|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

CONTENTS
I. Introduction 1
II. HaloTag™ Interchangeable Labeling Technology 1
A. Overview 1
B. Overview of Imaging Protocols Using the HaloTag™
Technology 3
C. Live-Cell Imaging Using the HaloTag™ Technology 4
D. Fixed-Cell Imaging Using the HaloTag™ Technology 6
E. Multiplexing Multicolor Live- and Fixed-Cell Imaging
Experiments 7

III. Monster Green® Fluorescent Protein phMGFP

Vector 7
A. Overview 7
IV. Antibodies and Other Cellular Markers 9
A. Antibodies and Markers of Apoptosis 9
B. Antibodies and Cellular Markers for Studying Cell
Signaling Pathways 13
C. Marker Antibodies 14
D. Other Antibodies 16
V. Fluorescent Dyes 16
A. Amine-Reactive Fluorophores 16
B. Benzofurazans 18
C. Calcein, Acetoxymethyl Ester 18
D. CTC—MaxxPure™ Grade 18
E. Quenchers 18
F. Stains 18
G. Thiol-Reactive Probes 19
H. UV-Excitable Fluorophores 19
I. Xanthenes 20
VI. References 20

Protocols & Applications Guide

www.promega.com
rev. 9/06
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

I. Introduction
Researchers are increasingly adding imaging analyses to
their repertoire of experimental methods for understanding
the structure and function of biological systems. New
methods and instrumentation for imaging have improved
resolution, signal detection, data collection and
manipulation for virtually every sample type. Live-cell and
in vivo imaging have benefited from the availability of
reagents such as vital dyes that have minimal toxicity, the
discovery of intrinsically fluorescent proteins (IFPs;
Stephens and Allan, 2003; Sullivan and Kay, 1999) and the
development of imaging systems that do not damage
biological samples (Stephens and Allan, 2003). Techniques
such as fluorescent analog chemistry have allowed scientists
to study the cytoskeleton in living cells, to follow the
recycling of cell-surface components, and to look at
movement and distribution of cellular proteins (Wang,
1989). Additionally, techniques for culturing cells on the
microscope stage and maintaining a constant focal plane
continue to improve (Stephens and Allan, 2003; McKenna
and Wang, 1989) so that cellular processes can be followed
in real time. New technologies such as magnetic resonance
imaging have allowed in vivo imaging of processes, such
as brain activity, in whole organisms (Check, 2005).
Here we present a summary of reagents that researchers
can use for imaging studies in virtually any field of
investigation. The HaloTag™ Interchangeable Labeling
Technology allows researchers to image live cells, multiplex
live-cell imaging experiments with immunocytochemistry,
or potentially immunohistochemistry, and perform
multicolor imaging experiments. We also present the
Monster Green® Fluorescent Protein, an engineered
intrinsically fluorescent protein (IFP) with improved signal
and expression for imaging studies in mammalian cells.
Additionally, we describe antibody reagents that label
protein markers of specific cell types, such as the Anti-βIII
Tubulin mAb. For researchers dissecting apoptosis, imaging
reagents for multiple points in the pathway are also
discussed, including phosphorylation-specific antibody to
caspase-3, an antibody against the p85-fragment of PARP,
and an anti-cytochrome c. Phosphorylation-specific
antibodies for studying a variety of cell-signaling pathways
are also presented. Finally, we describe the wide variety of
fluorescent dyes available from Promega.
conditions, is highly specific and essentially irreversible,
yielding a complex that is stable even under denaturing
conditions. The ability to create labeled HaloTag™ fusion
proteins with a wide range of optical properties allows
researchers to image and localize labeled HaloTag™ fusion
proteins in live- or fixed-cell populations.
Components of the HaloTag™ Technology
The HaloTag™ Protein is a genetically engineered
derivative of a hydrolase that efficiently forms a covalent
bond with the HaloTag™ Ligands. This 33kDa monomeric
protein can be used to generate N- or C-terminal fusions
that can be expressed in a variety of cell types (Figure 10.1).
Since the HaloTag™ Protein is of prokaryotic origin,
endogenous activities are absent from mammalian cells.
The HaloTag™ Protein is encoded by a variety of vectors
that allow construction of fusion proteins.
The HaloTag™ pHT2 Vector (Cat.# G8241) contains: a CMV
enhancer/promoter for strong, constitutive expression, a
chimeric intron to minimize the use of cryptic 5´-donor
splice sites, a T7 promoter for use with in vitro transcription
and/or translation systems, the sequence encoding the
HaloTag™ Protein, and an SV40 late polyadenylation signal
(Figure 10.2).
In addition to the features above, the pFC8A and pFC8K
(HaloTag™) CMV Flexi® Vectors (Cat.# C3631, C3641)
contain a linker sequence for N-terminal fusion to the
HaloTag™ Proteins. Additionally, the convenience of the
Flexi® Vector design allows users to easily transfer the
construct between a variety of vector backbones (Figures
10.3 and 10.4).
HaloTag™ Ligands are small chemical tags that are capable
of covalently labeling the HaloTag™ Protein. These ligands
contain two crucial components: 1) a common HaloTag™
Reactive Linker that ensures formation of a covalent bond
with the HaloTag™ Protein, and 2) a functional group such
as the fluorescent dyes TMR, diAcFAM and Coumarin.
Affinity tags such as biotin are available too (Figure 10.5).

II. HaloTag™ Interchangeable Labeling Technology

A. Overview
The HaloTag™ Interchangeable Labeling Technology
provides new options for rapid, site-specific labeling of
HaloTag™ fusion proteins in living cells and in vitro
(Animation (www.promega.com
/paguide/animation/selector.htm?coreName=halotag01)).
The technology is based on the formation of a covalent
bond between the HaloTag™ Protein and synthetic ligands
that carry a variety of functionalities, including fluorescent
labels, affinity tags and attachments to solid phase. The
covalent bond forms rapidly under physiological

Protocols & Applications Guide

www.promega.com
10-1
rev. 9/06
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

CMV Enhancer/
Promoter
cer

intron T7
ori
Sgf I
pFC8A (HaloTag™) CMV
Flexi® Vector Barnase
(5044bp)
EcoICR I
Factor Xa

Ampr
SV40 Late
poly(A)
HaloTag™ Open

5291MA
Reading Frame

Figure 10.3. pFC8A (HaloTag™) Flexi® Vector Map.

CMV Enhancer/
Promoter
cer

intron T7
ori
Sgf I
pFC8K (HaloTag™) CMV
Flexi® Vector Barnase
(5039bp)
EcoICR I
Factor Xa

Kanr
SV40 Late
poly(A)
HaloTag™ Open

5292MA
Figure 10.1. Molecular model of the HaloTag™ Protein with a Reading Frame
covalently bound HaloTag™ TMR Ligand. Overview of the
protein structure (top right) with close-up of the ligand tunnel Figure 10.4. pFC8K (HaloTag™) Flexi® Vector Map.
outlined by a mesh Connolly surface (lower left). The HaloTag™
TMR Ligand (fluorescent moiety in red, reactive linker in orange)
is shown covalently bound to the aspartate nucleophile (shown in
blue). Replacement of the catalytic base (histidine) with a
phenylalanine (also shown in blue) makes the protein incapable
of hydrolyzing the formed covalent bond, leading to the formation
of a stable covalent bond.

CMV Immediate Early

Enhancer/Promoter
ori
Chimeric
Intron T7 EEV (forward)
HaloTag™ sequencing primer
pHT2 Vector binding site
(4924bp) T7 Promoter
HT2 Nhe I
Ampr coding
sequence
Pvu II
EcoR V
HaloTag™
Start Codon
BamH I
SV40 Late Nae l
poly(A) signal HaloTag™ Stop Codon
Pac I
4785MA

Not I

Figure 10.2. HaloTag™ pHT2 Vector Map.

Protocols & Applications Guide

www.promega.com
10-2
rev. 9/06
||||||||||
Figure 10.5. Structure of the HaloTag™ Ligands showing the Functional Reporter and Reactive Linker. All HaloTag™ Ligands have the
same chloroalkane Reactive Linker. The ligands differ in the Functional Reporter and in the distance of the reporter from the linker.
B. Overview of Imaging Protocols Using the HaloTag™ Technology
The interchangeability of the ligands facilitates imaging at
different wavelengths without changing the underlying
genetic construct. Figure 10.6 outlines the HaloTag™
Labeling strategy. This example protocol is intended to
serve as a guide. You should empirically optimize the cell
culture protocol, transfection conditions, ligand
concentration and labeling protocol for your experimental
system.
• HaloTag™ pHT2 Vector or HaloTag™ Flexi® Vectors
and desired ligands: diAcFAM Ligand, TMR Ligand
or Coumarin Ligand and protocol #TM260
(www.promega.com/tbs/tm260/tm260.html)
• chambered cover glass
• transfection reagent
• endotoxin-free (transfection-grade) plasmid DNA
• fetal bovine serum (FBS)
• serum-free cell culture medium
• PBS (37°C)
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
• wide-field fluorescent microscope equipped with
standard FITC, TRITC and/or DAPI/AMC filters or
confocal microscope equipped with a lasers and filter
sets appropriate for each fluorescent ligand
• 37°C cell culture incubator
• 4% paraformaldehyde containing 0.5M sucrose
• Triton® X-100
• 0.1% sodium azide/PBS solution
The following protocol was used for HeLa cells (ATCC
#CCL-2) cultured in DMEM/F12 containing 10% FBS and
no antibiotic (growth medium) on 8-well Lab-Tek® II
chambered cover glass (Nalge Nunc Cat.# 155409) at 37°C,
5% CO2. The cells were transfected with the HaloTag™
pHT2 Vector using a lipid transfection reagent according
to the manufacturer’s directions.
10-3
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

Day 1: Plating Cells
1. Plate cells at a seeding density of 7.5–10 × 103 cells/cm2
(9–12 × 103 cells/well) in 400µl growth medium. Allow
cells to grow using standard conditions (37°C, 5% CO2)
to ~85% confluence (~24–48 hours).
Day 2: Transfecting Cells
2. Transfect cells according to the manufacturer’s
instructions for the transfection reagent that you are
using. See the Technical Manual for more information
about standard transfection methods.
3. Proceed with labeling protocol 24 hours after adding
transfection reagent.
Day 3: Labeling Cells with HaloTag™ TMR, diAcFAM, or
Coumarin Ligand
4. Prepare a 1:500 dilution of HaloTag™ TMR, diAcFAM
or Coumarin Ligand stock solution in 37°C growth
medium.
5. Remove all but 100µl of the growth medium from each
well. Cells should still be covered by medium.
6. Add 100µl medium containing the HaloTag™ Ligand
to each well. The final recommended working
concentration for the HaloTag™ TMR Ligand is 5µM.
For HaloTag™ diAcFAM it is 1–10µM, and for the
Coumarin Ligand the recommended working
concentration is 10µM.
Note: To avoid serum-induced hydrolysis of the
HaloTag™ diAcFAM Ligand, add the Ligand to the
cells immediately after diluting it.
Imaging Live Cells (Day 3 continued)
Note: Incubation temperatures are critical for sucessful
live-cell imaging experiments.
2. Remove the ligand-containing medium and rinse the
cells with 400µl/well PBS (37°C).
3. Replace the PBS with 400µl freshly prepared 4%
paraformaldehyde containing 0.5M sucrose at 37°C.
4. Incubate for 10 minutes at 37°C, 5% CO2 in the dark.
5. Replace the fixative with PBS containing 0.1% Triton®
X-100. 6. Incubate for 30 minutes at 37°C, 5% CO2 in
the dark.
6. Replace the Triton® X-100 solution with 400µl PBS.
7. Transfer the chambered cover glass to a microscope
stage and capture images. (Labeled cells may be stored
in a 0.1% sodium azide/PBS solution and protected
from light. Mounted slides may be stored at room
temperature, protected from light.)
C. Live-Cell Imaging Using the HaloTag™ Technology
The HaloTag™ Ligands and expression of the HaloTag™
Protein have shown no detectable toxicity or morphological
side effects at the recommended labeling conditions in the
cell lines tested (e.g., HeLa and CHO-K1). This characteristic
allows imaging of live cells over long periods of time,
including times required for studying phenomena such as
the cell cycle, cell differentiation, long-term effects of drugs
and other applications. Figure 10.7 presents images of HeLa
cells transiently transfected with the p65-HaloTag™ fusion
protein and labeled with the TMR Ligand. Images were
taken every 20 minutes for eight hours and 20 minutes.
Cells have unaltered morphology at all time points
examined. Importantly, expression of the p65-HaloTag™
fusion protein and treatment with the HaloTag™ TMR
Ligand have no effect on complex cellular functions. For
example, the images in Figure 10.7 clearly depict a cell in
the process of dividing (arrow).

1. Incubate the cells with the desired HaloTag™ Ligand

for 15 minutes at 37°C, 5% CO2 in the dark.

2. Remove the ligand-containing medium.

3. Rinse the cells with 0.5ml/well PBS (37°C). Repeat two

times for a total of three rinses.

4. Replace the PBS with fresh growth medium (37°C), and

return the cells to the incubator for 30 minutes (37°C,
5% CO2).

5. Replace the growth medium with 400µl of PBS (37°C)

or growth medium without phenol red (37°C).

6. Transfer the chambered cover glass to a microscope

and capture images.

Imaging Fixed Cells (Day 3 continued)

1. Incubate the cells with the desired HaloTag™ Ligand
for 15 minutes at 37°C, 5% CO2 in the dark.

Protocols & Applications Guide

www.promega.com
10-4
rev. 9/06
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

Day 1: Plate cells.
Day 2: Introduce HaloTag™
genetic construct into
cells using standard
transfection techniques.

