Vdoc - Pub Animal Cell Culture Methods

Series Editors
Leslie Wilson
Department of Biological Sciences
University of California, Santa Barbara
Santa Barbara, California
Paul Matsudaira
Whitehead Institute for Biomedical Research and
Department of Biology
Massachusetts Institute of Technology
Cambridge, Massachusetts
Methods in Cell Biology
Prepared under the Auspices of the American Society for Cell Biology
VOLUME 57
Animal Cell Culture Methods
Edited by
Jennie P. Mather
Genentech, Inc.
South San Francisco. California
and
David Barnes
Division of Cell, Developmental, and
Molecular Biology/Genetics
American Type Culture Collection
Manassas, Virginia
ACADEMIC PRESS
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Cover photo (combbound): Human Schwann cell in culture.
Immunofluorescent staining of passage 4 cultures for the
marker GFAP (see Chapter 9 for further details).
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CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Shahabuddin Alam (69), Laboratory of Cellular Regulation Technology, Gradu-

ate School of Genetic Resources Technology, Kyushu University, Hakozaki,
Higashi-ku, Fukuoka 812-81, Japan
David Barnes (3), Division of Cell, Developmental, and Molecular Biology/
Genetics, American Type Culture Collection, Manassas, Virginia 20110
Kenneth D. Bauer (265), Genentech, Inc., South San Francisco, California 94080
Christopher J. Bayne (187), Department of Zoology and Evironmental Health
Sciences Center, Oregon State University, Corvallis, Oregon 97331
Christopher J. Donahue (265), Genentech, Inc., South San Francisco, Califor-
nia 94080
Michael G. Gabridge (49), University Technology Corporation, Boulder, Colo-
rado 80466
Anna-Katerina Hadjantonakis (279), Samuel Lunenfeld Research Institute,
Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1 x 5
Robert J. Hay (31), Cell Culture Department, American Type Culture Collection,
Rockville, Maryland 20852
Angela Helmrich (3), Division of Cell, Developmental, and Molecular Biology/
Genetics, American Type Culture Collection, Manassas, Virginia 20110
Marie-Claude C. Hofmann (93), Department of Biology, The University of Day-
ton, Dayton, Ohio 45469
Bharati Hukku (203), Cell Culture Laboratory, Children’s Hospital of Michigan,
Department of Pediatrics, Wayne State University School of Medicine, Detroit,
Michigan 48201
Joseph Kaplan (203), Cell Culture Laboratory, Children’s Hospital of Michigan,
Department of Pediatrics, Wayne State University School of Medicine, Detroit,
Michigan 48201
Yoshinori Katakura (69, 11l), Laboratory of Cellular Regulation Technology,
Graduate School of Genetic Resources Technology, Kyushu University, Hako-
zaki, Higashi-ku, Fukuoka 812-81, Japan
Ronghao Li (167), Signal Pharmaceuticals, Inc., San Diego, California 92121
Carolyn Kay Lincoln (49), Bionique Testing Laboratories, Inc., Saranac Lake,
New York 12983
Deryk T. Loo (251), Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543
Jennie P. Mather (19,219,265), Genentech, Inc., South San Francisco, Califor-
nia 94080
xi
xii Contributors
Hildegard Meissner (147), Department of Neurosurgery, Laboratory for Brain

Tumor Biology, University Hospital Hamburg Eppendorf, 20246 Hamburg,
Germany
Gary F. Merrill(229), Department of Biochemistry and Biophysics, Oregon State
University, Corvallis, Oregon 97331
Jose Luis Millan (93), The Burnham Institute, La Jolla Cancer Research Center,
La Jolla, California 92037;and Department of Medical Genetics, Umed Univer-
sity, Umei, Sweden
Alison Moore (265), Amgen, Inc., Thousand Oaks, California 91320
Andrhs Nagy (279), Samuel Lunenfeld Research Institute, Mount Sinai Hospital,
Toronto, Ontario, Canada M5G 1x5
David M. Phillips (297), The Population Council, New York, New York 10021
Melinda Pirity (279), Samuel Lunenfeld Research Institute, Mount Sinai Hospi-
tal, Toronto, Ontario, Canada M5G 1x5
Jill R. Rillema (251), Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, New Jersey 08543
Sanetaka Shirahata (69, l l l ) , Laboratory of Cellular Regulation Technology,
Graduate School of Genetic Resources Technology, Kyushu University, Hako-
zaki, Higashi-ku, Fukuoka 812-81, Japan
Kiichiro Teruya (1l l ) , Laboratory of Cellular Regulation Technology, Graduate
School of Genetic Resources Technology, Kyushu University, Hakozaki,
Higashi-ku, Fukuoka 812-81, Japan
Yu-li Wang (313), Cell Biology Group, Worcester Foundation for Biomedical
Research, Shrewsbury, Massachusetts 01545
Manfred Westphal (147), Department of Neurosurgery, Laboratory for Brain
Tumor Biology, University Hospital Hamburg Eppendorf, 20246 Hamburg,
Germany
Sally P. Wheatley (313), Cell Biology Group, Worcester Foundation for Biomedi-
cal Research, Shrewsbury, Massachusetts 01545
Teresa K. Woodruff (333), Northwestern University, Departments of Medicine
and Neurobiology and Physiology, Chicago, Illinois 60611
PREFACE
In this volume we provide a broad introduction to animal cell culture tech-

niques and applications. We also try to integrate into this presentation the concep-
tual framework from which the techniques are derived. The book is divided
into four sections: (I) Principles of Cell Culture; (11) Establishing Cell Lines;
(111) Specialized Culture Techniques; and (IV) Microscopy and Morphology. In
each case, detailed technical treatment is balanced with the succinctness necessary
to contain the work to a single volume. It is our hope that each section and
chapter provide sufficient information to enlighten a reader new to the field
while providing direction to additional sources of information in the primary
literature as well as to relevant previous volumes of this series.
