 108, 45. (CHECK THIS ONE OUT-page number
showed as 4)
6. Cohen, J. J. (1993) Apoptosis. Immunol. Today 14, 126.
7. Savill, J. S. et al. (1989) Macrophage phagocytosis of aging neutrophils in inflammation. Programmed

cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83, 865.
8. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous

endonuclease activation. Nature 284, 555.
9. Leist, M. et al. (1994) Application of the Cell Death Detection ELISA for the Detection of Tumor
Necrosis Factor-induced DNA Fragmentation in Murine Models of Inflammatory Organ Failure
Biochemica No. 3, 1820.
10. Fraser, A. and Evan, G. (1996) A license to kill. Cell 85, 781784.
11. Duke, R. C. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cell-
mediated cytolysis. Proc. Natl. Acad. Sci. USA 80, 6361.
12. Duke, R. C. & Cohen, J. J. (1986) IL-2 addiction: withdrawal of growth factor activates a suicide
program in dependent T cells. Lymphokine Res. 5, 289.
13. Trauth, B. C. et al. (1994) Eur. J. Cell. Biol. 63, 32, Suppl 40.
14. Matzinger, P. (1991) The JAM test. A simple assay for DNA fragmentation and cell death. J. Immunol.
Methods 145, 185.
15. Kaeck, M. R. (1993) Alkaline elution analysis of DNA fragmentation induced during apoptosis. Anal.
Biochem. 208, 393.
16. Prigent, P. et al. (1993) A safe and rapid method for analyzing apoptosis-induced fragmentation of
DNA extracted from tissues or cultured cells. J. Immunol. Methods 160, 139.
17. Huang, P. & Plunkett, W. (1992) A quantitative assay for fragmented DNA in apoptotic cells.
Anal. Biochem. 207, 163
18. Bortner , C. D. et al. (1995) Trends Cell Biol. 5, 21.
19. Gold, R. et al. (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in
situ tailing and nick translation techniques. Lab. Invest. 71, 219.
C
20. Sgonc, R. et al. (1994) Simultaneous determination of cell surface antigens and apoptosis.
Trends Genet. 10, 41-42.
21. Darzynkiewicz, Z. et al. (1994) Assays of cell viability: discrimination of cells dying by apoptosis.
Methods Cell Biol. 41, 15.
22. Darzynkiewicz, Z. et al. (1992) Features of apoptotic cells measured by flow cytometry.
Cytometry 13, 795.
23. Duvall, E. et al. (1985) Macrophage recognition of cells undergoing programmed cell death
(apoptosis). Immunology 56, 351358.

References
General references
24. Savill, J. S. et al. (1993) Phagocyte recognition of cells undergoing apoptosis. Immunol.
Today 14, 131136.
25. Asch, A. S. et al. (1987) Isolation of the thrombospondin membrane receptor.

J. Clin. Invest. 79, 10541061.
26. Fadok, V. A. et al. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes
triggers specific recognition and removal by macrophages. J. Immunol. 148, 22072216.
27. Vermes, I. et al. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine
expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39.
28. Homburg, C. H. E. et al. (1995) Human neutrophils lose their surface Fc gamma RIII and acquire
Annexin V binding sites during apoptosis in vitro. Blood 85, 532.
29. Verhoven, B. et al. (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition

signal, on apoptotic T lymphocytes. J. Exp. Med. 182, 1597.
30. Dive, C. et al. (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death)
by multiparameter flow cytometry. Biochem. Biophys. Acta 1133, 275.
31. Schmid, I. et al. (1994) Sensitive method for measuring apoptosis and cell surface phenotype in human
thymocytes by flow cytometry. Cytometry 15, 12.
32. Afanasev, V. N. et al. (1986) FEBS Letts. 194, 347.
33. Ormerod, M. G. (1992) Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two

flow cytometric methods. J. Immunol. Methods 153, 57.
34. Meyaard, L. et al. (1992) Programmed death of T cells in HIV-1 infection. Science 257, 217.
35. Gavrieli, Y. et al. (1992) Identification of programmed cell death in situ via specific labeling of nuclear
DNA fragmentation. J. Cell. Biol. 119, 493.
36. Manning, F. C. R. and Patierno, S. R. (1996) Apoptosis: inhibitor or instigator of carcinogenesis?