Day 3: For PEG-Biotin Ligand or

HaloLink™ Resin, lyse
cells prior to labeling. Lyse cells.

Label cells with

appropriate HaloTag™
Ligand.

Wash unbound ligand

from sample.
Add HaloLink™
Resin and wash,
or
Add HaloTag™
PEG-Biotin Ligand.

Fix cells. Lyse cells.

Image fixed cells. Image live cells.

Excitation light Excitation light Proceed with

Capture protein. downstream
Fluorescence Fluorescence
applications:
Protein:protein
interactions,
enzyme assays
or purification of
nontagged protein.
Fixed cell imaging Live cell imaging Capturing of biotinylated
of fluorescently of fluorescently HaloTag™ Fusion Protein
labeled HaloTag™ labeled HaloTag™ and protein complexes on
Fusion Protein Fusion Protein a solid substrate such as
a streptavidin-coated particle

Proceed with
other analyses
HaloTag™ Protein
(e.g., cell-to-gel,
5495MA

pulse-chase). HaloTag™ Ligand

Figure 10.6. Overview of protocol for live-cell and fixed-cell imaging using the HaloTag™ Technology.

Protocols & Applications Guide

www.promega.com
10-5
rev. 9/06
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

The HaloTag™ gene can be fused to another gene encoding
a protein or target sequence of interest that directs the
fusion protein to a specific subcellular compartment. To
0 minutes
demonstrate this capability, we generated constructs
encoding a p65-HaloTag™ Protein chimera.
HeLa cells were transfected with the plasmid encoding the
p65 HaloTag™ fusion protein and labeled with the
4 hours HaloTag™ TMR Ligand (Figure 10.8). The p65 protein (also
20 minutes known as RelA and NF-κB3) is a member of the eukaryotic
nuclear factor κB (NF-κB) protein family. The NF-κB factor
is expressed in many cell types and plays an important role
in inflammation, autoimmune response and apoptosis by
regulating the expression of genes containing the
4 hours localization sequence (NLS), which is rendered inactive in
40 minutes nonstimulated cells through the binding of specific NF-κB
inhibitors known as the IκB proteins. Binding of IκB masks
the NLS, which leads to retention of the NF-κB proteins
(including p65) in the cytoplasm of the cell (Ghosh et al.
1998; Karin et al. 2004; Burstein and Duckett, 2003). As
5 hours expected in the nonstimulated cells, the p65-HaloTag™
20 minutes fusion protein is excluded from the nucleus and shows a
diffuse staining. Figure 10.11 further demonstrates the use
of HaloTag™ Technology to localize a protein at the
subcellular level. In this case, HaloTag™ Protein was
targeted to the nucleus by an NLS.
6 hours
20 minutes

6 hours
40 minutes

4882TA
Figure 10.8. Cytosolic localization of the p65-HaloTag™ fusion
protein labeled with HaloTag™ TMR Ligand. HeLa cells
transiently transfected with vector encoding the p65-HaloTag™
7 hours fusion protein were labeled with 5µM HaloTag™ TMR Ligand
(Left panel) for 15 minutes at 37°C according to the protocol
described in Technical Manual #TM260. Images were generated
on an Olympus FV500 confocal microscope using filter sets for
TMR fluorescence or transmitted light (Right panel).

8 hours D. Fixed-Cell Imaging Using the HaloTag™ Technology

20 minutes The stability of the covalent bond between the HaloTag™
Protein and the HaloTag™ Ligands allows users to image
4884TA

fixed cells. The resistance of the fluorescent signal to cell

Figure 10.7. HeLa cells expressing the p65-HaloTag™ fusion
protein and labeled with the HaloTag™ TMR Ligand. HeLa
cells transiently transfected with plasmid encoding the
p65-HaloTag™ fusion protein were labeled with 5µM HaloTag™
TMR Ligand for 15 minutes at 37°C according to the protocol
described in Technical Manual #TM260. Images were generated
on an Olympus FV500 confocal microscope using filter sets for
TMR or transmitted light. Images were collected every 20 minutes
over a period of 8 hours and 20 minutes.
fixatives also allows users to multiplex the HaloTag™
Technology with different immunocytochemical and
immunohistochemical techniques (see Section III.E). Figure
10.9 shows HeLa cells transiently expressing a
p65-HaloTag™ fusion protein labeled with the HaloTag™
TMR Ligand, fixed and counterstained with Anti-βIII
Tubulin Antibody. The transfected cells labeled with the
TMR Ligand remain brightly fluorescent after fixation.

Protocols & Applications Guide

www.promega.com
10-6
rev. 9/06
||||||||||
A B
4883TA
C D
Figure 10.9. Fixed cells expressing p65-HaloTag™ Protein labeled
with HaloTag™ TMR Ligand. HeLa cells transiently transfected
with plasmid encoding the p65-HaloTag™ TMR Ligand for 15
minutes at 37°C according to the protocol described in the
Technical Manual (#TM260). Cells were fixed with 3.7%
paraformaldehyde, stained with mouse Anti-βIII Tubulin Antibody
at 1µg/ml followed by incubation with Alexa
Fluor™-488-conjugated goat-antimouse IgG (Molecular Probes).
Images were generated on an Olympus FV500 confocal microscope
in sequential mode using appropriate filter sets for TMR, Alexa
Fluor™-488 fluorescence or transmitted light. Panel A. TMR
fluorescence. Panel B. Alexa Fluor™-488 fluorescence. Panel C.
Overlaid Alexa Fluor ™ -488 and TMR fluorescence and
transmitted light. Panel D. Overlaid AlexaFluor™-488 and TMR
fluorescence.
E. Multiplexing Multicolor Live- and Fixed-Cell Imaging
Experiments
The HaloTag™ Technology can simplify
multicolor/multiplex labeling experiments. The HaloTag™
Protein is not an intrinsically fluorescent protein (IFP), and
the choice of fluorescent labels, including secondary and
tertiary fluorophores, can be made after creating the
HaloTag™ fusion protein. This feature allows flexibility in
experimental design for multicolor labeling experiments.
We labeled cells expressing HaloTag™-α-tubulin with the
TMR Ligand or the diAcFAM Ligand and processed the
cells for ICC using Anti-βIII Tubulin mAb (Cat.# G7121)
and Alexa Fluor™-488 or Cy®3-conjugated secondary
antibodies (Figure 10.10). All HeLa cells expressed
βIII-tubulin in the cytoplasm. The HaloTag™-α-tubulin
reporter was localized to the cytoplasm in the
subpopulation of successfully transfected cells.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
Additional Resources for HaloTag™ Interchangeable
Labeling Technology
Technical Bulletins and Manuals
TM260 HaloTag™ Interchangeable Labeling
Technology Technical Manual
(www.promega.com
/tbs/tm260/tm260.html)
TM254 Flexi® Vector Systems Technical Manual
(www.promega.com
/tbs/tm254/tm254.html)
Promega Publications
PN089 HaloTag™ Interchangeable Labeling
Technology for cell imaging, protein
capture and immobilization
(www.promega.com
/pnotes/89/12416_02/12416_02.html)
CN011 HaloTag™ Interchangeable Labeling
Technology for cell imaging and protein
capture
(www.promega.com
/cnotes/cn011/cn011_02.htm)
CN012 Perform multicolor live- and fixed-cell
imaging applications with the HaloTag™
Interchangeable Labeling Technology
(www.promega.com
/cnotes/cn012/cn012_04.htm)
Vector Maps
HaloTag™ pHT2 Vector Map (www.promega.com
/figures/popup.asp?fn=4785MA)
pFC8A (HaloTag™) CMV Flexi® Vector Map
(www.promega.com/figures/popup.asp?fn=5291ma)
pFC8K (HaloTag™) CMV Flexi® Vector Map
(www.promega.com/figures/popup.asp?fn=5292ma)
HaloTag™ FAQ (www.promega.com/faq/halotag.html)
III. Monster Green® Fluorescent Protein phMGFP Vector
A. Overview
With the discovery of intrinsically fluorescent proteins
(IFPs) such as the green fluorescent protein (GFP) and the
subsequent creation of a full-color spectrum of these IFPs,
researchers can now fuse a protein of interest to IFPs with
a variety of properties. IFPs are commonly used reporter
molecules that can be visualized without cell lysis using
standard fluorescence microscopy. They are often used to
monitor gene expression but also can be used to monitor
intracellular protein trafficking by creating C- and
N-terminal protein fusions.
The Monster Green® Fluorescent Protein (Cat.# E6421) is
an improved synthetic version of the green fluorescent
protein gene originally cloned from Montastrea cavernosa
(Great Star Coral). The synthetic gene (hMGFP) expresses
a 26kDa protein that shows improved fluorescence intensity
compared to the native gene. Peak excitation occurs at
505nm, and peak emission occurs at 515nm. Standard FITC
filters may be used to visualize hMGFP fluroescence. The
10-7
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

A. B. C.

D. E. F.

4986TA
Figure 10.10. Multiplexing HaloTag™ labeling and immunocytochemistry. HeLa cells were transfected with HaloTag™-α-tubulin fusion
protein, labeled with HaloTag™ diAcFAM (Panels A–C) or TMR Ligand (Panels D–F), washed and fixed as described in Los et al. 2005.
Cells were permeabilized with 0.1% Trition® X-100 and immunolabeled with Anti-βIII tubulin mAb (1:5,000 dilution, Cat.# G7121). Cells
were incubated with 1:500 dilutions of Alexa Fluor™ -488-conjugated secondary antibody (Panels A–C) or Cy®3-conjugated secondary
antibody (Panels D–F). Panels A and D show cells labeled with the HaloTag™ Ligands only; Panel A, diAcFAM Ligand; Panel D, TMR
Ligand. Panels B and E show labeling for βIII tubulin. Panels C and F show double staining for the HaloTag™ Protein and βIII tubulin.

hMGFP gene is codon optimized and cleared of most

consensus sequence transcription factor binding sites to
ensure reliability and high levels of expression.
The Monster Green® Fluorescent Protein encoded by the
hMGFP gene is an ideal fluorescent reporter, providing
high-level fluorescence and reduced cytotoxicity. Monster
Green® Protein generally fluoresces at least 20% brighter
than other commercially available green fluorescent
proteins (GFPs) and also reduces cytoxicity, offering
flexibility when working with transient and stable
expression assays (Figure 10.11).
Additional Resources for the Monster Green® Fluorescent
Protein
Technical Bulletins and Manuals
TB320 Monster Green® Fluorescent Protein phMGFP
Vector Technical Bulletin
(www.promega.com/tbs/tb320/tb320.html)
Promega Publications
CN007 Transfecting a human neuroblastoma cell
line with Monster Green® Fluorescent
Protein
(www.promega.com
/cnotes/cn007/cn007_14.htm)
PN084 Monster Green® Protein: a brighter,
longer-expressing green fluorescent
protein
(www.promega.com
/pnotes/84/10705_12/10705_12.html)
FAQ Monster Green® Fluorescent Protein
phMGFP Vector FAQ
(www.promega.com
/faq/monstergreen.html)
Vector Maps
phMGFP Vector Map (www.promega.com
/figures/popup.asp?fn=3898ma)
Citations
Inman, M. et al. (2004) Identification of a novel bovine
herpesvirus 1 transcript containing a small open reading
frame that is expressed in trigeminal ganglia of latently
infected cattle. J. Virol. 78, 5438–47.
A fusion protein construct was made using the Monster
Green® Fluorescent Protein phMGFP Vector and the
PCR-amplified Open Reading Frame E (ORF-E) from Bovine
herpesvirus 1. Transfected human neuroblastoma
(SK-N-SH) cells, rabbit skin cells and bovine kidney cells
were visualized using an Olympus FV500/BX60 confocal
microscope with 488nm excitation laser and 522nm
emission filter set. The ORF-E-MGFP protein was localized
in discrete domains within the nucleus of Neuro-2A and
SK-N-SH cells. In rabbit skin cells and bovine kidney cells
the ORF-E-MGFP protein was detected in the cytoplasm
and nucleus.
PubMed Number: 15113922