The first section is devoted to the basics of cell culture: laboratory setup,
critical skills, and information with which to make choices of the appropriate
cell culture system. Particular attention is given to the rationale behind routine
cell culture approaches. This includes issues such as why and how commonly
used cell culture medium formulations were developed and the advantages and
disadvantages of using both familiar cell lines and some less frequently used
lines. Our intention is to provide the reader with a basis for troubleshooting as
well as for choosing the best system for the intended purpose. In some cases
the best choice may not be the system that is most commonly used or easiest
to propagate.
An investigator exploring cell culture models should not presume that the
possibilities are limited to available cell lines. In Section I1 we present techniques
and rationale for developing cell culture model systems to fit individual needs.
Specific examples have been chosen to illustrate general principles, and several
of the more common and useful cell culture manipulations are presented. These
include introduction and expression of exogenous DNA (transfection), cell fusion
(hybridoma derivation), cell line characterization (e.g., karyotyping), serum-free
cell culture, and derivation of cell lines of human origin.
Section 111,on specialized culture techniques, provides direction in laboratory-
scale culture for isolation of recombinant products, as well as techniques for
manipulation and measurement of cell proliferation and death, the cell cycle,
and cell differentiation. Emphasis is also placed on cell culture techniques and
applications associated with embryonal stem cells.
The last section points out the critical role of light and electron microscopy
in cell-culture-related work. Also included in this section are various means for
cellular-molecular localization of both proteins and nucleic acids.
xiii
XlV Preface
The editors thank the contributors to this volume for their contributions and
the editors of the series for helpful suggestions. We hope this volume not only
will provide a starting point for new researchers in the field who wish to apply
cell culture techniques to their particular scientific interests, but also will provide
useful additional information and viewpoints to those already expert in cell
culture methodology.
Jennie P. Mather
David Barnes
SECTION I
Principles of Cell Culture
The chapters in the first section are designed to present a brief review
of the basic principles of cell culture. The first chapter provides guidance
for those who are setting up a tissue culture facility or a tissue culture
space in their laboratory. Many people lose a great deal of time and
expend unnecessary effort through not taking sufficient time and thought
to choose the correct cell type, medium, and culture configuration to
achieve their goals. Chapter 2 reviews the role of tissue culture media in
an in vitro system and the different media that are available, whereas
Chapter 3 reviews the cell lines available and the culture repositories
where they can be obtained. Finally, Chapter 4 reviews the different
types of physical, chemical, and biological contamination that can destroy
experiments and/or cause artifactual results. Gross bacterial or fungal
contamination is by far the easiest type of contamination to deal with
because it is so obvious. However, chemically contaminated media or
mycoplasma contamination can be difficult to detect yet cause real prob-
lems in a cell culture laboratory, leading to invalid experimental data.
Section I should provide a good introduction to the special aspects of
2 Section I
laboratory practice that are unique to cell culture and a good review of
the most up to date information in these areas.
The following chapters deal with specific aspects of cell culture that
are deemed to be most important to the cell or molecular biologist wishing
to use cell culture as a tool in hidher work. Many of the chapters emphasize
general principles that will help the investigator select the appropriate
and most efficient tools to reach a desired goal, such as localizing a specific
protein, scaling up cell culture, or establishing a cell line from a normal
or transformed cell. The reader is referred to other volumes in this series,
where applicable, for more detailed protocols concerning some of the
individual techniques discussed here.