Cancer Invest. 14, 455465.
37. Stewart, B. W. (1994) Mechanisms of apoptosis: integration of genetic, biochemical, and cellular
indicators. J. Natl. Cancer Inst. 86, 12861296.
38. Ellis, H. M. and Horvitz, H. R. (1986) Genetic control of programmed cell death in the nematode C.
elegans. Cell 44, 817829.
39. Yuan, J. Y. and Horvitz, H. R. (1990)

The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell
death. Dev. Biol. 138, 3341.
40. Hentgartner, M. O., Ellis, R. E. and Horvitz, H. R. (1992) Caenorhabditis elegans gene ced-9 protects
cells from programmed cell death. Nature 356, 494499.
41. Baffy, G. et al. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent
hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by
enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 65116519.
42. Miyashita, T. and Reed, J. C. (1993) Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a
human leukemia cell line. Blood 81, 151157.
C
43. Oltvai, Z. N., Milliman, C. L. and Korsmeyer, S. J. (1993) Bcl-2 heterodimerizes in vivo with a conserved
homolog, Bax, that accelerates programmed cell death. Cell 74, 609619.
44. Yonish-Rouach, E. et al. (1991) Wild-type p53 induces apoptosis of myeloid leukaemic cells that is
inhibited by interleukin-6. Nature 352, 345347.
45. Bissonnette, R. P. et al. (1992) Apoptotic cell death induced by c-myc is inhibited by bcl-2.
Nature 359, 552554.
46. Wagner, A. J., Small, M. B. and Hay, N. (1993) Myc-mediated apoptosis is blocked by ectopic
expression of Bcl-2. Mol. Cell. Biol. 13, 24322440.
Appendix 155
References
General references
47. Curnow, S. J. (1993) The role of apoptosis in antibody-dependent cellular cytotoxicity. Cancer
Immunol. Immunother. 36, 149.
48. Danks, A. M. et al. (1992) Cellular alterations produced by the experimental increase in
intracellular calcium and the nature of protective effects from pretreatment with nimodipine.
Mol. Brain Res. 16, 168172.
49. Kolber, M. A. et al. (1988) Measurement of cytotoxicity by target cell release and retention of the
fluorescent dye bis-carboxyethyl-carboxyfluorescein (BCECF). J. Immunol. Methods 108, 255264.
50. Oldham, R. K. et al. (1977) Direct comparison of three isotopic release microtoxicity assays as
measures of cell-mediated immunity to Gross virus-induced lymphomas in rats. J. Natl.
Cancer Inst. 58, 10611067.
51. Decker, T. and Lohmann-Matthes, M.-L. (1988) A quick and simple method for the quantitation of
lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor
(TNF) activity. J. Immunol. Methods 15, 6169. (PubMed MedLine shows this as 115)
52. Martin, A. and Clynes, M. (1991) Acid phosphatase: endpoint for in vitro toxicity tests.
In Vitro Cell. Dev. Biol. 27A, 183184.
53. Mosmann, T. R. (1983) Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55.
54. Mosmann, T. R. and Fong, T. A. T. (1989) Specific assays for cytokine production by T cells.
J. Immunol. Methods 116, 151.
55. Sanderson, C. J. (1981) The mechanism of lymphocyte-mediated cytotoxicity. Biol. Rev. Camb. Philos.
Soc. 56, 153.
56. Keilholz, U. et al. (1990) A modified cytotoxicity assay with high sensitivity. Scand.
J. Clin. Lab. Invest. 50, 879.
57. Cook, J. A. and Mitchell, J. B. (1989) Viability measurements in mammalian cell system.
Anal. Biochem. 179, 1.
58. Roehm, N. W. et al. (1991) An improved colorimetric assay for cell proliferation and viability utilizing
the tetrazolium salt XTT. J. Immunol. Methods 142, 257.
59. Slater, T. F. et al. (1963) Biochem. Biophys. Acta 77, 383.
60. Berridge, M. V. and Tan, A. S. (1993) Characterization of the cellular reduction of 3-(4,5-dimethyltiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence and
involvement of mitchondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303, 474.
61. Cory, A. H. et al. (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays
in culture. Cancer Commun. 3, 207.
62. Jabbar, S. A. B. et al. (1989) The MTT assay underestimates the growth inhibitory effects of interferons.
Br. J. Cancer 60, 523.
63. Scudiero, E. A. et al. (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and
drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48, 4827.
64. Vistica, D. T. et al. (1991) Tetrazolium-based assays for cellular viability: a critical examination of
selected parameters effecting formazan production (published erratum appears in Cancer Res. 1991
Aug 15; 51 (16): 45et). Cancer Res. 51, 2515.
C
65. Magaud, J. P. et al. (1988) Detection of human white cell proliferative responses by immunoenzymatic
measurement of bromodeoxyuridine uptake. J. Immunol. Methods 106, 95.
66. Porstmann et al. (1985) Quanitation of 5-bromo-2-deoxyuridine incorporation into DNA:

an enzyme immunoassay for the assessment of the lymphoid cell proliferative response.
J. Immunol. Methods 82, 169.
67. Konttinen, Y. T. et al. (1988) An immunoperoxidase-autoradiography double labeling method for

analysis of lymphocyte activation markers and DNA synthesis. J. Immunol. Methods 110, 19.