Protocols & Applications Guide

www.promega.com
10-8
rev. 9/06
||||||||||
A. B.
C. D.
E. F.
4990TA
Figure 10.11. Expression of Monster Green® Fluorescent Protein
in HeLa Cells. HeLa cells were transiently cotransfected with
either HaloTag™-(NLS)3 plus hMGFP-α-tubulin (Panels A–D) or
HaloTag™-α-tubulin plus hMGFP-(NLS)3 (Panels E and F).
Twenty-four hours later, cells expressing HaloTag™-(NLS)3 were
incubated with either 25µM Coumarin Ligand (Cat.# G8581, Panels
A and B) or 5µM HaloTag™ TMR Ligand (Panels C and D) for
15 minutes; cells expressing HaloTag™-α-tubulin were incubated
with 5µM TMR Ligand for 15 minutes at 37°/5%CO2 (Panels E
and F). Cells were washed and incubated for 30 minutes. In Panels
A and B, cells were imaged with an Olympus IX81 epifluorescent
microscope equipped with Chroma filter sets (#31000 DAPI for
the Coumarin Ligand and #41001 FITC for hMGFP), a Hamamatsu
Orca CCD camera and environmental controls. Images in Panels
C–F were captured with the Olympus FV500 confocal attachment
with sequential two-laser scanning and filters appropriate for TMR
and FITC fluorescence.
IV. Antibodies and Other Cellular Markers
Promega offers a variety of antibodies for detecting markers
of apoptosis, assessing activation of cellular signaling
pathways, and monitoring indicators of cell type. These
antibodies include antibodies raised against phosphorylated
proteins including kinases as well as antibodies against
growth factors and growth-factor receptors.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
A. Antibodies and Markers of Apoptosis
In Situ Marker for Caspase-3: FITC-VAD-FMK
CaspACE™ FITC-VAD-FMK In Situ Marker (Cat.# G7461)
is a fluorescent analog of the pan caspase inhibitor
Z-VAD-FMK
(carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-
fluoromethylketone). The fluorescein isothiocyanate (FITC)
group has been substituted for the carbobenzoxy (Z)
N-terminal blocking group to create the fluorescent
apoptosis marker. This structure allows delivery of the
inhibitor into the cell where it irreversibly binds to activated
caspases. The FITC label allows for a single-reagent addition
to assay for caspase activity in situ. The FITC-VAD-FMK
Marker is supplied as a 5mM solution in DMSO and is
intended for in situ monitoring of caspase activity by
fluorescence detection. The suggested concentration for
use in anti-Fas-treated Jurkat cell culture is 10µM.
Method for Detecting Apoptosis in Jurkat Cells
Materials Required:
• CaspACE™ FITC-VAD-FMK In Situ Marker (Cat.#
G7461, G7462)
• poly-L-lysine-coated slides
• anti-Fas mAb (Clone CH-11 MBL International Cat.#
SY-100)
• PBS
• formalin
• mounting medium
• fluorescence microscope
1. Seed Jurkat cells at 1 × 105 cells/ml and grow in
RPMI-1640 + 10% FBS in a 37°C, 5% CO2 incubator for
2–3 days until they reach a density of 5 × 105 cells/ml.
2. Prepare poly-L-lysine-coated slides. Coat each chamber
of multichamber slides with 0.01% poly-L-lysine
solution. When partially dry, rinse the slides in
NANOpure® water and then air-dry.
Poly-L-lysine-coated slides can be prepared in advance
and stored at 4°C for up to 7 days before use.
3. To induce apoptosis, add anti-Fas mAb (Clone CH-11,
MBL International Cat.# SY-100) to a final concentration
of 0.1µg/ml. Do not add to controls. Incubate for 3–4
hours at 37°C.
4. Add CaspACE™ FITC-VAD-FMK In Situ Marker to
the Jurkat cells at a final concentration of 10µM. Protect
cells from light and incubate for 20 minutes in the
incubator. Keep cells protected from light for the
remaining steps.
5. Centrifuge at 300 × g for 5 minutes.
6. Wash cells with PBS, then centrifuge at 300 × g for 5
minutes.
7. Suspend cells in PBS to 1.5 × 106 cells/ml.
10-9
||||||||||
8. Add cells to poly-L-lysine-coated slides and incubate
at room temperature for 5 minutes to allow the cells to
adhere to the poly-L-lysine.
9. Fix in 10% buffered formalin for 30 minutes at room
temperature (protected from light).
10. Rinse 3 times for 5 minutes each time in PBS.
11. Add mounting medium and coverslips to the slides.
Analyze under a fluorescence microscope.
Additional Resources for the CaspACE™ FITC-VAD-FMK
In Situ Marker
Technical Bulletins and Manuals
9PIG746 CaspACE™ FITC-VAD-FMK In Situ Marker
Product Information
(www.promega.com
/tbs/9pig746/9pig746.html)
Promega Publications
eNotes CaspACE™ FITC-VAD-FMK In Situ
Marker as a probe for flow cytometry
detection of apoptotic cells
(www.promega.com
/enotes/applications/ap0020_tabs.htm)
PN075 CaspACE™ FITC-VAD-FMK In Situ
Marker for Apoptosis: Applications for
flow cytometry
(www.promega.com
/pnotes/75/8554_20/8554_20.html)
NN016 Live/Dead Assay: In situ labeling of
apoptotic neurons with CaspACE™
FITC-VAD-FMK Marker
(www.promega.com
/nnotes/nn503/503_14.htm)
Online Tools
Apoptosis Assistant (www.promega.com/apoasst/)
Citations
Allombert-Blaise, C. et al. (2003) Terminal differentiation
of human epidermal keratinocytes involves mitochondria-
and caspase-dependent cell death pathway. Cell Death
Differ. 10, 850–2.
The CaspACE™ FITC-VAD-FMK In Situ Marker was used
at a concentration of 5µM in primary human epidermal
keratinocyte culture to visualize active caspases during cell
differentiation induced with calcium. In this experiment,
the authors cultured primary human epidermal
keratinocytes for 48 hours in 1.2mM calcium with or
without 100mM z-VAD-FMK to demonstrate specific
caspase activation and cell differentiation in
calcium-induced keratinocytes upon labeling with the
CaspACE™ FITC-VAD-FMK In Situ Marker.
PubMed Number: 12815468
Qi, H. et al. (2003) Inactivation of Cdc13p triggers
MEC-1-dependent apoptotic signals in yeast. J. Biol. Chem.
278, 15136–41.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
Apoptosis in yeast cells was detected using the CaspACE™
FITC-VAD-FMK In Situ Marker. Yeast cells were stained
with the marker at room temperature, washed and
resuspended. FACS® analysis of cells was performed with
excitation at 488nm and emission of 520–550nm.
PubMed Number: 12569108
Elbaz, M. et al. (2002) Constitutive caspase-like machinery
executes programmed cell death in plant cells. Cell Death
Differ. 9, 726–33.
In this article, the CaspACE™ FITC-VAD-FMK In Situ
Marker was used to stain tobacco plant cells induced to
undergo apoptosis.
PubMed Number: 12058273
Detecting Active Caspase-3 Using an Antibody
Anti-ACTIVE® Caspase-3 pAb (Cat.# G7481) is intended
for use as a marker of apoptosis; it specifically stains
apoptotic human cells without staining nonapoptotic cells.
All known caspases are synthesized as pro-enzymes
activated by proteolytic cleavage. Anti-ACTIVE® Caspase-3
pAb is an affinity-purified rabbit polyclonal antibody
directed against a peptide from the p18 fragment of human
caspase-3. The antibody is affinity purified using a peptide
corresponding to the cleaved region of caspase-3.
General Immunochemical Staining Protocol
Materials Required:
• Anti-ACTIVE® Caspase-3 pAb (Cat.# G7481)
• prepared, fixed samples on slides
• Triton® X-100
• PBS
• blocking buffer (PBS/0.1% Tween® 20 + 5% horse serum)
• donkey anti-rabbit Cy®3 conjugate secondary antibody
(Jackson Laboratories Cat.# 711-165-152)
• mounting medium
• humidified chamber
1. Permeabilize the fixed cells by incubating in PBS/0.2%
Triton® X-100 for 5 minutes at room temperature. Wash
three times in PBS, in Coplin jars, for 5 minutes at room
temperature.
2. Drain the slide and add 200µl of blocking buffer
(PBS/0.1% Tween® 20 + 5% horse serum). Use of serum
from the host species of the conjugate antibody (or
closely related species) is suggested. Lay the slides flat
in a humidified chamber and incubate for 2 hours at
room temperature. Rinse once in PBS.
3. Add 100µl of the Anti-ACTIVE® Caspase-3 pAb diluted
1:250 in blocking buffer. Prepare a slide with no
Anti-ACTIVE® Caspase-3 pAb as a negative control.
Incubate slides in a humidified chamber overnight at
4°C.
4. The following day, wash the slides twice for 10 minutes
in PBS, twice for 10 minutes in PBS/0.1% Tween® 20
and twice for 10 minutes in PBS at room temperature.
10-10
||||||||||
5. Drain slides and add 100µl of donkey anti-rabbit Cy®3
conjugate diluted 1:500 in PBS. (We recommend Jackson
ImmunoResearch Cat.# 711-165-152.) Lay the slides flat
in a humidified chamber, protected from light, and
incubate for 2 hours at room temperature. Wash twice
in PBS for 5 minutes, once in PBS/0.1% Tween® 20 for
5 minutes and once in PBS for 5 minutes, protected
from light.
6. Drain the liquid, mount the slides in a permanent or
aqueous mounting medium and observe with a
fluorescence microscope.
Additional Resources for the Anti-ACTIVE® Caspase-3
pAb
Technical Bulletins and Manuals
9PIG748 Anti-ACTIVE® Caspase-3 pAb Product
Information
(www.promega.com
/tbs/9pig748/9pig748.html)
Promega Publications
CN001 Immunohistochemical staining using
Promega Anti-ACTIVE® and apoptosis
antibodies
(www.promega.com
/cnotes/cn001/cn001_4.htm)
PN075 Anti-ACTIVE® Caspase-3 pAb for the
detection of apoptosis
(www.promega.com
/pnotes/75/8554_17/8554_17.html)
Online Tools
Apoptosis Assistant (www.promega.com/apoasst/)
Antibody Assistant (www.promega.com
/techserv/tools/abasst/)
Citations
Kommers, G.D. et al. (2004) Pathogenesis of six
pigeon-origin isolates of Newcastle disease virus for
domestic chickens. Vet. Pathol. 39, 353–62.
The Anti-ACTIVE® Caspase-3 polyclonal antibody was
used to immunohistochemically stain Newcastle Disease
Virus (NDV)-infected chicken spleens. Sections were
deparaffinized, peroxidase-treated and microwaved for 10
minutes to retrieve antigens. The Anti-ACTIVE® Caspase-3
polyclonal antibody was utilized and detected with a
biotinylated anti-rabbit antibody, steptavidin-phosphatase
and DAB.
PubMed Number: 12014499
Using an Antibody Against a Cleaved Caspase-3 Substrate
(Anti-PARP p85 Fragment pAb)
Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme
involved in DNA repair, is a well-known substrate for
caspase-3 cleavage during apoptosis. Anti-PARP p85
Fragment pAb (Cat.# G7341) is a rabbit polyclonal antibody
specific for the p85 fragment of PARP that results from
caspase cleavage of the 116kDa intact molecule and thus
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
provides an in situ marker for apoptosis. Each batch of
antibody is tested for use in immunostaining applications
and contains sufficient antibody for 50
immunocytochemical reactions at a working dilution of
1:100.
General Immunocytochemistry Protocol
Materials Required:
• Anti-PARP p85 Fragment pAb (Cat.# G7341)
• cells fixed on slides
• PBS
• blocking buffer (PBS/0.1% Tween® 20 + 5% horse serum)
• donkey anti-rabbit biotin conjugate (Jackson Cat.#
711-065-152) or donkey anti-rabbit Cy®3 conjugate
(Jackson Cat.# 711-165-152)
• H2O2 (if using biotin conjugate)
• DAB solution (if using biotin conjugate)
• ultrapure water
• humidified chamber
• peroxidase-labeled streptavidin (eg., KPL Cat.#
14-300-00, diluted 1µg/ml in PBS)
1. Permeabilize cells fixed on slides in 0.2% Triton®
X-100/PBS for 5 minutes at room temperature.
2. Wash in 1X PBS in coplin jars for 5 minutes at room
temperature. Repeat twice for a total of 3 washes.
3. Drain the slides and add blocking buffer (PBS/0.1%
Tween® 20 + 5% normal serum). Cover cells with
blocking buffer (200µl per slide). Lay the slides flat in
a humidified chamber and incubate for 2 hours at room
temperature.
4. Rinse once in PBS.
5. Add 100µl of the Anti-PARP p85 Fragment pAb diluted
in blocking buffer. We recommend a starting dilution
of 1:100. Include a slide with no Anti-PARP p85
Fragment pAb as a negative control. Incubate slides in
a humidified chamber overnight at 4°C.
6. The following day, wash the slides twice for 10 minutes
in 1X PBS, twice for 10 minutes in PBS/0.1% Tween®
20, and twice for 10 minutes in 1X PBS at room
temperature.
7. If the secondary antibody is a horseradish peroxidase
(HRP) conjugate, block endogenous peroxidases by
incubating with 0.3% hydrogen peroxide for 4–5
minutes at room temperature. If you are using a
different method of detection with a secondary
antibody, proceed to Step 9.
8. Wash in 1X PBS in Coplin jars for 5 minutes. Repeat
twice for a total of 3 washes.
9. Drain slides and add 100–200µl of diluted secondary
antibody to each slide. We recommend donkey
anti-rabbit biotin conjugate (Jackson Cat.# 711-065-152)
or donkey anti-rabbit Cy®3 conjugate (Jackson Cat.#
10-11
||||||||||
711-165-152) diluted 1:500 in PBS/0.1% Tween® 20. Lay
the slides flat in a humidified chamber and incubate
for 2 hours at room temperature.
10. Wash several times in 1X PBS.
11. For the biotin conjugate, drain the slides and add
100–200µl of Streptavidin-HRP solution to each slide.
Lay the slides flat in a humidified chamber and incubate
for 45 minutes at room temperature. For
HRP-conjugated secondary antibodies, proceed to Step
13. For other secondary antibodies, proceed to Step 15.
12. Wash in 1X PBS in Coplin jars for 5 minutes. Repeat
twice for a total of 3 washes.
13. Add 100–200µl of freshly made diaminobenzidine
(DAB) solution to each slide. Lay the slides flat and
incubate for ~10 minutes at room temperature.
14. Rinse the slides in NANOpure® water. Bleach is
frequently used to inactivate the DAB before disposal;
however, local requirements for hazardous waste
should be followed.
15. Drain the liquid and mount the slides in a permanent
or aqueous mounting medium (slides mounted in 70%
glycerol can be stored for several weeks at 4°C or
–20°C).
Method for Staining Postnatal Day 0 Mouse Brain,
Paraffin-Embedded Sections. (All steps are performed at
room temperature.)
Materials Required:
• Anti-PARP p85 Fragment, pAb (Cat.# G7341)
• paraffin-embedded, fixed sample
• Hemo-De® (Fisher Scientific) or xylene
• ethanol (100, 95 and 70%)
• PBS
• Triton® X-100
• H2O2
• biotin-conjugated donkey anti-rabbit pAb
• RTU ABC reagent (Vector Laboratories)
• DAB substrate kit (Vector Laboratories)
• VECTASHIELD® DAPI anti-fade Reagent (Vector
Laboratories)
1. Embed tissue in paraffin after fixation in 4%
paraformaldehyde. Six micron sections are used for
this protocol.
Note: Best results will be obtained if the animal is
perfused with fix and postfixed after dissection.
2. Deparaffinize by washing tissue 3 times for 5 minutes
each in Hemo-De® (Fisher Scientific) or xylene. Rinse
tissue sections for 2 minutes in 100% ethanol. Transfer
sections to 95% ethanol for 2 minutes, then transfer
them to 70% ethanol for 2 minutes. Finally, rinse tissue
sections 2 times for 2 minutes each in PBS.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
3. Permeabilize for 10 minutes in PBS + 0.1% Triton®
X-100.
4. Wash sections 2 times for 5 minutes each in PBS.
5. Block endogenous peroxide activity by incubating
sections in 0.3% H2O2 in PBS for 30 minutes.
6. Wash sections 2 times for 5 minutes each in PBS.
7. Block for 45 minutes in PBS + 5% donkey serum
8. Incubate with Anti-PARP p85 Fragment pAb diluted
1:50 in PBS + 1.0 % donkey serum for 60 minutes.
9. Wash sections 3 times for 5 minutes each in PBS.
10. Incubate with biotin-conjugated donkey anti-rabbit
pAb (Jackson ImmunoResearch) diluted 1:500 in PBS
for 60 minutes.
11. Wash sections 3 times for 5 minutes each in PBS.
12. Incubate in RTU (Ready To Use) ABC reagent (Vector
Laboratories) for 60 minutes.
13. Wash sections 3 times for 5 minutes each in PBS.
14. Develop with DAB substrate kit (Vector Laboratories)
for 10 minutes.
15. Wash 3 times for 5 minutes each in water.
16. Mount in VECTASHIELD® + DAPI anti-fade reagent
(Vector Laboratories).
17. Analyze samples immediately using a fluorescence
microscope.
Additional Resources for the Anti-PARP p85 Fragment
pAb
Technical Bulletins and Manuals
TB273 Anti-PARP p85 Fragment pAb Technical
Bulletin
(www.promega.com/tbs/tb273/tb273.html)
Promega Publications
PN072 Cleaved PARP as a marker for apoptosis
in tissue sections
(www.promega.com
/pnotes/72/8094_07/8094_07.html)
CN001 Immunohistochemical staining using
Promega Anti-ACTIVE® and apoptosis
antibodies
(www.promega.com
/cnotes/cn001/cn001_4.htm)
Online Tools
Apoptosis Assistant (www.promega.com/apoasst/)
Antibody Assistant (www.promega.com
/techserv/tools/abasst/)
10-12
||||||||||
Citations
Davidson, B. et al. (2003) Expression and activation of the
nerve growth factor receptor TrkA in serous ovarian
carcinoma. Clin. Cancer Res. 9, 2248–59.
Anti-PARP p85 Fragment pAb was used to stain human
peritoneal and pleural effusions.
PubMed Number: 12796393
O'Brien, M.A., Moravec, R.A. and Riss, T. (2001) Poly
(ADP-ribose) polymerase cleavage monitored in situ in
apoptotic cells. Biotechniques. 30, 886–91.
The authors demonstrate specificity of an affinity-purified
polyclonal antibody to the p85 fragment of PARP with
Western blots that show that the antibody recognizes the
85kDa (p85) fragment of PARP but not full-length PARP.
PubMed Number: 11314271
B. Antibodies and Cellular Markers for Studying Cell Signaling
Pathways
Promega provides a variety of phosphorylation-specific
antibodies for studying cell signaling pathways. These
antibodies and example protocols are discussed in detail
in the Cell Signaling Chapter (Chapter 7)
(www.promega.com/paguide/chap7.htm#title4) of this
online Protocols and Applications Guide. Brief descriptions
of these products are provided below.
Anti-ACTIVE® Phosphorylation-Specific Antibodies
The Anti-ACTIVE® phosphorylation-specific antibodies
were developed to provide an accurate measure of enzyme
activation. These antibodies specifically recognize the
active, phosphorylated form of a given kinase. The
Anti-ACTIVE® Antibodies are raised against
phosphorylated peptide sequences present in the activating
loop of a number of protein kinases. Whether used in
Western analysis, immunocytochemistry or
immunohistochemical staining, the Anti-ACTIVE® MAPK,
JNK, p38 and CaM KII Antibodies will recognize only the
active form of the enzyme.
Anti-ACTIVE® MAPK, pAb, Rabbit, (pTEpY)
This antibody is an affinity-purified polyclonal antibody
that specifically recognizes the dually phosphorylated,
active form of MAPK. The antibody is raised against a
dually phosphorylated peptide sequence representing the
catalytic core of the active ERK enzyme and recognizes the
active forms of ERK1, ERK2 and ERK7.
Anti-ACTIVE® JNK pAb, Rabbit, (pTPpY)
Anti-ACTIVE® JNK pAb is an affinity-purified polyclonal
antibody that recognizes the dually phosphorylated, active
form of cJun N-terminal protein Kinase (JNK).
Anti-ACTIVE® JNK pAb is raised against a dually
phosphorylated peptide sequence representing the catalytic
core of the active JNK enzyme. The antibody recognizes
the active forms of JNK1, JNK2, and JNK3 isoforms.
Anti-ACTIVE® p38 pAb, Rabbit, (pTGpY)
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
Anti-ACTIVE® p38 Ab, Rabbit, is an affinity-purified
polyclonal antibody that recognizes the active form of p38
kinase. The Anti-ACTIVE® p38 pAb is raised against the
dually phosphorylated peptide sequence representing the
catalytic core of the active p38 enzyme. The Anti-ACTIVE®
p38 pAb recognizes the active forms of p38α, γ, and δ
isoforms.
Additional Resources for the Anti-ACTIVE® Antibodies
Technical Bulletins and Manuals
TB262 Anti-ACTIVE® MAPK, p38 and JNK
Polyclonal Antibodies and Anti-ACTIVE®
Qualified Secondary Antibody Conjugates
(www.promega.com/tbs/tb262/tb262.html)
Promega Publications
PN069 New Anti-ACTIVE® MAPK and 'pan ERK
1/2' antibodies for Western analysis
(www.promega.com
/pnotes/69/7542_09/7542_09.html)
PN076 Technically speaking: Anti-ACTIVE®
Antibodies and MAPK signaling pathways
(www.promega.com
/pnotes/76/8840_23/8840_23.html)
PN080 Demonstration of immunohistochemical
staining using Promega's Anti-ACTIVE®
and apoptosis Antibodies
(www.promega.com
/pnotes/80/9748_20/9748_20.html)
FAQ MAPK FAQ
(www.promega.com/faq/MAPKFAQ.html)
Online Tools
Antibody Assistant (www.promega.com
/techserv/tools/abasst/)
Citations
Hsu, C.Y. et al. (2004) Characterization of active
mitogen-activated protein kinase in ovarian serous
carcinomas Clin. Can. Res. 10, 6432–6.
The Anti-ACTIVE® MAPK polyclonal antibody was used
to immunohistochemically stain and type patient ovarian
serous carcinomas using paraffin-fixed tissue sections on
tissue microarrays. Western blots were also performed on
tissue lysates using a 1:3,000 dilution of the antibody.
PubMed Number: 15475429
Le'Negrate, G. et al. (2003) Downregulation of caspases and
Fas ligand expression, and increased lifespan of neutrophils
after transmigration across intestinal epithelium Cell Death
Differ. 10, 153–62.
Anti-ACTIVE® JNK pAb was used in immunoblot analysis
of human polymorphonuclear leukocyte protein lysates.
PubMed Number: 12700643
Aballay, A. et al. (2003) Caenorhabditis elegans innate immune
response triggered by Salmonella enterica requires intact LPS
and is mediated by a MAPK signaling pathway Curr. Biol.
13, 47–52.
10-13
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