CHAPTER 1
Animal Cell Culture Equipment

and Techniques
Angela Helmrich and David Barnes
Division of Cell, Developmental, and Molecular Biology/Genetics
American Type Culture Collection
Manassas, Virginia 201 10
~~
I. Introduction
11. Equipment
A. Hoods
B. Incubators
C. Microscopes
D. Autoclaves
E. Water Purification Equipment and Medlum Filtration Devices
F. Cell Counter
G. Liquid Nitrogen Storage Tanks
H. Water Baths, Centrihges, Freezers, and Refrigerators
111. Laboratory Design
IV. Materials
A. Reagents, Media, and Serum
B. Cell Culture Plasticware and Glassware
V. Cell Culture Methods
A. Sterile Technique and Routine Procedures
B. Primary Culture
C. Multipassage Culture and Cloning
D. Freezing Cells
References
I. Introduction
This chapter is devoted to some of the basics of cell culture equipment and
techniques. It is based largely on innovations, observations, realizations, acci-
METHODS IN CELL BIOLOGY. VOL. 57
Copynghr 0 1998 by Acadermc Press. All ngho of reproductmn in any fonn reserved. 3
00YI-h79X/90 S25.00
4 Angela Helmrich and David Barnes
dents, mistakes, and misunderstandings encountered since the mid-1970s in cell

culture laboratories, representing knowing and unknowing contributions from
dozens of individuals. Some suggestions may seem painfully obvious, but a failure
to understand the concepts behind these suggestions will lead to even more
painful results. Many of the statements and suggestions might be challenged by
investigators at least as competent as the author, and it remains the responsibility
of each investigator to “take what you need and leave the rest,” as much of the
technical specifics of experimental design using cell culture technology must be
determined by the distinctive nature of the questions asked and cell types used.
The first section discusses cell culture equipment: hoods, incubators, micro-
scopes, autoclaves, water purification and medium filtration devices, cell counters,
liquid nitrogen storage tanks, water baths, centrifuges, refrigerators, and freezers.
This is followed by a section on laboratory design. A section on materials deals
with reagents, media and serum, cell culture plasticware, and glassware. The
subsequent section on cell culture methods covers sterile technique and routine
procedures, primary culture, multipassage culture, cloning, and freezing cells.
The chapter concludes with a list of reference books that deal with these subjects
in more detail.
11. Equipment
A. Hoods
Animal cell culture can be surprisingly successful when carried out on the
unprotected laboratory bench top, especially when antibiotics are used in the
medium. However, a commitment to cell culture techniques over the long term
requires a hood that provides a sterile environment for the manipulation of cells,
solutions, and culture vessels.
Horizontal flow hoods are simple devices for maintaining a sterile working
area in which filtered air is blown through a contained space directly at the
investigator. Anything in the hood that impedes air flow compromises the capabil-
ity of the system. To operate properly, these hoods require a substantial air flow
rate, and it usually is not feasible to use a burner to provide a sterilizing flame
in these hoods. The high air flow rate also often contributes to rapid alkalization
of culture medium that is buffered with bicarbonate only.
It is not wise to work with poorly characterized transformed human cells,
potentially infectious microorganisms, radioactivity, or toxic or volatile solutions
in horizontal flow hoods, as the investigator is unprotected from vapor or liquid
droplets that might be generated in the hood and then blown out. Appropriate
tasks for horizontal hoods include sterile filtration or dispensing of nontoxic
solutions, sterile microdissections requiring a microscope in the work space, and
culture of cells considered “safe.”
“Safe cultures” must be defined by each investigator; any culture could in
principle be contaminated with a potential human pathogen, and human-derived
1. Animal Cell Culture Equipment and Techniques 5
material is considered by some to represent a risk uniformly. Even under pre-

sumed safe conditions, a hand-held pipetting device is essential. The most popular
style is small enough to be placed inside the hood so that it draws in (and
therefore pushes out) sterile air, and various sizes of pipettes can be attached.
These are available as both house current-driven and battery-powered models.
Laminar flow hoods utilize a sterile air curtain blowing vertically in front of
the investigator, usually with a glass barrier between the investigator and the
hood work space below which is an opening for the investigators hands to enter
the work space. Higher protection for work with infectious agents can be provided
by a glove box in place of this opening. Hoods are also available that have
incubators built into the side, so cultures need not pass through open laboratory
space when moving between hood and incubator.
Additional options are available for exhausting the hood completely out of
the room through building ducts for work with volatile or otherwise hazardous
materials. The commonly used laminar flow hoods exhaust a fraction of the air
through a filter and back into the room, and recycle the rest. This feature has
the added attractions of producing a more sterile environment in the room itself
and prolonging the life of the hood filters. Most hoods of a particular design are
generally comparable in functionality because all are built to satisfy standard
specifications developed by the National Institutes of Health.
It is possible to use a sterilizing flame in these hoods, but manufacturers warn
that the flame disturbs the air flow and may thus jeopardize sterility in the hood
work space. If a flame is used, one approach is to use a gas burner with a pilot
flame that can be activated to the full flame when needed. Remote control foot
pedals are available for these burners, freeing the investigators hands during
operation. Gas fires can occur in these hoods, especially if the burners, tubing,
or remote control devices malfunction. Inflammable, gas-tight tubing is recom-
mended for connecting the gas outlet inside the hood to the burner.