References
General references
68. Steel, G. G. (1977) In: Growth Kinetics of Tumours, Clarendon Press, Oxford, UK.
69. Takagi, S. et al. (1993) Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal
anti-BrdUrd antibody after deoxyribonuclease treatment. Cytometry 14, 640.
70. Gerdes, J. et al. (1984) Cell cycle analysis of a cell proliferation-associated human unclear antigen
defined by the monoclonal antibody Ki-67. J. Immunol. 133, 1710.
71. Hall, P. A. and Levison, D. A. (1990) Review: assessment of cell proliferation in histological material.
J. Clin. Pathol. 43, 184.
72. Hall, P. A. et al. (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin
sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms.
J. Pathol. 162, 285.
73. Kreipe, H. et al. (1993) Determination of the growth fraction in non-Hodgkins lymphomas by
monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen.
Am. J. Pathol. 142, 1689.
74. Scott, R. J. et al. (1991) A comparison of immunohistochemical markers of cell proliferation with
experimentally determined growth fraction (see comments). J. Pathol. 165, 173.
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Appendix 157
General abbreviations
4. General abbreviations
ABTS 2,2-azino-di-[3-ethylbenzthiazoline-sulfonate (6)]
Ac N-acetyl
ActD actinomycin D
ALT alanine aminotransferase
AP alkaline phosphatase
APAAP alkaline phosphatase anti-alkaline phosphatase
APES aminopropyl-triethoxysilane
BCIP 5-bromo-4-chloro-3-indolyl phosphate
B-CLL chronic lymphocytic leukemia (B-type)
Bio biotin
BrdU 5-bromo-2-deoxyuridine
BSA bovine serum albumin
CAM campothecin
Con A concanavalin A
cpm counts per minute
CTL cytotoxic T lymphocytes
DAB 3,3-diaminobenzidine
DES diethylstilbestrol
DX dexamethasone
ELISA enzyme-linked immunosorbent assay
Fab protease-generated antibody fragments
F(ab)2 protease-generated antibody fragment
FACS fluorescence activated cell sorter
FAQs frequently asked questions
FITC fluorescein isothiocyanate
FLUOS 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester
FSC forward light scatter
G0 resting phase
G1 gap between mitosis and DNA synthesis
G2 gap between DNA synthesis and mitosis
h hour
HMW DNA high molecular weight DNA
HSV herpes simplex virus type I antigen
[3H]-TdR tritiated thymidine (2-deoxy)
ICE interleukin-1-converting enzyme
INT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride
INV-A influenza A virus antigen
INV-B influenza B virus antigen
INV-KA influenza control antigen
C
ISNT in situ nick translation
kD kilodalton
LAK cells lymphokine-activated killer cells
LDH lactate dehydrogenase
LMW DNA low molecular weight DNA
LSC liquid scintillation counting
M-phase mitosis
MTP microtiter plate
MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