spinal cord, human and rat fetal CNS progenitor cell
Activation of the p38 homolog in the worm was monitored
cultures and adult human paraffin-embedded brain (Figure
by Western analysis using the Anti-ACTIVE® p38 pAb.
10.12).
PubMed Number: 12526744

Phosphorylation-Specific CaM KII Antibody

This antibody recognizes calcium/calmodulin-dependent
protein kinase, CaM KII, that is phosphorylated on
threonine 286. The Anti-ACTIVE® CaM KII pAb (Cat.#
V1111) was raised against the phosphothreonine-containing
peptide derived from this region.

Additional Information for the Anti-ACTIVE® CaM KII

pAb
Technical Bulletins and Manuals

2137TA
TB264 Anti-ACTIVE® CaM KII pAb, (pT286) and
Anti-ACTIVE® Qualified Secondary Antibody
Figure 10.12. Immunostaining for βIII tubulin in rat cerebellum
Conjugates Technical Bulletin using Anti-βIII Tubulin mAb. Paraffin-embedded rat brain section
(www.promega.com/tbs/tb264/tb264.html) double-immunofluorescence- labeled with the primary antibody
Promega Publications and detected using an anti-mouse Cy®3-conjugated secondary
PN067 Anti-ACTIVE® Antibody for specific antibody (yellow-green). Nuclei were stained with DAPI (blue).
detection of phosphorylated CaM KII Protocols developed and performed at Promega.
protein kinase
• Immunogen: Peptide corresponding to the C-terminus
(www.promega.com
(EAQGPK) of βIII tubulin.
/pnotes/67/7201_09/7201_09.html)
• Antibody Form: Mouse monoclonal IgG1 (clone 5G8),
FAQ MAPK FAQ 1mg/ml in PBS containing no preservatives.
(www.promega.com/faq/MAPKFAQ.html) • Specificity: Cross-reacts with most mammalian species.
BR095 Signal Transduction Resource Does not label nonneuronal cells (e.g., astrocytes).
(www.promega.com • Suggested Working Dilutions: Immunocytochemistry
/guides/sigtrans_guide/default.htm) (1:2,000), Immunohistochemistry (1:2,000), Western
Online Tools blotting (1:1,000 dilution).
Antibody Assistant (www.promega.com
/techserv/tools/abasst/) Additional Resources for β-III Tubulin MAb
Citations Promega Publications
Matsumoto, Y. and Maller, J.L. (2002) Calcium, calmodulin CN012 Perform multicolor live- and fixed-cell
and CaM KII requirement for initiation of centrosome imaging applications with the HaloTag™
duplication in Xenopus egg extracts Science 295, 499–502. Interchangeable Labeling Technology
CaM KII (281-309) was added to metaphase-arrested (www.promega.com
extracts. After adding calcium, the extracts were incubated /cnotes/cn012/cn012_04.htm)
at room temperature. Anti-ACTIVE® CaM KII pAb and NN020 CellTiter-Glo® Luminescent Cell Viability
Anti-ACTIVE® Qualified HRP secondary antibodies were Assay: Primary neurons and human
used to probe immunoblots for phospho-T286 CaM KIIα. neuroblastoma SH-SY5Y cells
PubMed Number: 11799245 (www.promega.com
/nnotes/nn020/20_05.htm)
C. Marker Antibodies NN019 Rat neurospheres express mRNAs for
Anti-βIII Tubulin mAb TrkB, BDNF, NT-3 and p75
Anti-βIII Tubulin mAb (Cat.# G7121) is a protein G-purified (www.promega.com
IgG1 monoclonal antibody (from clone 5G8) raised in mice /nnotes/nn019/19_18.htm)
against a peptide (EAQGPK) corresponding to the NN018 Specific labeling of neurons and glia in
C-terminus of βIII tubulin. It is directed against βIII tubulin, mixed cerebrocortical cultures
a specific marker for neurons. The major use of this (www.promega.com
antibody is for labeling neurons in tissue sections and cell /nnotes/nn018/018_10.htm)
culture. The antibody performs in frozen and NN018 Imaging with Promega Reagents: Anti-βIII
paraffin-embedded sections of rat brain, cerebellum and Tubulin mAb (1,439kb PDF)
(www.promega.com
/nnotes/nn018/18_8.pdf)