Many hoods have gas cutoff valves inside the work space. This design is of
little use if the fire is also inside the work space. A better design includes an
easily accessible gas cutoff valve outside the hood. House vacuum also is routinely
plumbed into cell culture hoods to facilitate medium removal and vacuum filtra-
tion, and the vacuum cutoff valve is also routinely placed inside the hood work-
place. The combination of an open vacuum line and a gas fire inside a contained
hood space can create some interesting phenomena that might be best avoided.
A gas fire in a cell culture hood may represent a larger danger than a comparable
bench-top gas fire because of the increased air flow in the vicinity of the fire.
Most laminar flow hoods are available with ultraviolet (UV) light fixtures for
sterilization of the work space when not in use. This is effective with a new bulb,
but the bulbs may rapidly lose intensity in the UV range, while continuing to
provide a pleasant, deceiving blue light that is of reduced value for sterility
protection. Use of a UV also will hasten the destruction of many types of
plastics, causing them to crack or depolymerize to organic goo. Hand-held plastic
micropetters routinely left under these lights to sterilize them will exhibit a
shortened life span.
As with the simpler hood types, cramming unnecessary stuff into the hood
work space will compromise sterile operation. Filter integrity verification and
replacement can be accomplished by the investigator, but professional personnel
are also available to do this for a fee. Periodic inspection from an outside
professional may be worth the cost, especially if hazardous materials are being
used. Replacing filters is often not as straightforward as it might seem. Profes-
sional evaluations might be especially comforting if an externally vented, 100%
exhaust laminar flow hood is used because these hoods introduce additional air
flow and pressure considerations that may require careful balancing between
intake and exhaust in both the hood and the room containing the hood.
Laminar flow hoods may introduce a false sense of security to the point that
an investigator may conclude that the normal rules of sterile technique need no
longer apply inside the hood work space. The open space at the bottom of the
hood window is designed for the insertion of hands, but it will also allow the
insertion of other less desirable appendages. Difficulties in manipulations inside
the hood space or an impeded view through the hood glass may lead one to
stick all or part of one’s head inside the hood. This is undesirable from a number
of points of view.
Another problem is a tendency of personnel to use the laminar flow hood for
procedures in which this level of protection is not needed, simply because it is
conveniently plumbed with gas and vacuum. If sufficiently unsupervised, the
most unsterile of laboratory components, including antibiotic-resistant bacterial
or fungal cultures, could find their way into the hood. For these and other reasons,
it is advisable to spray the inside of the hood with 70% ethanol and wipe away
the excess before hood use. Allow the hood to run for a few minutes after this
before the flame is lit on the burner, especially if long hair, a beard, or flammable
clothing are involved.
B. Incubators
The simplest reliable COz incubator is a water-jacketed chamber with remov-
able shelves inside and a control for gas flow, a pan in the bottom for water, a
water jacket heater, and a thermostat with overheating protection. Both shelving
and the frame that holds the shelves inside the incubator should be removable
for sterilization, and an antimicrobial detergent should be added routinely to
the water pan. More sophisticated incubators with considerable gadgetry can be
purchased, including sensing and automatic control of gas and humidity levels,
copper walls, chamber fans, and individual compartmentation inside the chamber.
Eventually, a C02-sensing and control device will pay for itself in gas savings,
but this will take longer than might be expected because a major fraction of the
cost of C 0 2 is cylinder rental.
Fans in incubators are useful when precise temperature control is required,

as in work with temperature-sensitive mutants, because an undisturbed incubator
chamber develops a humidity and temperature gradient from bottom to top. A
disadvantage of a fan in the chamber is the potential for increased spread of
microbial contamination throughout the incubator because of the increased air
circulation. Copper incubator walls are argued to be antimicrobial, but are expen-
sive. Incubators without water jackets, commonly used with roller bottle or
spinner cultures, generally have fans but usually do not have gas flow control.
These incubators rapidly return to ambient temperature if power is interrupted.
Some incubators are designed so that external pressured air is unnecessary.
In incubators that require continuous air flow, pressurized air derived from a
central building source may be undesirable because the compressor introduces
oil into the system each time it engages. Air can be supplied by simple, electrically
powered aquarium pumps. Incubators can be modified or purchased to use a
three gas mixture (e.g., oxygen, nitrogen, carbon dioxide) instead of the routine
air-carbon dioxide mixture. Humidity is best maintained by bubbling the entering
gas through the water pan at the bottom of the chamber. Because of the humidity
gradient, it may be useful to routinely place cell culture plates on the bottom
shelves and flasks on the top shelves to minimize evaporation from the plates.
An effective approach for carbon dioxide gas delivery is a system in which
three 50-lb standard grade carbon dioxide tanks are secured to a wall, with two
tanks connected to an electronic switch box. These boxes are commercially
available and activate an audible alarm if they sense no gas pressure. The switch
box automatically switches from an empty tank to a full tank, and the gas supply
also can be switched from tank to tank manually using a toggle switch on the
front of the unit. One of the connected tanks is a full backup, while the other
supplies carbon dioxide to the incubators.