NBT 4-nitro-blue tetrazolium chloride

NK cells natural killer cells
OKT3 anti-CD3 monoclonal antibody
PARP poly(ADP-ribose) polymerase
PBL peripheral blood lymphocytes
PBS phosphate buffered saline
PFA paraformaldehyde
PHA phytohemagglutinin
PI propidium iodide
PMS phenazine methosulfate
pNA 4-nitranilide
POD peroxidase
PS phosphatidylserine
PVDF polyvinylidene difluoride
PWM pokeweed mitogen
ref. reference
rlu/s relative light units/second
RT room temperature
RUV rubella virus antigen
SA streptavidin
SAC Staphylococcus aureus Cowan I
SN supernatant
SOD superoxide dismutase
S-phase DNA synthesis (replication)
SSC side light scatter
TdR thymidine
TdT terminal deoxynucleotidyltransferase
TMB tetramethylbenzidine
TNF tumor necrosis factor
TRITC tetramethylrhodamine isothiocyanate
TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
WST-1 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate
X-dUTP hapten-labeled deoxyuracil triphosphate
X-dNTP hapten-labeled deoxynucleoside triphosphate
XTT 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
Z carbobenzoxy
C
Appendix 159
Amino acids
Name 3-letter 1-letter
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic Acid Asp D
Cysteine Cys C
Glutamic Acid Glu E
Glutamine Gln Q
Glycine Gly G
Histidine His H
Homoserine Hse
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Methionine sulfoxide Met (O)
Methionine methylsulfonium Met (S-Me)
Norleucine Nle
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
-aminoisobutyric acid Aib
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Ordering Guide
5. Ordering Guide
Products for Measuring Apoptosis in Cell Populations Cat. No. Pack Size
Apoptotic DNA Ladder Kit 11 835 246 001 1 kit (20 tests)
PLUS
Cell Death Detection ELISA 11 774 425 001 1 kit (96 tests)
Cell Death Detection ELISAPLUS, 10x 11 920 685 001 10 x 96 tests
Cell Death Detection ELISA 11 544 675 001 1 kit (96 tests)
Anti-PARP 11 835 238 001 100 l
Caspase 3 Activity Assay 12 012 952 001 1 kit (96 tests)
Homogeneous Caspase Assay, fluorimetric 03 005 372 001 100 tests
12 236 869 001 1000 tests
Products for Measuring Apoptosis in Cat. No. Pack Size

Individual Cells
In Situ Cell Death Detection Kit, Fluorescein 11 684 795 001 1 kit (50 tests)
In Situ Cell Death Detection Kit, TMR red 12 156 792 001 1 kit (50 tests)
In Situ Cell Death Detection Kit, AP 11 684 809 001 1 kit (50 tests)
TUNEL kits and related reagents
In Situ Cell Death Detection Kit, POD 11 684 817 001 1 kit (50 tests)
TUNEL Label Mix 11 767 291 001 3 x 550 l (30 tests)
TUNEL Enzyme 11 767 305 001 2 x 50 l (20 tests)
TUNEL POD 11 772 465 001 3.5 ml (70 tests)
TUNEL AP 11 772 457 001 3.5 ml (70 tests)
TUNEL Dilution Buffer 11 966 006 001 2 x 10 ml
DAB substrate, precipitating (POD substrate) 11 718 096 001 1 pack
NBT/BCIP Stock Solution (AP substrate) 11 681 451 001 8 ml
Fast Red Tablets (AP substrate) 11 496 549 001 20 Tablets
Propidium iodide solution* 11 348 639 001 20 ml
Fluorescence
labels
DAPI 10 236 276 001 10 mg
Annexin-V-FLUOS 11 828 681 001 250 tests

Annexin-V and related
Annexin-V-FLUOS Staining Kit 11 858 777 001 1 kit (50 tests)

reagents
Annexin-V-Biotin 11 828 690 001 250 tests

Annexin-V-Alexa 568 03 703 126 001 250 tests
Streptavidin-POD 11 089 153 001 500 U (1 ml)
Streptavidin-AP 11 089 161 001 1000 U (1 ml)
M30 CytoDEATH 12 140 322 001 50 tests
12 140 349 001 250 tests
Antibodies
M30 CytoDEATH, Fluorescein 12 156 857 001 250 tests

Anti-Fas (CD95/Apo-1) 11 922 432 001 100 g
C
p53 pan ELISA 11 828 789 001 1 kit (96 tests)
* only sold in the US.
Products for Measuring Cytotoxicity Cat. No. Pack Size

Cytotoxicity Detection Kit (LDH) 11 644 793 001 1 kit (2000 tests)
Cellular DNA Fragmentation ELISA 11 585 045 01 1 kit (500 tests)
Cell Proliferation Kit I (MTT) 11 465 007 001 1 kit (2500 tests)
Cell Proliferation Kit II (XTT) 11 465 015 001 1 kit (2500 tests)
Cell Proliferation Reagent WST-1 11 644 807 001 2500 tests
Appendix 161
Ordering Guide
Products for Measuring Cell Proliferation in Cat. No. Pack Size

Cell Populations
Cell Proliferation Kit I (MTT) 11 465 007 001 1 kit (2500 tests)
Cell Proliferation Kit II (XTT) 11 465 015 001 1 kit (2500 tests)
Cell Proliferation Reagent WST-1 11 644 807 001 2500 tests
Cell Proliferation ELISA, BrdU (colorimetric) 11 647 229 001 1 kit (1000 tests)
Cell Proliferation ELISA, BrdU (chemiluminescent) 11 669 915 001 1 kit (1000 tests)
FixDenat 11 758 764 001 4 x 100 ml (2000
tests)
BrdU Labeling and Detection Kit III 11 444 611 001 1 kit (1000 tests)
Products for Measuring Cell Proliferation in Cat. No. Pack Size