Protocols & Applications Guide

www.promega.com
10-14
rev. 9/06
||||||||||
NN016 Imaging with Promega Reagents: Anti-βIII
Tubulin mAb (293kb PDF)
(www.promega.com
/nnotes/nn503/503_10.pdf)
NN015 Imaging with Promega Reagents: Anti-βIII
Tubulin mAb (417kb PDF)
(www.promega.com
/nnotes/nn502/502_08.pdf)
NN014 Imaging with Promega Reagentts:
Anti-βIII Tubulin mAb (187kb PDF)
(www.promega.com
/nnotes/nn501/501_10.pdf)
Online Tools
Antibody Assistant (www.promega.com
/techserv/tools/abasst/antibodypages/antib3tubmab.htm)
Citations
Brunelli, G. et al. (2005) Glutamatergic reinnervation
through peripheral nerve graft dictates assembly of
glutamatergic synapses at rat skeletal muscle Proc. Natl.
Acad. Sci. USA 102, 8152–7.
PubMed Number: 15937120
Citations
Walker, K. et al. (2001) mGlu5 receptors and nociceptive
function II. mGlu5 receptors functionally expressed on
peripheral sensory neurons mediate inflammatory
hyperalgesia Neuropharmacology 40, 10–19.
Rat skin sections were subjected to immunohistochemistry
with the Anti-βIII Tubulin mAb to detect metabolic
glutamate receptor expressing neurons. Twenty micron
sections were fixed in acetone, permeabilized with 0.1%
Triton® X-100, and incubated with the Anti-βIII Tubulin
mAb at a final concentration of 1µg/ml.
PubMed Number: 11077066
Anti-GFAP pAb
Anti-GFAP pAb (Cat.# G5601) is a polyclonal antibody
against glial fibrillary acidic protein (GFAP), a specific
marker of astrocytes in the central nervous system and is
qualified for immunostaining applications (Figure 10.13)
• Immunogen: Purified glial fibrillary acidic protein from
bovine spinal cord.
• Antibody Form: Purified rabbit IgG; supplied at
1mg/ml in PBS containing 50µg/ml gentamicin.
• Specificity: Human, bovine and rat GFAP; not
recommended for mouse.
• Suggested Dilutions: 1:1,000 for Western blotting,
immunocytochemistry and immunohistochemistry.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
2773TA
Figure 10.13. Anti-GFAP-labeled astrocytes in mixed-rate neural
progenitor cultures. DAPI staining (blue) and Anti-GFAP pAb
with Cy®-3-conjugated secondary (red) were used. Protocols
developed and performed at Promega.
Additional Resources for Anti-GFAP pAb
Promega Publications
NN018 Specific labeling of neurons and glia in
mixed cerebrocortical cultures
(www.promega.com
/nnotes/nn018/018_10.htm)
CN001 Immunohistochemical staining using
Promega Anti-ACTIVE® and apoptosis
antibodies
(www.promega.com
/cnotes/cn001/cn001_4.htm)
NN016 Imaging with Promega Reagents:
Anti-GFAP pAb (293kb pdf)
(www.promega.com
/nnotes/nn503/503_10.pdf)
Citations
Moreno-Flores, M.T. et al. (2003) Immortalized olfactory
ensheathing glia promote axonal regeneration of rat retinal
ganglion neurons. J. Neurochem. 85, 861–71.
This paper describes the development of an immortalized
line of olfactory bulb ensheathing glia (OEG) from rat
olfactory bulbs. Immortalized lines were established by
transfection of primary OEG with the plasmid pEF321-T,
which expressed the viral oncogene SV40 large T antigen.
The starting primary cell culture was characterized by
immunocytochemistry for OEG-specfic markers such as
p75-NGFr, S100, neuroligin, vimentin and GFAP. Western
blotting of p75-NGFr and GFAP was performed on the
established cell lines to determine levels of these markers.
The Anti-GFAP pAb was used at a concentration of 1:200
for immunocytochemistry and at 1:1,000 for Western
blotting.
PubMed Number: 12716418
10-15
||||||||||
Anti-VAChT pAb
The purified Anti-VAChT (Vesicular Acetylcholine
Transporter) pAb (Cat.# G4481) is raised in goats against
a peptide (CSPPGPFDGCEDDYNYYSRS) corresponding
to amino acids 511–530 of the carboxy terminus of the
cloned rat VAChT. It is a novel tool to identify functional
cholinergic neurons in the central and peripheral nervous
system where the antibody stains fibers and neuronal cell
bodies. This antibody has application for the study of the
pathophysiology of neurodegenerative diseases involving
the cholinergic system and for mapping cholinergic neurons
in the nervous system.
• Immunogen: Carboxy-terminal peptide sequence
511–530 corresponding to cloned rat VAChT protein.
• Antibody Form: Purified goat polyclonal IgG; 0.5mg/ml
in PBS containing no preservatives.
• Specificity: Cross-reacts with VAChT in rat and mouse,
but not in human, guinea pig, rabbit or cat.
• Suggested Dilution: 1:500 for immunohistochemistry.
Additional Resources for Anti-VAChT Antibody
Promega Publications
NN010 Immunostaining with Promega Reagents:
Anti-VAChT pAb (514kb pdf)
(www.promega.com
/nnotes/nn303/303_12.pdf)
Online Tools
Antibody Assistant (www.promega.com
/techserv/tools/abasst/antibodypages/antivachtpab.htm)
D. Other Antibodies
Promega offers a variety of antibodies against growth
factors, neurotrophic factor receptors and other molecules.
Usage information for these antibodies is summarized in
Table 11.1. For additional information on these antibodies,
please visit antibody assistant (www.promega.com
/techserv/tools/abasst/).
V. Fluorescent Dyes
A. Amine-Reactive Fluorophores
The uses of amine-reactive fluorophores have been
extensively described and reviewed by Hermanson (1996).
The fluoresceins are the brightest in this family with high
extinction coefficients (>90,000) and quantum yields (>0.8);
however fluorescein is also more prone to photobleaching
and environmental effects (Chitarra et al. 2000). Proteins
labeled with these dyes (usually on lysines) have been
studied extensively by a variety of instruments (Brunner
et al. 1998; Holmes and Lantz, 2001; Truneh and Machy,
1987). Although it is not critical to use isomerically pure
carboxyxanthenes, it is usually advisable in order to
simplify interpretation of the data. It is especially critical
when labeling oligonucleotides, since even using pure dye
results in multiple oligo conformers (Vámosi et al. 1996).
Both 5-FITC (also known as "isomer I") and 6-FITC (also
known as "isomer II") are the most widely used fluorescent
amine-reactive reagents, with 5-FITC being by far the most
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
common. A literature search for "FITC" results in over 8,000
hits with uses such as FITC-labeled avidin (Yoo and
Regnier, 2004), release of encapsulated drugs using
FITC-dextran (Kim and Park, 2004) and FITC-labeled
MHCII (Van Rijt et al. 2004). A comparison of FITC and
FAM labeling showed that the degree of conjugation was
easier to control with FITC, but the stability of the conjugate
was better with FAM (Banks and Paquette, 1995).
5(6)-CFDA, SE, is a nonpolar reagent that readily crosses
cell membranes and then is subject to acetate cleavage by
intracellular esterases. The resulting carboxyfluorescein,
SE, then irreversibly labels amine groups. This reagent has
been widely used in cell division and proliferation studies
of various lymphocytes (Hodgkin et al. 1996; Fazekas de
St. Groth et al. 1999; Lyons, 1999; Parish, 1999; Fulcher and
Wong, 1999; Tesfa et al. 2003). It has also been used to study
adherence of H. pylori to gastric epithelial cells (Logan et
al. 1998), HPV particle internalization by epithelial cells
(Drobni et al. 2003), viability of cardiomyocytes
(Mueller-Stahl et al. 2003), and nerve cell grafting techniques
(Li et al. 2003).
Chemical Names
5-FAM, SE: 5-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
(6-hydroxy-3-oxo-3H-xanthen-9-yl)
benzoic acid (Cat.# P4171).
6-FAM, SE: 4-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
(6-hydroxy-3-oxo-3H-xanthen-9-yl)
benzoic acid (Cat.# P4181).
5(6)-FAM, SE: 5(or
4)-[[(2,5-Dioxo-1-pyrrolidinyl)oxy]carbonyl]-2-
(6-hydroxy-3-oxo-3H-xanthen-9-yl)
benzoic acid (Cat.# P4191).
5-TAMRA, SE:
9-[2-Carboxy-4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]phenyl]-
3,6-bis(dimethylamino)xanthylium salt (Cat.# P4201).
6-TAMRA, SE:
9-[2-Carboxy-5-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]phenyl]-
3,6-bis(dimethylamino)xanthylium salt (Cat.# P4211).
5-ROX, SE:
9-[2-Carboxy-4-[[2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]
phenyl]-2,3,6,7,12,13,16,17-octahydro-1H,5H,11H,15H-
xantheno[2,3,4-ij:5,6,7-i´j´]diquinolizin-18-ium inner salt
(Cat.# P4221).
6-ROX, SE:
9-[2-Carboxy-5-[[2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]
phenyl]-2,3,6,7,12,13,16,17-octahydro-1H,5H,11H,15H-
xantheno[2,3,4-ij:5,6,7-i´j´]diquinolizin-18-ium inner salt
(Cat.# P4231).
5-FITC: 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-
5-isothiocyanatobenzoic acid (Cat.# P4341).
6-FITC: 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-
4-isothiocyanatobenzoic acid (Cat.# P4351).
5(6)-CFDA, SE:
1-[[[3´,6´-Bis(acetyloxy)-3-oxospiro[isobenzofuran-
10-16
 Table 10.1. Additional Antibodies Available From Promega.

rev. 9/06
Application and Known Species
Antibody Species (subclass) Recommended Dilution1 Cross-Reactivity Catalog Number
Anti-Human BNDF Chicken (IgY) Western 1µg/ml; ELISA Human, mouse, rat, rabbit2 Cat.# G1641

www.promega.com
pAb 1µg/ml; ICC 1–10µg/ml; IHC and quail2
10–15µg/ml; BN 10µg/ml
||||||||||

Anti-Rat CNTF pAb Chicken (IgY) Western 1µg/ml; ELISA Rat, mouse, human and cow2 Cat.# G1631
1µg/ml; ICC 1–10µg/ml

Protocols & Applications Guide

Anti-Human GDNF Chicken (IgY) Western 1µg/ml; ELISA Human, mouse, rat and Cat.# G2791
pAb 1µg/ml; ICC2 Rhesus monkey
Anti-NGF mAb Rat (IgG) Western 1µg/ml; ELISA Human, mouse, guinea pig, Cat.# G1132; Cat.# G1131
10.5µg/ml; ICC, IHC rat, rabbit, goat, sheep, cow,
0.5–1.0µg/ml; BN not pig, horse, cat2
recommended
Anti-Human NT-3 pAb Chicken (IgY) Western 1µg/ml; ELISA Human, mouse, rat and cat2 Cat.# G1651
1µg/ml; ICC 1–10µg/ml; IHC2;
BN 1–10µg/ml
Anti-TBFβ1 pAb Rabbit (IgG) Western 1:1,000 dilution; Human, mouse, rat and cat2 Cat.# G1221
ELISA not recommended; IHC
1:50 diltuion; BN2
Anti-Human p75 pAb Rabbit (IgG) Western not recommended; IP Human, mouse, rat and Cat.# G3231
1–10µg/ml; ICC, IHC chicken (species cross-
1–10µg/ml; BN 1–10µg/ml reactivity due to high
conservation of cytoplasmic
domain)
Anti-TrkB In pAb Chicken (IgY) ICC, IHC 1–10µg/ml; BN not Human, rat and mouse2 Cat.# G1561
recommended
Anti-Pan Trk pAb Chicken (IgY) Western 1µg/ml; ELISA TrkA, TrkB and TrkC due to Cat.# G1581
1µg/ml; ICC, IHC 1–10µg/ml; the highly conserved
IP 0.5µg/ml; BN not ALAQAPPVYLDVL sequence
recommended of human, rat and mouse
Anti-Human Tryptase Mouse Western 1:10,000 dilution; Human and nonhuman Cat.# G3361
mAb Biotin ELISA 1:2,000–1:5,000 dilution; primates; marker for mast cells
ICC, IHC 1:1,000 dilution
1
Investigators should optimize contentrations for their specific applications and conditions.
2
This information was not generated by Promega scientists but has been published in the scientific literature. Please contact Promega Technical Services or refer to the Promega
Antibody Assistant (www.promega.com/techserv/tools/abasst/) for additional information.
3
Key: ICC = immunocytochemistry; IHC = immunohistochemistry; BN = biological neutralization; IP = immunoprecipitation
10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