Tanks are received with about 900 lbs of pressure and are stepped down to
about 15 lbs of pressure at the switch box with a two-step regulator. A single
tank can supply gas to two double incubators (four chambers) for approximately
2 weeks. When a tank approaches empty, the tank pressure gauge will fall from
900 to 0 over 3 to 5 days. At zero pressure the electronic switch will automatically
transfer supply from the now empty tank to the backup tank. The third tank
then replaces the empty one, and a new one is ordered. Occasionally, the gas
line tubing used inside the electronic switches will become cracked or unseated
on its fittings. The escaping gas may be audible, and the tank will empty much
more rapidly than normal. These problems can be remedied by dismantling the
box (unplug it first!), trimming, or replacing the faulty tubing and reseating.
C . Microscopes
An inverted-phase microscope is essential, and the best one that the budget
allows is recommended. Some are designed to require external lubrication of
moving parts; these are made of hard metal and last longer. The other design is
made of softer metal and relies on small particles of metal scuffed from the
apparatus by everyday movement for lubrication. Eventually these models lose
the ability to maintain a set position against gravity or become imprecise in
settings. Previously, specificationswere sufficiently common among manufactur-
ers that lenses and other parts were interchangeable to a surprising degree. This
situation has reversed in recent years to the point that lenses for some models
from a single microscope manufacturer are not exchangeable even with earlier
models from that manufacturer.
D. Autoclaves
House steam is commonly used for sterilization by an autoclave. Such a source
can be quite dirty, and an autoclave that generates its own steam from deionized
water is recommended. Dirty steam may be obvious as a layer of scum on
autoclaved glassware. Otherwise, the usual rules of autoclaving apply: using
autoclave tape does not guarantee sterility (especially with large volumes of
liquids), do not pack the autoclave completely full, place glass bottles in a pan
of water, do not seal containers before autoclaving, and do not autoclave full con-
tainers.
E. Water Purification Equipment and Medium Filtration Devices

For all cell culture reagents, HPLC grade water is recommended. A number
of filtration systems that produce HPLC grade water are available commercially.
Triple glass distillation is also fine, but is less used these days. Storage of water
purified earlier is not recommended, as even minimal microbial growth upon
storage can lead to pyrogen contamination of the water. Algae can grow any-
where, and an ecosystem in which other microorganisms benefit from the algae
can develop quickly. For large-scale filtration, pump-driven or pressure-driven
devices are available. These require some degree of assembly or sterilization
and may be considered worthwhile if the volume of medium to filter routinely
exceeds 4 liters. Otherwise, disposable, sterile, plastic vacuum filtration devices
may be used.
F. Cell Counter
Patent and market considerations dictate that the automated cell counter
available probably will be a Coulter counter. These are sufficiently complicated
to require routine maintenance and occasional troubleshooting. Service contracts
are available for this instrument, but depending on your budget, it may prove
beneficial to become an expert on this machine as an alternative. The author
and colleagues have observed that on occasions when counters mysteriously
malfunction (especially the older ones), they can be cured by simply taking them
apart and putting them back together. Keep your mouth shut, and laboratory
observers to the process will be duly impressed by your intricate knowledge of
cell counter operation.
Phosphate-buffered solutions used for counting cells require filtration before
use. These should be free of particulates that may interfere with counting, but
need not be strictly sterile. If the background count is greater than 50-60, flush
the system and check for debris in the reservoir, dispenser, probe, etc. Cell
counts should be maintained between 1000 and 35,000 per 0.5 ml counted, with
corrections applied when counts exceed 10,000. Always keep the electrode in
an appropriate solution. The pump should be oiled weekly and glass stopcocks
greased monthly. Splitting of the mercury column indicates that the mercury
should be changed or the mercury and glassware cleaned. Using new mercury
may be preferable to acid cleaning.
G. Liquid Nitrogen Storage Tanks

Tanks for storage of frozen cells vary from relatively small to very large, with
and without an automatic nitrogen level sensing and filling capability. It is easier
to retrieve cells from small tanks, but they are less conservative of liquid nitrogen.
It has been argued that cells should be stored in vapor-phase nitrogen above
the liquid. Advantages are that the vials are less likely to fill with liquid and
then explode when warmed up and that cross-contamination of vials by microor-
ganisms via the liquid is minimized. These advantages must be weighed against
the greater potential for the tanks to go dry because little liquid exists in the
tank to compensate for warming.
H. Water Baths, Centrifuges, Freezers, and Refrigerators

Routine laboratory water baths are fine, and precise temperature control is
usually not necessary. Water baths are a major source of contamination in a
cell culture laboratory and should be periodically cleaned and an antimicrobial
detergent added. After warming or thawing a container that will end up in a
sterile hood, spray it with 70% ethanol and wipe clean before placing into a
hood. Secure the thermostat setting on water baths located in tissue culture
rooms so that they cannot be changed easily from 37°C. Microwave ovens and
custom-built d r y warmers are also fine, but care must be taken not to overheat
with the microwave oven.