Individual Cells
In Situ Cell Proliferation Kit, FLUOS 11 810 740 001 1 kit (100 tests)
BrdU Labeling and Detection Kit I 11 296 736 001 1 kit (100 tests)
BrdU Labeling and Detection Kit II 11 299 964 001 1 kit (100 tests)
Anti-BrdU (clone BMC 9318)
unlabeled, formalin grade 11 170 376 001 50 g (500 l)
fluorescein-labeled, formalin grade 11 202 693 001 50 g (500 l)
Anti-BrdU, POD-labeled 11 585 860 001 15 U
(clone BMC 6H8), Fab fragments, formalin grade
Additional Products for Cell Death/Cell Cat. No. Pack Size

Proliferation Studies
Calpain inhibitor I 11 086 090 001 25 mg
Calpain inhibitor II 11 086 103 001 25 mg
DNase I, RNase free 10 776 785 001 10 000 units
DNase I, grade I 10 104 132 001 20 000 units
DNase II, grade II 10 104 159 001 100 mg
Interleukin-1, human, recombinant (E. coli) 11 457 756 001 10 0000 units
Interleukin-1, mouse recombinant (E. coli) 11 444 590 001 10 0000 units
Nuclease S7 10 107 921 001 15 000 units
Nuclease P1 10 236 225 001 1 mg
Proteinase K, lyophilizate 03 115 836 001 25 mg
Staurosporine 11 055 682 001 500 g
TNF-, human, recombinant (E. coli) 11 371 843 001 10 g
(1 000 000 units)
TNF-a, human, recombinant (yeast) 11 088 939 001 10 g
(1 000 000 units)
TNF-, mouse, recombinant (E. coli) 11 271 156 001 5 g
(2 000 000 units)
TNF-ELISA, human 11 425 943 001 1 kit (96 tests)
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Ordering Guide
Trademarks
ABTS is a registered trademark of a Member of the Roche Group
Alexa is a trademark of Molecular Probes, Inc., Eugene, OR, USA
BOBO is a trademark of Molecular Probes, Inc., Eugene, OR, USA
C
Appendix 163
Index
6. Index
A C
A0 cells ................................................................... 49 CAM, see Campothecin
Acridine orange ....................................................... 49 Campothecin ............................................... 15, 46, 48
Alkaline elution analysis of DNA ............................ 30 Caspases ................................................................... 18
Annexin V Caspase 3 Activity Assay ......................................... 22
-Alexa 568 ........................................................... 44 Ced-3 ..................................................................... 123
assays for ............................................................. 45 ced-9 gene .............................................................. 123
binding of phosphatidylserine .......................... 43 Cell cycle, overview of ............................................. 75
-Biotin ................................................................ 47 Cell death
-FLUOS .............................................................. 44 accidental ............................................................. 2
-FLUOS Staining Kit .......................................... 44 and cytotoxicity ................................................... 2
Anti- programmed ........................................................ 2
BrdU ................................................................. 105 Cell Death Detection ELISAPLUS ............................ 15
DNA .................................................................... 15 Cell-mediated cytotoxicity ...................................... 58
PARP ................................................................... 27 Cell proliferation
Antibody to, see Anti-Apopain ............................. 123 assays for cell populations ................................. 82
Apoptosis assays for individual cells ................................ 100
assays for cell populations ................................. 10 assays that use tetrazolium salts ....................... 82
assays for individual cells ................................... 32 ELISA, BrdU (chemiluminescent) ................... 94
biochemical characteristics of ............................. 2 ELISA, BrdU (colorimetric) ............................. 94
definition of ......................................................... 2 Kit I (MTT) ....................................................... 85
difference between cytotoxicity and ................. 58 Kit II (XTT) ....................................................... 86
difference between necrosis and ......................... 3 overview of ........................................................ 74
hallmark of ......................................................... 10 Reagent WST-1 .................................................. 87
inducers of .......................................................... 11 Cell viability assays, see Cell proliferation assays
morphological characteristics of ......................... 3 Cellular
overview of ........................................................... 2 DNA Fragmentation ELISA .............................. 64
pathways ............................................................... 5 Ceramide, role in apoptosis .................................. 123
proteases, role of ................................................ 18 Chemiluminescent cell proliferation ELISA .......... 94
simultaneous detection of necrosis and ........... 15 Chromatin aggregation ............................................. 3
surface morphology changes during .................. 5 c-jun gene ............................................................... 123
Apoptotic DNA Ladder Kit ..................................... 13 c-myc gene .............................................................. 123
Aspartate at proteolysis site ..................................... 18 Colorimetric assays
for cytotoxicity ............................................ 61, 64
for proliferation ................................................. 94
CPP32 .................................................................... 123
B Crm A .................................................................... 123
bad gene .................................................................. 123 Cr release assay, radioactive .................................... 74
bax .......................................................................... 123 Cyclin ....................................................................... 75
BCIP ......................................................................... 42 Cysteine proteases ..................................................... 6
bcl-2 gene ................................................................ 123 Cytotoxic T cells ........................................................ 2
bcl-xL gene .............................................................. 123 Cytotoxicity
bcl-xS gene .............................................................. 123 assays ............................................................ 61, 64
Beads on a string .................................................. 11 cell-mediated ..................................................... 58
Bisbenzimidazole dye, see Hoechst dye definition of ....................................................... 58
C
5-Bromo-2-deoxy-uridine Detection Kit (LDH) ......................................... 61
Labeling and Detection Kit I ........................... 101 effectors of ......................................................... 58
Labeling and Detection Kit II .......................... 101 overview of ........................................................ 58
Labeling and Detection Kit III .......................... 91
labeling of DNA ............................................... 100
incorporation assay .......................................... 100
release assay ........................................................ 74
Bromo-deoxy-uridine, see 5-Bromo-2-deoxy-uridine