10-17
||||||||||
1(3H),9´-[9H]xanthen]-5(or 6)-yl]carbonyl]oxy]-
2,5-pyrrolidinedione (Cat.# P5631).
B. Benzofurazans
Benzofurazans are commonly used to label amine groups
when a strong environmental sensitivity is required,
although the fluorescence quantum yields of amine adducts
in water are quite low. These compounds react with
secondary amines and are useful for labeling proline or
hydroxyproline. The compounds also react with thiols and
tend to give the same products, even though NBD fluoride
is significantly more reactive (Imai and Watanabe, 1981).
The thiol derivatives have shorter absorption and much
weaker fluorescence than the amine conjugates (Birkett et
al. 1970) and tend to migrate to nearby lysines in a
time-dependent manner (Houk et al. 1983).
Chemical Names:
NBD-Cl: 4-Chloro-7-nitro-2,1,3-benzoxadiazole (Cat.#
P4381)
NBD-F: 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (Cat.# P4391).
C. Calcein, Acetoxymethyl Ester
Calcein AM (Cat.# P1901) is a nonfluorescent molecule that
is able to penetrate cellular membranes. Nonspecific
esterases in the cytoplasm then cleave the compound to
the highly fluorescent and nonpermeable calcein. These
two characteristics make the compound well suited for
determination of cell viability using a fluorometer (Wang
et al. 1993; Lichtenfels et al. 1994) or a flow cytometer
(Weston and Parish, 1990; Papadopoulos et al. 1994). Calcein
AM is more sensitive than the annexin-phosophatidyl serine
detection reagent for detecting early apoptotic events (Gatti
et al. 1998). Calcein AM has also been used to determine
labile iron pools in mammalian cells (Epsztejn et al. 1997).
Chemical Name:
N,N´-[[3´,6´-bis(acetyloxy)-3-oxospiro[isobenzofuran-1
(3H),9´-[9H]xanthene]- 4´,5´diyl]bis(methylene)]
bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-glycine,
bis[(acetyloxy)methyl] ester.
D. CTC—MaxxPure™ Grade
CTC—MaxxPure™ (Cat.# P5511) is a tetrazolium redox
dye that produces an insoluble fluorescent formazan upon
reduction. It has been used to assess the redox activity of
various cells and organisms, including but not limited to,
Giardia cysts (Iturriaga et al. 2001), ascites tumor cells
(Stellmach, 1984; Severin et al. 1985), Micrococcus luteus
(Kaprelyants and Kell, 1993), E. coli (Severin and Seidler,
1998) and microorganisms from aquatic sediments (Gruden
et al. 2003). Techniques for visualizing fluorescence include
microscopy (Stellmach, 1984; Pyle et al. 1999), flow
cytometry (Severin et al. 1985; Kaprelyants and Kell, 1993;
Severin and Seidler, 1998; Gruden et al. 2003) and laser
cytometry (Pyle et al. 1999).
Chemical Name:
5-Cyano-2,3-bis(4-methylphenyl)-2H-tetrazolium chloride.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
E. Quenchers
These materials are widely used as quenchers in FRET
applications. The amine-reactive DABSYL chloride or
DABCYL SE can be used interchangeably as the quenching
partner (Pennington and Thornberry, 1994; Ermolieff et al.
2000; Beebe and Pei, 1998) to EDANS or 1,5-IAEDANS
because of the significant spectral overlap of the emission
of the latter with the absorption of the former. Kramer
(Marras et al. 2002) has shown that, in the case of DNA
probes, quenching may occur by more than energy transfer,
and spectral overlap is not always necessary. DABCYL
must be activated with EDAC or as the mixed anhydride
(Beebe and Pei, 1998) to react with an amine, the latter being
especially suitable when the amine is a poor nucleophile.
Chemical Names:
DABCYL Acid: 4-[[4-(Dimethylamino)phenyl]azo]benzoic
acid (Cat.# P4281).
DABCYL, SE:
1-[[4-[[4-(Dimethylamino)phenyl]azo]benzoyl]oxy]-
2,5-pyrrolidinedione (Cat.# P4291).
DABSYL Cl:
4-[[4-(Dimethylamino)phenyl]azo]benzenesulfonyl chloride
(Cat.# P4301).
F. Stains
DiI (Cat.# P5551), also known as DiIC18(3), is a member of
a large family of lipophilic dyes that show very large
extinction coefficients although relatively low quantum
yields, especially in water. The distinguishing feature
among the various DiI compounds is the length of the alkyl
side chains. These compounds are useful for staining
membranes, where they become highly fluorescent. The
DiI family is especially well suited for use with standard
rhodamine optical filters. The octadecanyl member of this
family is the most widely used (more than 150 publications)
to study molecular movement in monolayers (Czikkely et
al. 1969; Möbius, 1969), lipid probes on cell membranes
(Schlessinger et al. 1977), lipid rafts in model membranes
(Kahya et al. 2003) and liposome penetration through skin
(Verma et al. 2003; Verma et al. 2003b).
DAPI is used to stain double-stranded DNA (Dann et al.
1971; Kapuscinski and Skoczylas, 1977; Kapuscinski and
Skoczlas, 1978; Kapuscinski, 1995) and is routinely used as
a nuclear counterstain in microscopy (Sanna et al. 1992;
Netten et al. 1997). DAPI is also used in fluorescence
correlation spectroscopy (FCS) studies of DNA-DAPI
complexes (Barcellona et al. 2004).
Sometimes also known as bisbenzimide, Hoechst 33342
was originally designed as an anthelmintic agent (Loewe
and Urbanietz, 1974) but is also a sensitive stain for DNA
(Latt and Stetten, 1976), especially for intracellular
measurements (Lydon et al. 1980). More recently, the
compound has been used to determine the difference in
DNA content between X- and Y-bearing spermatozoa for
cell sorting (O'Brien et al. 2005) and to study apoptosis in
rat hepatocytes (Lee and Shukla, 2005).
10-18
||||||||||
JC-1. 5,6-Dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro
-2H- benzimidazol-2-ylidene)-1-propenyl]-1,3-
diethyl-1H-benzimidazolium iodide is a green fluorescent
molecule that exists as a monomer at low concentrations.
At higher membrane potentials, JC-1 forms J-aggregates
that are red fluorescent, showing a broad excitation
spectrum and an emission maximum nearer 590nm (Smiley
et al. 1991; Reers et al. 1991). This sensitivity to changes in
mitochondrial membrane potential allows use of JC-1 as a
standard measure of apoptotic events (Feeney et al. 2003;
Simeonova et al. 2004; Das et al. 2005).
Chemical Names, Formulas and Formula Weights:
DiI: Chemical Name:
2-[3-(1,3-Dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-
1-propenyl]-3,3-dimethyl-1-octadecyl-3H-
indolium perchlorate. Formula: C59H97ClN2O4. Formula
Weight: 933.87 (Cat.# P5491).
DAPI: Chemical Name:
2-[4-(Aminomethyl)phenyl]-1H-indole-6-carboximidamide,
dihydrochloride. Formula: C16H17Cl2N5. Formula Weight:
350.25 (Cat.# P5521).
Hoechst 33342: Chemical Name:
2´-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5´-
bi-1H-benzimidazole, trihydrochloride, hydrate. Formula:
C27H31Cl3N6O. Formula Weight: 561.93 (Cat.# P5541).
JC-1 : Chemical Name:
5-Cyano-2,3-bis(4-methylphenyl)-2H-tetrazolium chloride.
Formula: C25H27Cl4IN4 (Cat.# P5551).
G. Thiol-Reactive Probes
These products react readily with cysteines. The water
solubility of Fluorescein-5-maleimide at pH 7 makes it an
especially useful thiol-reactive probe (Stephens et al. 2003).
However, the increased photostability and pH insensitivity
of the rhodamine is especially useful in imaging
experiments (Sase et al. 1995). There have been reports that
some specificity of labeling is lost at very high dye:thiol
ratios (Tyagarajan et al. 2003). SBF-Cl is a water-soluble,
thiol-selective fluorescent labeling reagent (Andrews et al.
1982). It has been used to label GSH (Andrews et al. 1982;
Bolton et al. 1994; Chen et al. 1998), cysteine and its
derivatives (Chen et al. 1998) and insulins (Nanami et al.
1993).
Chemical Names:
Fluorescein-5-maleimide:
1-(3´,6´-Dihydroxy-3-oxospiro[isobenzofuran-1(3H),
9´-[9H]xanthen]-5-yl]-1H-pyrrole-2,5-dione (Cat.# P4361).
5-TMRIA: 9-[2-Carboxy-4-[(iodoacetyl)amino]phenyl]-3,6-
bis(dimethylamino)xanthylium salt (Cat.# P4371).
SBF-Cl: 7-Chloro-2,1,3-benzoxadiazole-4-sulfonic acid,
ammonium salt (Cat.# P1961).
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
H. UV-Excitable Fluorophores
These materials are the UV-excitable fluorophores used in
FRET applications with DABCYL or DABSYL as the
quencher. These dyes have significant spectral overlap of
emission with the absorption of the quencher.
EDANS is used to label the carboxyl group of aspartic or
glutamic acids (Ermolieff et al. 2000), of an N-succinoylated
amino terminus (Beebe and Pei, 1998) or of the C-terminus
(Pennington and Thornberry, 1994).
1,5-IAEDANS is used as the thiol-reactive fluorescent
partner (Liou and Fuchs, 1993). 1,5-IAEDANS is quite water
soluble above pH 4 and shows strong environmental effects
on its fluorescence, also making it useful for ligand binding
studies (Weber et al. 2002).
7-Hydroxycoumarin-3-carboxylic acid has been used to
study opening of connexin43 hemichannels (Koval et al.
1995 and Li et al. 1996), N-terminally label peptides directly
in a peptide synthesizer (Daugherty and Gellman, 1998;
Zhou and Ghosh, 2004) and label amino-modified sugars
to study glycosyltransferase activity (Higai et al. 1999).
7-Hydroxycoumarin-3-carboxylic acid, succinimidyl ester,
is a pH-sensitive, UV-excitable fluorophore that has been
used to study mannose-specific binding of labeled Crocus
sativus lectin (CSL) (Oda et al. 2000). It has been used to
study RNase L activation using labeled oligoadenylates
(Cole et al. 1997; Carroll et al. 1997) and to prepare novel
ratiometric FRET probes (Takakusa et al. 2003).
7-Methoxycoumarin-3-carboxylic acid was first described
and used as a fluorescent acylating agent (Baker and
Collins, 1949). Unlike the corresponding
7-hydroxycoumarin derivatives, the fluorescence of
methoxycoumarins is not pH-sensitive.
7-Methoxycoumarin-3-carboxylic acid, succinimidyl ester,
has been used to label short amino-modified oligomers in
multicolor hybridization studies (Eickhoff et al. 1996) and
to label peptides for use as quenched fluorogenic substrates
for neurolysin (Oliveira et al. 2001; Dauch et al. 1991). This
active ester has also been used extensively to label proteins
to study protein:protein interactions using FRET quenching
with DABSYL-labeled binding partners (Wang et al. 2000;
Wang et al. 2001).
Fluorescamine is a nonfluorescent reagent that rapidly
reacts with primary amines in amino acids, peptides, and
proteins (Udenfriend et al. 1972; Stein et al. 1973; Stein et al.
1974) to yield a blue-green fluorescent derivative. It has
also been used as a sensitive assay of plasma lipoproteins
despite the presence of bound lipids and turbidity (Funk
et al. 1986). Instrumentation used for analysis includes
microplate readers (Bantan-Polak et al. 2001),
high-performance liquid chromatography (Boppana et al.
1991) and capillary electrophoresis (Skelley and Mathies,
2003).
Chemical Names
EDANS: 5-[(2-Aminoethyl)amino]-1-naphthalenesulfonic
acid (Cat.# P4311).
10-19
||||||||||
EDANS, Na:
5-[(2-Aminoethyl)amino]-1-naphthalenesulfonic acid,
sodium salt (Cat.# P4321).
1,5-IAEDANS:
5-[[2-[(Iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic
acid (Cat.# P4331).
7-Hydroxycoumarin-3-carboxylic acid:
7-Hydroxy-2-oxo-2H-1-benzopyran-3-carboxylic acid (Cat.#
P5571).
7-Hydroxycoumarin-3-carboxylic acid, succinimidyl ester:
1-[[(7-Hydroxy-2-oxo-2H-1-benzopyran-3-yl)carbonyl]oxy]-
2,5-pyrrolidinedione (Cat.# P5581).
7-Methoxycoumarin-3-carboxylic acid:
7-Methoxy-2-oxo-2H-1-benzopyran-3-carboxylic acid (Cat.#
P5591).
7-Methoxycoumarin-3-carboxylic acid, succinimidyl ester:
1-[[(7-Methoxy-2-oxo-2H-1-benzopyran-3-yl)carbonyl]oxy]-
2,5-pyrrolidinedione (Cat.# P5601).
Fluorescamine:
4-Phenylspiro[furan-2(3H),1´(3´H)-isobenzofuran]-3,3´-dione
(Cat.# P1971).
I. Xanthenes
Xanthenes show strong fluorescence with Stokes' shifts of
approximately 20nm and excitation maxima coinciding
with readily available laser lines (FAM: Ar ion, 492nm;
TAMRA: HeNe, 543nm and ROX: Kr, 574nm). Filter sets
for these dyes are universally available. These dyes have
been used to label oligos directly (Vinayak, 1999) and for
histological staining of ganglia (Ammermüller et al. 1990).
Although labeling of proteins with succinimidyl esters is
more convenient, it is possible to use a protein coupling
reagent, such as EDAC, with these carboxylic acids. These
dyes are also a convenient starting material for the synthesis
of a variety of fluorescent chain-extended molecules.
Fluorescein Cadaverine. Amine-reactive labels are by far
the most common reactive dyes, but there are instances
wherein an acid-reactive dye has a role. Amines are known
to react with activated carboxylic acids, and when the acid
is a glutamic or aspartic acid residue of a protein, activation
can be performed using a water soluble carbodiimide such
as EDAC. A much more common labeling procedure,
however, is the enzyme-catalyzed transglutamination of
various proteins, such as Factor XIIIA (Lorand et al. 1983),
fibronectin (Lajemi et al. 1997) or actin (Miki et al. 1998;
Yengo et al. 2000). Transglutaminase activity in extracellular
tissue repair (Summey et al. 2002; Gross et al. 2003) has also
been studied with this probe. It is the synthetic chemistry
VI. References
of the label that has broadened its use to modify
methotrexate to study dihydrofolate reductase (Gapski et
al. 1975; Gaudray et al. 1986; Whiteley et al. 1986) and further
conversion of the resultant molecule to an affinity label of
folate transport proteins (Huennekens et al. 1992). More
significantly, fluorescein cadaverine has been used to
synthesize biopolymers to study cell surface receptor
interactions (Gordon et al. 2000; Owen et al. 2002), nanotube
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
transporters (Shi Kam et al. 2004), endocytosis of HPMA
copolymer drug carriers (Jensen et al. 2001) and in the
preparation of SAMs (Metallo et al. 2003). We have found
that the material is more stable as the hydrobromide salt.
However this leads to a slightly variable amount of HBr.
Please use the Promega Product Information sheet for an
accurate molecular weight.
5-Amino TMR. This weakly fluorescent material is used
as an intermediate in the synthesis of 5-TMRIA and
5-TRITC. It is readily acylated (with acid chlorides, for
example) to give fluorescent amides with unique physical
properties.
Chemical Names
5-FAM:
4-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-1,3-benzenedicarboxylic
acid (Cat.# P4091).
6-FAM:
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-1,4-benzenedicarboxylic
acid (Cat.# P4101).
5-TAMRA:
9-(2,4-Dicarboxyphenyl)-3,6-bis(dimethylamino)xanthylium
salt (Cat.# P4141).
6-TAMRA:
9-(2,5-Dicarboxyphenyl)-3,6-bis(dimethylamino)xanthylium
salt (Cat.# P4151).
5(6)-TAMRA: 9-(2,4(or 5)-Dicarboxyphenyl)-3,6-
bis(dimethylamino)xanthylium salt (Cat.# P4161).
6-ROX:
9-(2,5-Dicarboxyphenyl)-2,3,6,7,12,13,16,17-octahydro-
1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-i´j´]diquinolizin-
18-ium salt (Cat.# P4121).
5(6)-ROX: 9-(2,4(or 5)-Dicarboxyphenyl)-2,3,6,7,12,13,16,17-
octahydro-1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-
i´j´]diquinolizin- 18-ium salt (Cat.# P4131).
5-ROX, TEA Salt:
9-(2,4-Dicarboxyphenyl)-2,3,6,7,12,13,16,17-
ctahydro-1H,5H,11H,15H-xantheno[2,3,4-ij:5,6,7-i´j´]
diquinolizin-18-ium triethylammonium salt (Cat.# P4111).
Fluorescein Cadaverine:
N-(5-aminopentyl)-N´-(3´,6´-dihydroxy-3-oxospiro
[isobenzofuran-1(3H),9´-[9H]xanthen]-5-yl)-thiourea (Cat.#
P1911).
5-Amino TMR: 9-(4-Amino-2-carboxyphenyl)-
3,6-bis(dimethylamino)xanthylium salt (Cat.# P4401).
Ammermüller, J. et al. (1990) A "puff and advance" technique for
visually controlled staining of turtle retinal ganglion cells. J.
Neurosci. Methods 32, 235–43.
Andrews, J.L. et al. (1982) Ammonium
4-chloro-7-sulfobenzofurazan: A new fluorigenic thiol-specific
reagent. Arch. Biochem. Biophys. 214, 386–92.
10-20
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