Bench-top, clinical centrifuges without refrigeration are fine for routine cell
centrifugation. Centrifuges with timers are preferred because the investigator is
likely to be doing several things simultaneously at the time of centrifugation. A
low-temperature freezer is extremely useful in a cell culture laboratory. Self-
defrosting freezers should be avoided. Refrigeration should be in as dry an
atmosphere as possible.
111. Laboratory Design
Attention should be given to the placement of the cell culture hood in the
laboratory to minimize air flow that may interfere with hood function and to
minimize exposure to air- or personnel-borne contaminating particulates. This
might best be accomplished by relegating cell culture equipment to small rooms
that are not high traffic laboratory areas or by designating a particular corner
of a larger laboratory for cell culture purposes. In a small room with a standard
laminar flow hood, sterile exhaust from the hood itself will help maintain sterility
in the room.
It is best to place the cell culture hood outside the influence of any high-
velocity laboratory fume hoods that may compromise cell culture hood function.
The incubator, microscope, and centrifuge should be as close as possible to the
cell culture hood work space to minimize physical movement of the cultures.
Cell culture work even in the most efficient environment involves considerable
transfer of vessels from hood to microscope to incubator, and so on, and economy
of movement helps prevent disasters.
If all of these elements can be accommodated in a small, dedicated cell culture
room, then it may be worthwhile to plumb a carbon dioxide gas line to the
incubator from a larger laboratory room. This avoids the possibility of a poten-
tially dangerous gas leak in a small room and also makes the cylinders, regulators,
and alarms accessible to a larger number of people to prevent oversights and
emergencies. Although C02 itself is not a toxic gas, carbon dioxide is heavier
than air and will sink to the floor. A room suddenly filled with the gas can cause
asphyxiation, which is also true for nitrogen. If you enter a laboratory and hear
a rush of gas or have other suspicions that a gas line might be broken, the best
course of action may be to vacate the room immediately, leaving all doors open
behind you, and seek help before proceeding.
Often a sticky mat is placed at the entrance to a cell culture room to trap
particulates on the shoes of entering personnel. Some laboratories incorporate
air locks or anterooms, positive or negative pressure barriers, or intercoms for
communication between rooms, but these may be a serious consideration only
if experiments of a hazardous nature are contemplated. Malfunction alarms are
useful on freezers, liquid nitrogen tanks, and positive or negative pressure rooms.
Most laminar flow hoods, especially those designed for 100%exhaust, have alarms
to indicate insufficient air flow or exhaust.
Thought should be given to the default situation if electrical power fails in a
cell culture laboratory. For instance, using the carbon dioxide gas switch boxes
described earlier, the gas flow will stop when power fails because the regulator
boxes are controlled electrically. In this case it is also ideal to use electrically
pumped air to the incubators so that all air flow will also stop in the incubators.
Under these conditions, tolerable atmosphere and temperature will be main-
tained for hours in a water-jacketed incubator if incubator doors are not opened.
A roller bottle or spinner incubator without a water jacket will require more
attention. Battery-powered emergency systems are available for these incubators
that can keep the bottles turning, the spinners spinning, and the temperature
correct for a short period. In an emergency, flasks containing cells can simply
be screwed shut tightly and left at ambient temperature. Most mammalian cell
types will survive at room temperature as long as the proper pH is maintained.
Negative or positive pressure rooms or rooms with 100%exhaust laminar flow
cell culture hoods and fume hoods for use with hazardous materials require
special consideration regarding power interruption and configuration of supply
and exhaust air sources. This includes room supply and exhaust, fume hood
exhaust, and cell culture hood exhaust. For instance, a room in which the cell
culture hood ceases to operate but a fume hood in the room switches to emergency
power when routine power is interrupted can present a hazard, as potentially
hazardous material can be drawn out of the cell culture hood and into the room.
A similar situation may occur if the laminar flow cell culture hood continues
to operate on emergency power but the fume hood does not operate or operates at
reduced air flow. Furthermore, under these circumstances potentially hazardous
material could further escape the room and enter the building air supply, depend-
ing on how the room supply and exhaust is configured to respond when regular
power is interrupted. These serious issues require consultation with engineers
and institutional biosafety officers at the time of design and installation of proper
equipment and regular inspection thereafter.
IV. Materials
A. Reagents, Media, and Serum
Some cell culture-related chemicals appear in catalogues in two grades: a
cheaper standard grade and a more expensive “cell culture” or “tested for cell
culture” grade. This issue may have some merit; for example, early industrial
batches of HEPES buffer were inconsistent in levels of contaminants toxic to
cultured cells, but this particular problem has not been of recent concern. Each
investigator must decide in each case the degree to which the increased cost is
worthwhile and the degree to which any testing that may have been done is
relevant to the particular cell culture system that will be used. At the very least,
reagent grade materials should be used; contaminating levels of lead, for instance,
in poor quality NaCl or NaOH used for adjusting medium pH can contribute
to cell toxicity.