Index
D Flow cytometry
DAB substrate .......................................................... 42 assays for apoptotic cells ............................. 36, 44
Damage/leakage of plasma membrane, assays for . 49 Formazan
DAPI ......................................................................... 49 insoluble ............................................................ 84
Deoxynucleotidyltransferase, terminal .................. 33 soluble ................................................................ 84
Dexamethasone ....................................................... 35 fos gene .................................................................. 125
DNA cleavage, see DNA fragmentation Fragmentation of DNA ........................................... 30
DNA fragmentation
during apoptosis ............................................ 6, 10
DNA fragments, histone-associated ....................... 12
G
DNA end labeling .................................................... 33
DNA ladder Glucocorticoid receptor ........................................ 125
appearance of ..................................................... 11 Granzyme .............................................................. 125
assay for .............................................................. 13
size of fragments ................................................ 11
DNA polymerase ..................................................... 32
H
DNA synthesis
assays .................................................................. 89 Hallmark of apoptosis ............................................ 10
Dye HeLa cells .............................................................. 103
exclusion assays .................................................. 49 Histone-associated DNA fragments ....................... 12
uptake ................................................................. 50 Hoechst dye ............................................................. 49
Homeostasis, loss during cell death ......................... 3
Homogeneous Caspases Assay ............................... 25
E
Effector cells for inducing cell death ...................... 66
I
ELISA
kits .......................................................... 15, 91, 94 ICE ............................................................................. 6
End labeling of DNA ............................................... 33 ICH-I ..................................................................... 127
Epidermis tissue, in vivo labeling of ..................... 104 INT .......................................................................... 61
Ethidium bromide ................................................... 49 Interleukin converting enzyme (ICE) ...................... 6
Exclusion assays, see Dye exclusion assays In situ Cell Death Detection Kit
-AP ..................................................................... 39
-POD ................................................................. 39
-Fluorescein ....................................................... 36
F In situ Cell Proliferation Kit
FADD ..................................................................... 124 -FLUOS ............................................................ 102
False positive, TUNEL ........................................... 114 In situ nick translation ............................................ 33
Fast red ..................................................................... 42 ISNT method .......................................................... 33
FIENA ...................................................................... 22
FixDenat ................................................................... 94
Fluorochrome staining assays for measuring
J
DNA loss .................................................................. 49
Flow cytometric techniques, kits for, see JAM test ................................................................... 30
Annexin V-FLUOS Staining Kit ......................... 44
In situ Cell Death Detection Kit, Fluorescein ... 36
In situ Cell Proliferation Kit, FLUOS .............. 102
L
Flow cytometric measurement
C
of Annexin V-stained cells ................................ 44 Lactate dehydrogenase, see LDH
of apoptosis ........................................................ 32 LDH
of BrdU label .................................................... 103 Cytotoxicity Detection Kit ................................ 61
of cell cycle position ........................................ 107 release assay ....................................................... 74
of ISNT method ................................................. 33 Leakage/damage of plasma membrane, assays for 49
of normal and apoptotic cells ..................... 46, 49 LMW DNA .............................................................. 12
of peripheral blood lymphocytes ...................... 34 Lymphokine-activated killer cells .......................... 58
of total DNA .................................................... 103
of TUNEL method ............................................ 38
Appendix 165
Index
M P
M30 CytoDEATH .................................................... 19 p53 pan ELISA ......................................................... 56
M30 CytoDEATH, Fluorescein ............................... 19 PARP ........................................................................ 10
Mch2, Mch3, Mch4 ................................................ 127 Peripheral blood lymphocytes
MCL-1 .................................................................... 125 proliferation of .................................................. 97
Membrane symmetry during apoptosis ................. 43 stimulation of .................................................... 96
Method selection guide Phagocytic cells ......................................................... 3
for apoptosis assay ........................................... 8, 9 Phosphatidylserine .................................................. 43
for cell proliferation assay ........................... 80, 81 Phospholipid ........................................................... 43
Microwave pretreatment for TUNEL ................... 116 Phospholipid-binding protein,
Mononucleosomes ................................................... 11 see Annexin V .......................................................... 43
MORT-1 ................................................................. 125 Plasma membrane
M-phase ................................................................... 76 damage during apoptosis .................................... 3
MTT damage during necrosis ...................................... 3
assay kit .............................................................. 85 Poly-(ADP-ribose) polymerase,
biochemical basis for reduction of .................... 82 see PARP
cellular basis for reduction of ............................ 82 Positive, false, TUNEL .......................................... 114
comparison with other tetrazolium salts .......... 84 Proliferating cells
effect of superoxide dismutase on ................... 121 assays for .................................................... 85 87
structure of ......................................................... 83 increased metabolic activity in ......................... 82
use in cell proliferation assay ............................ 82 Propidium iodide
use in cytotoxicity assay ..................................... 59 exclusion assay ............................................. 44, 47
properties ........................................................... 49
Proteases in apoptosis ............................................. 18
Proteinase K pretreatment for TUNEL ................ 116
N Proto-oncogene ....................................................... 54
Natural killer cells ................................................ 2, 58
NBT .......................................................................... 42
Necrosis
definition of ......................................................... 2 Q
difference between apoptosis and ....................... 3 Questions frequently asked about cell death
difference between cytotoxicity and ................. 58 assays ...................................................................... 112
inducers of ............................................................ 3
inflammation during ........................................... 3
overview of ........................................................... 3
secondary ............................................................. 3 R
NEDD ..................................................................... 125 Radioactive assays
NF-kappa B ............................................................ 125 for apoptosis ...................................................... 30
Nick translation ....................................................... 33 for cell proliferation .......................................... 98
Nonradioactive assays for DNA fragmentation .................................... 30
for apoptosis ....................................................... 30 Ras .......................................................................... 126
for cell proliferation ........................................... 98 Reduced metabolic activity, assay for ..................... 68
for DNA fragmentation ..................................... 30 RIP ......................................................................... 126
Nucleosome quantification ELISA ......................... 30
C
Oligonucleosomes ................................................... 11