Baker, W. and Collins, C.B. (1949) Fluorescent acylating agents
derived from 7-hydroxycoumarin J. Chem. Soc. Suppl. 1, S12–5.
Banks, P.R. and Paquette, D.M. (1995) Comparison of three
common amine reactive fluorescent probes used for conjugation
to biomolecules by capillary zone electrophoresis. Bioconjug. Chem.
6, 447–58.
Bantan-Polak, T. et al. (2001) A comparison of fluorescamine and
napthalene-2,3-dicarboxaldehyde fluorogenic reagents for
microplate-based detection of amino acids. Anal. Biochem. 297,
128–36.
Barcellona, M.L. et al. ( 2004) Polarized fluorescence correlation
spectroscopy of DNA-DAPI complexes. Microsc. Res. Tech. 65,
205–17.
Beebe, K.D. and Pei, D. (1998) A continuous fluorimetric assay for
tail-specific protease. Anal. Biochem. 263, 51–6.
Birkett, D.J. et al. (1970) The reactivity of SH groups with a
fluorogenic reagent. FEBS Lett. 6, 346–8.
Bolton, R.M. et al. (1994) Ammonium 4-chloro-7-sulfobenzofurazan:
A fluorescent substrate highly specific for rat glutathione
S-transferase subunit 3. Anal. Biochem. 216, 418–23.
Boppana, V.K et al. (1991) High-performance liquid
chromatographic determination of peptides in biological fluids by
automated precolumn fluorescence derivatization with
fluorescamine J. Chromatogr. 548, 319–27.
Brunner, A. et al. (1998) Labelling peptides with fluorescent probes
for incorporation into degradable polymers. Eur. J. Pharm. Biopharm.
45, 265–73.
Burstein, E. and Duckett, C.S. (2003) Dying for NF-kappaB? Control
of cell death by transcriptional regulation of the apoptotic
machinery. Curr. Opin. Cell Biol. 15, 732–7.
Carroll, S.S. et al. (1997) Activation of RNase L by
2′5′-oligoadenylates. Kinetic characterization. J. Biol. Chem. 272,
19193–8.
Check, E. (2005) Ethicists urge caution over emotive power of brain
scans. Nature 435, 254–5.
Chen, X.P. et al. (1998) Chromatographic separation of fluorescent
thiol adducts of 4-chloro-7-sulphobenzofurazan. Use as substrates
for enzymes of the mercapturic acid xenobiotic pathway. J.
Chromatogr. Biomed. Sci. Appl. 709, 19–25.
Chitarra, L.G. et al. (2000) Rapid fluorescence assessment of
intracellular pH as a viability indicator of Clavibacter michiganensis
subsp. michiganensis. J. Appl. Microbiol. 88, 809–16.
Cole, J.L. et al. (1997) Activation of RNase L by 2′,5′-oligoadenylates.
Biophysical characterization. J. Biol. Chem. 272, 19187–92.
Czikkely, V. et al. (1969) Zeitschrift fuer Naturforschung, Teil A:
Astrophysik, Physik und Physikalische Chemie 24, 1821–30.
Dann, O. et al. (1971) Justus Liebigs Annalen der Chemie 749, 68–89.
Das, A. et al. (2005) Phosphodiesterase-5 inhibitor sildenafil
preconditions adult cardiac myocytes against necrosis and
apoptosis. Essential role of nitric oxide signaling. J. Biol. Chem. 280,
12944–55.
Dauch, P. et al. (1991) Fluorimetric assay of the
neurotensin-degrading metalloendopeptidase, endopeptidase 24.6
Biochem. J. 280, 421–6.
Daugherty, D.L. and Gellman, S.H. (1998) A fluorescence assay
for leucine zipper dimerization: Avoiding unintended
consequences of fluorophore attachment J. Amer. Chem. Soc. 121,
4325–33.
Drobni, P. et al. (2003) Carboxy-fluorescein diacetate, succinimidyl
ester labeled papillomavirus virus-like particles fluoresce after
internalization and interact with heparan sulfate for binding and
entry. Virology 310, 163–72.
Eickhoff, H. et al. (1996) Sequence verification by hybridisation
with fluorescent octanucleotides as a first step to a fluorescent
sequencing by hybridisation protocol. J. Biochem. Biophys. Methods
32, 59–68.
Epsztejn, S. et al. (1997) Fluorescence analysis of the labile iron pool
of mammalian cells. Anal. Biochem. 248, 31–40.
Ermolieff, J. et al. (2000) Proteolytic activation of recombinant
pro-memapsin 2 (pro-beta-secretase) studied with new fluorogenic
substrates. Biochemistry USA 39, 12450–6.
Fazekas de St. Groth, B. et al. (1999) Carboxyfluorescein diacetate
succinimidyl ester and the virgin lymphocyte: A marriage made
in heaven. Immunol Cell Biol. 77, 530–8.
Feeney, C.J. et al. (2003) A cuvette-based fluorometric analysis of
mitochondrial membrane potential measured in cultured astrocyte
monolayers. J. Neurosci. Methods 125, 13–25.
Fulcher, D.A. and Wong, S.W.J. (1999) Carboxyfluorescein
succinimidyl ester-based proliferative assays for assessment of T
cell function in the diagnostic laboratory. Immunol. Cell Biol. 77,
559–64.
Funk, G.M. et al. (1986) Use of a rapid and highly sensitive
fluorescamine-based procedure for the assay of plasma lipoproteins
J. Lipid Res. 27, 792–5.
Gapski, G.R. et al. (1975) Synthesis of a fluorescent derivative of
amethopterin. J. Med. Chem. 18 526–8.
Gatti, R. et al. (1998) Comparison of annexin V and calcein-AM as
early vital markers of apoptosis in adherent cells by confocal laser
microscopy. J. Histochem. Cytochem. 46, 895–900.
Gaudray, P. et al. (1986) Fluorescent methotrexate labeling and
flow cytometric analysis of cells containing low levels of
dihydrofolate reductase.. J. Biol. Chem. 261 6285–92.
Ghosh, S. et al. (1998) NF-kappa B and Rel proteins: Evolutionarily
conserved mediators of immune responses. Ann. Rev. Immunol. 16,
225–60.
Gordon, E.J. et al. (2000) Synthesis of end-labeled multivalent
ligands for exploring cell-surface-receptor-ligand interact. Chem.
Biol. 7 9–16.
Gross, S.R. et al. (2003) Importance of tissue transglutaminase in
repair of extracellular matrices and cell death of dermal fibroblasts
after exposure to solarium ultraviolet A source. J. Invest. Dermatol.
121, 412–13.