Powdered and liquid media formulations are available commercially. Com-
monly used basal medium formulations such as Ham’s F12, Dulbecco-modified
Eagle’s medium, RPMI 1640, MCDB media, and combinations of these media,
as well as sterile solutions of trypsin-EDTA, PBS, and so forth, are available from
multiple sources. The degree to which an investigator chooses to use commercially
prepared solutions depends on budgets and the degree of faith in the quality
and consistency of the product. Unusual medium formulations can be obtained

by special order, but usually in large lots only (e.g., 100 liters or more).
Making media fresh from a powdered formulation is preferable to buying
liquid media because the liquids have undergone a period of storage prior to
shipping. If medium is made from laboratory chemicals, it is imperative that the
original papers reporting these formulations be consulted because an understand-
ing of storage stability and solubility of stock components is essential. In general,
it is recommended to store liquid medium frozen in 100- to 200-ml aliquots if
possible. Most serum-containing media can be stored this way, but some serum-
free media can precipitate upon freezing because of relatively high calcium and
phosphate concentrations.
Liquid medium stored in the refrigerator may be warmed in a 37" water bath
for 10-15 min. If frozen, medium can be thawed in a microwave for a few minutes
on the defrost setting. It is good practice to minimize the time any cell culture
reagent is maintained in a warm environment prior to exposing to cells as some
of the relevant components are heat sensitive.
Serum is available from multiple companies, and batch-to-batch variation is
the rule. It is common practice to request samples of various serum batches for
testing with the particular cell system of interest. Serum can be stored long term
at -70 to -90°C. Some serum lots are provided with an analysis of components
of presumed general interest; of course this gives no insight regarding the compo-
nents that are not assayed. It is recommended that sterility of any commercial
solution, including serum, be treated with skepticism.
In situations in which serum-containing medium is used, a relatively safe
approach is to filter the serum-containing medium as the last step rather than
adding presumed sterile serum to medium that has been filtered. Medium can
be tested for sterility after filtration by inoculation of a small volume into a
larger volume of antibiotic-free medium and incubation for a few days or by
inoculation onto antibiotic-free LB agar plates. Glass bottles of serum stored at
very low temperatures can present a problem when thawing. To prevent the
bottle from breaking, first place the bottle at -20°C for 2 hr, then at 4°C for
1 hr, and then into a 37°C water bath.
Advances in cell culture since the mid-1970s have been made by supplementing
or replacing serum with purified growth factors or hormones. Although some of
the hormones are relatively inexpensive commercially, others, particularly pep-
tide growth factors, traditionally have been quite expensive. Progress in the large-
scale production of recombinant products and peptide synthesis has led to price
reductions for some of these.
Unlike the approach one might routinely take with a serum supplement, these
supplements generally should not be added directly to the medium, filtered, and
then the medium stored for later use. Stability problems dictate that most serum-
free supplements are best added directly to medium in individual plates or flasks
as small aliquots from concentrated stocks immediately after plating cells. Many
peptide growth factors may be obtained as sterile, lyophilized powders from
commercial sources and reconstituted with sterile water or buffered salt solutions
as indicated by the vendors. Store sterile stock solutions of supplements in the
refrigerator. Supplements may be stored long term in the freezer in aliquots.
Multiple freeze-thaws should be avoided.
B. Cell Culture Plasticware and Glassware

It is recommended to use plastic, disposable cell culture materials as much as
the budget will allow. This is particularly true for serum-free cell culture. It is
difficult and time-consuming to wash reusable glassware so that it is sufficiently
free of toxic detergent to guarantee reproducible success when using these in
cell culture, although detergents sold commercially for use with cell culture
glassware improve this situation. Some commercially available plasticware is
advertised to have been chemically or physically altered to improve certain
functions, such as adhesion or growth of primary cultures; these must be tested
individually for each cell culture system.
Cell culture vessels occasionally are damaged in shipping or manufactured
improperly so that integrity is compromised in a way that is not immediately
obvious visually. If microbial contamination suddenly appears, do not discount
the possibility that the plasticware is faulty. Plastic formulations used by the
commercial suppliers may change from time to time in ways that may be insignifi-
cant for the vast majority of users but may have unpredicted effects for some
cell culture systems.
It may be useful to impress upon laboratory personnel that sterile, cotton-
plugged, individually wrapped plastic pipettes are essential for sterile work in
the cell culture hood, but should only be used when necessary. Unwrapped plastic
pipettes or disposable glass pipettes are available for nonsterile manipulations.