Index
S U
S-phase ..................................................................... 76 Uptake of dyes by dead cells ................................... 50
Staining of DNA ...................................................... 52
Storage of samples for apoptosis assay ................. 112
Streptavidin conjugates ........................................... 48
Sub-G1 peak ......................................................... 49 V
Succinate-tetrazolium reductase ............................ 82 Viable cell number .................................................. 78
Surface glycoproteins .............................................. 43
Symmetry of membranes during apoptosis ........... 43
W
Water-insoluble formazan ...................................... 84
T Water-soluble formazan ......................................... 84
TdR proliferation assay ........................................... 98 WST-1
TdT ........................................................................... 33 assay ................................................................... 87
Terminal deoxynucleotidyl transferase .................. 33 biochemical basis for reduction of ................... 82
Tetrazolium salt cellular basis for reduction of ........................... 82
See also MTT, WST-1, XTT comparison with other tetrazolium salts ......... 84
mitochondrial reduction and effect of reducing agents on ............................ 121
use in cell proliferation assays ........................... 82 effect of superoxide dismutase on .................. 121
Thymidine release assay, radioactive ...................... 74 structure of ........................................................ 83
TRADD .................................................................. 126 use in cell proliferation assay ............................ 82
Transferase, terminal ............................................... 33 use in cytotoxicity assay .................................... 59
Trypan blue exclusion assay .................................... 49
Two-color assay for dead and viable
apoptotic cells ........................................................ 119
TUNEL X
AP ....................................................................... 42 XTT
definition of ....................................................... 33 assay kit .............................................................. 86
diminished staining during DNA biochemical basis for reduction of ................... 82
counterstaining ................................................ 116 cellular basis for reduction of ........................... 82
dilution buffer .................................................... 42 comparison with other tetrazolium salts ......... 84
effect of different fixatives on .......................... 115 effect of reducing agents on ............................ 121
effect of pretreatments on ............................... 116 effect of superoxide dismutase on .................. 121
enzyme ............................................................... 42 structure of ........................................................ 83
evaluation of, for in situ apoptotic cell use in cell proliferation assay ............................ 82
identification .................................................... 113 use in cytotoxicity assay .................................... 59
false positives in ............................................... 114
high background in ......................................... 115
improvement of, for in situ apoptotic cell
identification .................................................... 113 Y
kits for ........................................................ 36 39 YAMA .................................................................... 126
label .................................................................... 42
low labeling in .................................................. 116
nonspecific labeling in ..................................... 115
no signal in ....................................................... 116
optimization of ................................................ 115
overview of ......................................................... 32
C
POD .................................................................... 42
pretreatments for ............................................. 116
protocol for tissues which tend to give false
positives ............................................................ 114
single reagents for .............................................. 42
special applications of ..................................... 119
specificity of ....................................................... 33
tips for avoiding or eliminating potential
artifacts in ........................................................ 115
Appendix 167
Apoptosis on the Internet
http://www.roche-applied-science/apoptosis
C
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Apoptosis, Cell Death and