Protocols & Applications Guide

www.promega.com
10-21
rev. 9/06
||||||||||
Gruden, C.L. et al. (2003) Activity assessment of microorganisms
eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium
chloride: A quantitative comparison of flow cytometry to
epifluorescent microscopy. J. Microbiol. Methods 55, 865–74.
Hermanson, G.T. (1996) Tags and Probes. In: Bioconjugate Techniques
Academic Press, San Diego, CA, 297–364.
Higai, K. et al. (1999) A fluorometric assay for glycosultransferase
activities using sugars aminated and tagged with
7-hydroxycoumarin-3-carboxylic acid as substrates and high
performance liquid chromatography. Biol. Pharm. Bull. 22, 333–8.
His, K.-L. et al. (1998) Visualization of proteins by modification of
lysines, cysteines, and phosphorylated serines facilitates sample
preparation for microsequencing. Anal. Biochem. 258, 38–47.
Hodgkin, P.D. et al. (1996) B cell differentiation and isotype
switching is related to division cycle number J. Exp. Med. 184,
277–81.
Holmes, K.L. and Lantz, L.M. (2001) Protein labeling with
fluorescent probes. Methods Cell. Biol. 63, 185–204.
Houk, T. et al. (1983) pH and polymerization dependence of the
site of labeling of actin by 7-choloro-4-nitrobenzo-2-oxa-1,3,-diazole.
J. Biol. Chem. 258, 5419–23.
Huennekens, F.M. et al. (1992) Membrane transport of folate
compounds. J. Nutr. Sci. Vitaminol. Spec. No. 52–7.
Imai, K. and Watanabe, Y. (1981) Fluormetric determination of
secondary amino acid by 7-fluoro-4-nitorbenzo-2-oxa-1,3-diazole.
Analytica Chimica Acta 130, 377–83.
Iturriaga, R. et al. (2001) Detection of respiratory enzyme activity
in Giardia cysts and Cryptosporidium oocysts using redox dyes and
immunofluorescence techniques. J. Microbiol. Methods 46, 19–28.
Jensen, K.D. et al. (2001) The cytoplasmic escape and nuclear
accumulation of endocytosed and microinjected HPMA copolymers
and a basic kinetic study in Hep G2 cells.. AAPS Pharm. Sci. 3 e32.
Kahya, N. et al. (2003) Probing lipid mobility of raft-exhibiting
model membranes by fluorescence correlation spectroscopy. J.
Biol. Chem. 278, 28109–15.
Kaprelyants, A.S. and Kell, D.B. (1993) The use of
5-cyano-2,3-ditolyl tetrazolium chloride and flow cytometry for
the visualisation of respiratory activity in individual cells of
Micrococcus luteus. J. Microbiol. Methods 17, 115–22.
Kapuscinski, J. (1995) DAPI: A DNA-specific fluorescent probe.
Biotech. Histochem. 70, 220–33.
Kapuscinski, J. and Skoczylas, B. (1978) Fluorescent complexes of
DNA with DAPI 4',6-diamidine-2-phenyl indole.2HCl or DCI
4′,6-dicarboxyamide-2-phenyl indole. Nucl. Acids Res. 5, 3775–99.
Kapuscinski, J. and Skoczylas, B. (1977) Simple and rapid
fluorimetric method for DNA microassay. Anal. Biochem. 83, 252–7.
Karin, M. et al. (2004) The IKK NF-kappa B system: A treasure
trove for drug development. Nat. Rev. Drug Discov. 3, 17–26.
Kim, T.H. and Park, T.G. (2004) Critical effect of
freezing/freeze-drying on sustained release of FITC-dextran
encapsulated within PLGA microspheres. Int. J. Pharm. 271, 207–14.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
Koval, M. et al. (1995) Transfected connexin 45 alters gap junction
permeability in cells expressing endogenous connexin 43. J. Cell
Biol. 130, 987–95.
Lajemi, M. et al. (1997) The use of Fluoresceincadaverine for
detecting amino acceptor protein substrates accessible to active
transgultaminase in living cells. Histochem. J. 29 593–606.
Latt, S.A. and Stetten, G. (1976) Spectral studies on 33258 Hoechst
and related bisbenzimidazole dyes useful for fluorescent detection
of deoxyribonucleic acid synthesis. J. Histochem. Cytochem. 24,
24–33.
Lee, Y.J. and Shukla, S.D. (2005) Pro- and anti-apoptotic roles of
c-Jun N-terminal kinase (JNK) in ethanol and acetaldehyde exposed
rat hepatocytes. Eur. J. Pharmacol. 508, 31–45.
Li, H. et al. (1996) Properties and regulation of gap junctional
hemichannels in the plasma membranes of cultured cells. J. Cell
Biol. 134, 1019–30.
Li, X. et al. (2003) Labeling Schwann cells with CFSE-an in vitro
and in vivo study J. Neurosci. Meth. 125, 83–91.
Lichtenfels, R. et al. (1994) CARE-LASS (calcein-release-assay), an
improved fluorescence-based test system to measure cytotoxic T
lymphocyte activity. J. Immunol. Methods 172, 227–39.
Liou, Y.M. and Fuchs, F. (1993) Energy transfer measurements of
the Cys35-Cys85 distance in bovine cardiac troponin C. Biochim.
Biophys. Acta 1202, 92–8.
Loewe, H. and Urbanietz, J. (1974) Basic-substituted
2,6-bisbenzimidazole derivates, a novel class of substances with
chemotherapeutic activity. Arzneimittelforschung 24, 1927–33.
Logan, R.P.H. et al. (1998) A novel flow cytometric assay for
quantitating adherence of Helicobacter pylori to gastric epithelial
cells. J. Immunol. Methods 213, 19–30.
Lorand, L. et al. (1983) New colored and fluorescent amine
substrates for activated fibrin stabilizing factor (Factor XIIIA) and
for transglutaminase. Anal. Biochem. 131 416–25.
Los, G.V. et al. (2005) HaloTag™ Interchangeable Labeling
Technology for cell imaging and protein capture. Cell Notes 11,
2–6.
Lydon, M.J. et al. (1980) Vital DNA staining and cell sorting by
flow microfluorometry. J. Cell Physiol. 102, 175–81.
Lyons, A.B. (1999) Divided we stand: Tracking cell proliferation
with carboxyfluorescein diacetate succinimidyl ester. Immunol.
Cell Biol. 77, 509–15.
Marras, S.A. et al. (2002) Efficiencies of fluorescence resonance
energy transfer and contact-mediated quenching in oligonucleotide
probes. Nucl. Acids Res. 30, e122.
McKenna, N.M. and Wang, Y-l. (1989) Culturing cells on the
microscope stage. Methods in Cell Biol. 29, 1–12. San Diego, CA:
Academic Press.
Metallo, S.J. et al. (2003) Using bifunctional polymers presenting
vancomycin and fluorescein groups to direct anti-fluorescein
antibodies to self-assembled monolayers presenting
D-alanine-D-alanine groups. J. Am. Chem. Soc. 125 4534–40.
10-22
||||||||||
Miki, M. et al. (1998) Fluorescence resonance energy transfer
between points on tropomyosin and actin in sketetal muscile thin
filaments: Does tropomyosine move? J. Biolchem. (Tokyo) 123,
1104–115.
Möbius, D. (1969) Zeitschrift fuer Naturforschung, Teil A: Astrophysik,
Physik und Physikalische Chemie 24, 251–3.
Mueller-Stahl, K. et al. (2003) Carboxyfluorescein diacetate
succinimidyl ester facilitates cell tracing and colocalization studies
in bioartificial organ engineering. Int. J. Artif. Organs 26, 235– 40.
Nanami, M. et al. ( 1993) Preparation of fluorescence labeled
insulins, sulfobenzoxadiazolyl-insulins, for fluorescence
immunoassay. Biol. Pharm. Bull. 16, 99–102.
Netten, H. et al. (1997 ) FISH and chips: Automation of fluorescent
dot counting in interphase cell nuclei. Cytometry 28, 1–10.
O'Brien, J.K. et al. (2005) Flow cytometric sorting of non-human
primate sperm nuclei. Theriogenology 63, 246–59.
Oda, Y. et al. (2000) Crocus sativus lectin recognizes Man3GlcNAc
in the N-Glycan core structure. J. Biol. Chem. 275, 26772–9.
Oliveira, V. et al. (2001) Selective neurotensin-derived internally
quenched fluorogenic substrate for neruolysin (EC 3.4.24.16):
Comparison with thimet oligo peptidase (EC 3.4.24.75) and
neprilysin (EC 3.4.34.11). Anal. Biochem. 292, 257–65.
Owen, R.M. et al. (2002) Synthesis and applications of end-labeled
neoglycopolymers. Org. Lett. 4 2293–6.
Papadopoulos, N.G. et al. (1994) An improved fluorescence assay
for the determination of lymphocyte-mediated cytotoxicity using
flow cytometry. J. Immunol. Methods 177, 101–11.
Parish, C.R. (1999) Fluorescent dyes for lymphocyte migration and
proliferation studies. Immunol. Cell Biol. 77, 499–508.
Pennington, M.W. and Thornberry, N.A. (1994) Synthesis of a
fluorogenic interleukin-1 beta converting enzyme substrate based
on resonance energy transfer. Peptide Research 7, 72–6.
Pyle, B.H. et al. (1999) Sensitive detection of Escherichia coli O157:H7
in food and water by immunomagnetic separation and solid-phase
laser cytometry. Appl. Environ. Microbiol. 65, 1966–72.
Reers, M. et al. (1995) Mitochondrial membrane potential monitored
by JC-1 dye. Meth. Enzymol. 260, 406–17.
Reers, M. et al. (1991) J-aggregate formation of a carbocyanine as
a quantitative fluorescent indicator of membrane potential.
Biochemistry 30, 4480–6.
Sanna, P.P. et al. (1992) Applications of DAPI cytochemistry to
neurobiology. Biotech. Histochem. 67, 346–50.
Sase, I. et al. (1995) Real time imaging of single fluorophores on
moving actin with an epifluorescence microscope. Biophys. J. 69,
323–8.
Schlessinger, J. et al. (1977) Lateral transport of a lipid probe and
labeled proteins on a cell membrane. Science 195, 307–9.
Severin, E. and Seidler, E. (1998) Flow cytometric assay of
cytochemically demonstrated NAD(P)H oxidoreductase
(diaphorase) activities. J. Histochem. Cytochem. 46, 761–5.
Protocols & Applications Guide
www.promega.com
rev. 9/06
10Cell Imaging
PROTOCOLS & APPLICATIONS GUIDE
Severin, E. et al. (1985) Fluorimetric assay of redox activity in cells.
Analytica Chimica Acta 170, 341–46.
Shi Kam, N.W. et al. (2004) Nanotube molecular transporters:
Internalization of carbon nanotube-protein conjugates into
mammalian cells. J. Biol. Chem. 126 6850–1.
Simeonova, E. et al. (2004) Monitoring the mitochondrial
transmembrane potential with the JC-1 fluorochrome in
programmed cell death during mesophyll leaf senescence.
Protoplasma 223, 143–53.
Skelley, A.M. and Mathies, R.A. (2003) Chiral separation of
fluorescamine-labeled amino acids using microfabricated capillary
electrophoresis devices for extraterrestrial exploration. J.
Chromatogr. A. 1021, 191–9.
Smiley, S.T. et al. (1991) Intracellular heterogeneity in mitochondrial
membrane potentials revealed by a J-aggregate-forming lipophilic
cation JC-1. Proc. Natl. Acad. Sci. USA. 88, 3671–5.
Stein, S. et al. (1974) Studies on the kinetics of reaction hydrolysis
of fluroescamine. Arch. Biochem. Biophys. 163, 400–3.
Stein, S. et al. (1973) Amino acid analysis with fluorescamine at the
picomole level. Arch. Biochem. Biophys. 155, 202–12.
Stellmach, J. (1984) Fluorescent redox dyes. 1. Production of
fluorescent formazan by unstimulated and phorbol ester- or
digitonin-stimulated Ehrlich ascites tumor cells. Histochemistry 80,
137–43.
Stephens, A.N. et al. (2003) The molecular neighborhood of subunit
8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine
scanning mutagenesis and chemical modification. J. Biol. Chem.
278, 17867–75.
Stephens, D.J. and Allan, V.J. (2003) Light microscopy techniques
for live cell imaging. Science 300, 82–86.
Sullivan, K.F. and Kay, S.A. (1999) Preface. Methods in Cell Biol. 58,
xv–xvi.
Summey, B.T. et al. (2002) Tissue transglutaminase localization
and activity regulation in the extracellular matrix of articular
cartiledge. J. Orthop. Res. 20 76–82.
Takakusa, H. et al. (2003) A novel design method of ratiometric
fluorescent probes based on fluorescence resonance energy transfer
switching by spectral overlap integral. Chemistry–A European Journal
9, 1479–85.
Tesfa, L. et al. (2003) A protocol for combining proliferation,
tetramer staining and intracellular cytokine detection for the
flow-cytometric analysis of antigen specific T-cells J. Biol. Reg.
Homeost. Agents 17, 366–70.
Truneh, A. and Machy, P. (1987) Detection of very low receptor
numbers on cells by flow cytometry using a sensitive staining
method. Cytometry 8, 562–7.
Tyagarajan, K. et al. (2003) Thiol-reactive dyes for fluorescence
labeling of proteomic samples. Electrophoresis 24, 2348–58.
Udenfriend, S. et al. (1972) Fluorescamine: A reagent for assay of
amino acids, peptides, proteins and primary amines in the picamole
range. Science. 178, 871–2.
10-23
|||||||||| 10Cell Imaging

PROTOCOLS & APPLICATIONS GUIDE

Vámosi, G. et al. (1996) Fluorescence characteristic of Technology Corporation. Tween is a registered trademark of ICI Americas,
Inc. VECTASHIELD is a registered trademark of Vector Laboratories, Inc.
5-carboxytetramethylrhodamine linked covalently to the 5´ end
Products may be covered by pending or issued patents or may have certain
of oligonucleotides multiple conformers of single-stranded and limitations. Please visit our Web site for more information.
double-stranded dye-DNA complexes. Biophys. J. 71, 972–94. All prices and specifications are subject to change without prior notice.
van Rijt, L.S. et al. (2004) A rapid flow cytometric method for Product claims are subject to change. Please contact Promega Technical
Services or access the Promega online catalog for the most up-to-date
determining the cellular composition of bronchoalveolar lavage
information on Promega products.
fluid cells in mouse models of asthma. J. Immunol. Methods 288,
© 2004–2006 Promega Corporation. All Rights Reserved.
111–21.
Verma, D.D. et al. (2003b) Particle size of liposomes influences
dermal delivery of substances into skin. Int. J. Pharm. 258, 141–51.
Verma, D.D. et al. (2003) Liposomes increase skin penetration of
entrapped and non-entrapped hydrophilic substances into human
skin: A skin penetration and confocal laser scanning microscopy
study. Eur. J. Pharm. Biopharm. 55, 271–7.
Vinayak, R. (1999) A convenient, solid-phase coupling of
rhodamine dye acids to 5′ amino-oliognucleotides. Tetrahedron
Letters 40, 7611–3.
Wang, T. et al. (2001) Gulatinone S-transferase P1-1(GSTP1-1)
inhibits c-Jun N-terminal kinase (JNK 1) signaling through
interaction witht he c-terminus. J. Biol. Chem. 276, 20999–1003.
Wang, T. et al. (2000) The pleckstrin homology domain of
phospholipase C-beta(2) links the binding of gbetagamma to the
activation of the catalytic core. J. Biol. Chem. 275, 7466–9.
Wang, X.M. et al. (1993) A new microcellular cytotoxicity test based
on calcein AM release. Hum. Immunol. 37, 264–70.
Wang, Y-l. (1989) Fluorescent analog cytochemistry: Tracing
functional protein components in living cells. Methods in Cell Biol.
29, 1–12.
Weber, J. et al. (2002) Cysteine-reactive fluorescence probes of the
catalytic site of ATP synthase Arch. Biochem. Biophys. 397, 1–10.
Weston, S.A. and Parish, C.R. (1990) New fluorescent dyes for
lymphocyte migration studies. Analysis by flow cytometry and
fluorescence microscopy. J. Immunol. Methods 133, 87–97.
Whiteley, J.M. et al. (1986) Synthesis and efficient isolation
procedure for gamma-linked fluorescein methotrexate. Prep.
Biochem. 16, 143–54.
Yengo, C.M. et al. (2000) INteraction of myosin LYS-553 with the
C-terminus and DNase I binding loop of actin examined by
fluorescence resonance energy transfer. J. Struct. Biol. 131 187–96.
Yoo, B.S. and Regnier, F.E. (2004) Proteomic analysis of
carbonylated proteins in two-dimensional gel electrophoresis using
avidin-fluorescein affinity staining. Electrophoresis 25, 1334–41.
Zhou, M. and Ghosh, I. (2004) Noncovalent multivalent assembly
of jun peptides on a leucine ziper dendrimer displaying fos
peptides. Org. Lett. 6, 3561–4.

Anti-ACTIVE, CellTiter-Glo and Monster Green are registered trademarks

of Promega Corporation. CaspACE, HaloTag, MaxxPure and TransFast are
trademarks of Promega Corporation.
Alexa Fluor is a trademark of Molecular Probes, Inc. Axiovert is a registered
trademark of Carl Zeiss, Inc. Cy is a registered trademark of Amersham
Biosciences, Ltd. FACS is a registered trademark of Becton, Dickinson and
Company. Hemo-De is a registered trademark of Scientific Safety Solvents.
NANOpure is a registered trademark of Barnstead/Thermolyne Corporation.
Triton is a registered trademark of Union Carbide Chemicals & Plastics

Protocols & Applications Guide

www.promega.com
10-24
rev. 9/06