Preparations with hazardous substances

Preparations of human origin

Cover and Section Dividers

Apoptosis, Cell Death, and

A Cell Death Apoptosis and Necrosis Page

II Apoptosis, Cell Death, and Cell Proliferation Manual

B Cell Proliferation and Viability Page

1. Technical Tips ................................................................................................................................................. 112

IV Apoptosis, Cell Death, and Cell Proliferation Manual

Overview of this Guide

CED3 is the prototype of a family of around a dozen mammalian proteases, called

VI Apoptosis, Cell Death, and Cell Proliferation Manual

A second mitochondrial protein of enormous significance in apoptosis is BCL2, a mam-

Impressive confirmation of the significance of these pathways to apoptosis is available

Andrew H. Wyllie FRS,

VIII Apoptosis, Cell Death, and Cell Proliferation Manual

Necrosis and apoptosis

Apoptosis (normal or programmed cell death) is the physiological process by which

Figure 1: Illustration of the morphological features of necrosis and apoptosis.

2 Apoptosis, Cell Death, and Cell Proliferation Manual

1.2 Differences between necrosis and apoptosis

Cells undergoing apoptosis show characteristic morphological and biochemical features6.

Cell Death Apoptosis and Necrosis 3

4 Apoptosis, Cell Death, and Cell Proliferation Manual

1.3 Apoptotic Pathways

Key elements of the apoptotic pathway include:

Cell Death Apoptosis and Necrosis 5

6 Apoptosis, Cell Death, and Cell Proliferation Manual

2 Apoptosis Assay Methods

Radioactive and non-radioactive assays that measure increases in plasma membrane

Colorimetric assays that measure reduction in the metabolic activity of mitochondria;

However, as more information on apoptosis became available, researchers realized that

Fragmentation of DNA in populations of cells or in individual cells, in which apop-

Alterations in membrane asymmetry. Phosphatidylserine translocates from the cyto-

Release of cytochrome C and AIF into cytoplasm by mitochondria.

Cell Death Apoptosis and Necrosis 7

Are you studying

Are you studying

cell populations single cells

Do you analyze data by

Fluorescence microplate Reader

Do your cells proliferate in vitro?

Do your cells proliferate in vitro?

Cytotoxicity Detection Kit (LDH) 11 644 793 001L

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cell cultures, tissue sections

DNA fragmentation Proteolytic activity

2.1 Methods for studying apoptosis in cell populations

2.1.1 Assays that measure DNA fragmentation

The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, an

Radioactive as well as non-radioactive methods to detect and quantify DNA fragmenta-

10 Apoptosis, Cell Death, and Cell Proliferation Manual

Note: Because cell-mediated cytotoxicity (CMT) proceeds, at least in part, by apoptotic

Cell Death Apoptosis and Necrosis 11

Apoptosis Cell mediated Note: In the early phases of apopto-

Table 2: Distribution of HMW and LMW DNA in cells under-

An alternative method which circumvents the isolation and electrophoretic analysis of

12 Apoptosis, Cell Death, and Cell Proliferation Manual

Apoptotic DNA Ladder Kit

Type DNA purification kit

Figure 8: Sensitivity of Cell Death Detection

Figure 9: Dose-response experiment analyzed by