Similarly, the appropriate pipette size should be used, as the cost goes up with
the increasing size of the pipette. Sterile, disposable, cotton-plugged glass Pasteur
pipettes are inexpensive and extremely versatile for small volume work. It is
recommended that flasks, graduated cylinders, stir bars, and so on used in making
up medium and glass bottles used to store medium be rinsed immediately after
use and washed with HPLC grade water without soap. Glassware used for cell
culture work should never have been used previously for other purposes.
V. Cell Culture Methods

A. Sterile Technique and Routine Procedures
Use of a flame in hoods is a matter of individual choice. This investigator finds
it useful to flame autoclaved, disposable glass Pasteur pipettes and flask or bottle
caps and lips routinely. To decrease the potential for contamination, make sure
that the necks of flasks and lips of dishes do not have medium on them or leaking
out of them and clean up spilled medium in a hood, incubator, or bench top
immediately, washing with 70% ethanol. Spots of dried medium are a source of
microbial growth. Do not use tape to label shelves or culture vessels in an
incubator as microbial growth occurs on the glue of the tape. In general, the
best way to maintain sterile technique is by employing foresight and economy
of motion.
A vacuum flask hooked to the house vacuum or a small vacuum pump contain-
ing a decontamination solution (e.g., 50 ml Virex in a 1-or 2-liter flask) becomes
convenient for removing medium from culture dishes when connected to a tube
with a pipette on the end for removing the medium. Do not use bleach in the
vacuum flask, as the volatile bleach will destroy the pump. Disposable plastic
pipettes and other cultureware contaminated with live cells should be disposed
of in biohazard bags and autoclaved. To make biohazard bags ready for autoclav-
ing, do not completely seal by tying or taping top shut. Loosely fold top over
and tape, leaving room for pressure exchange.
Some investigators leave hoods running constantly, helping to maintain a
sterile environment in the general laboratory, whereas others turn them off when
not in use, conserving the lifetime of the motor and filter. These issues only
become critical with 100% exhaust hoods and biohazardous work, in which it is
recommended that the safest mode be maintained constantly.
B. Primary Culture
In general, one may expect that routine cell types derived from normal tissues
and cultured in conventional, serum-containing media will grow for a limited
period, lose proliferative potential, and undergo crisis. Depending on the cell
type and culture conditions, this phenomenon may be followed by the appearance
of abnormal, immortalized lines. Initial, or primary, culture is the first step in
this process. The basic principles for initiating primary cultures from abnormal
(e.g., tumor) tissue are the same, but the growth pattern may not conform to
the growth-crisis-immortalization steps outlined earlier.
Animals from which tissue is to be obtained may be best killed by C 0 2
asphyxiation or cervical dislocation, as anesthesia may affect the cells to be
cultured. The outside of the animal can be swabbed with 70% ethanol to sterilize
before removing the tissues. Flaming is discouraged, particularly on alcohol-
soaked, hairy animals. Remove tissue with sterile instruments (autoclaved or
dipped in 70% ethanol) under a tissue culture hood with sterile instruments. For
usual jobs, several pairs of sharp scissors and forceps are adequate.
Place tissues in a culture dish, trim unwanted material (fat, membranes, other
tissues, bone, blood clots, parasites, hair), and wash with a suitable solution (e.g.,
phosphate-buffered saline without calcium or magnesium). Mince tissues with
scissors and incubate with an appropriate disaggregation solution. A trypsin
solution might be the simplest [0.25% crude trypsin with 1mM ethylenediamine-
tetraacetate (EDTA) in phosphate-buffered saline without calcium or magne-
sium]. If PBS is used as the buffer, do not incubate the samples in a carbon
dioxide incubator as PBS is not bicarbonate buffered.
A primary culture of some tissues may call for additional collagenase, hyaluron-
idase, DNase, or other proteases exposed to cells in a defined sequence. DNase
is sometimes used because dead cells will release chromatin and the protease
activity of the trypsin solution will destroy the DNA-associated proteins, leading
to hydration of the freed DNA and a noticeable increase in the viscosity of the
suspension. DNase will digest the released material. Some crude trypsin solutions
may contain sufficient contaminating DNase to prevent this problem.
The progress of disaggregation can be monitored with a microscope, and the
suspension should be pipetted or agitated periodically. The point at which the
incubation is terminated depends on the cell type to be cultured. For some cell
types, the appropriate point is reached when the major portion of the cells are
single cells; for other cell types one should stop when the cells exist primarily
as aggregates of a dozen or less cells. In general, the initial incubation should
not be extended for long periods in an attempt to obtain an entirely homogeneous
single cell suspension, as lengthy incubations will lead to cell death.
Larger chunks of tissue may be allowed to settle for a few seconds in a
centrifuge tube, and the cell suspension removed and centrifuged in a bench-
top centrifuge. Cells are resuspended in the appropriate culture medium, counted,
and plated. Cells from the larger chunks that settled from the suspension may
be harvested further by repeating the procedures described earlier.
Cells for primary culture are best counted with a hemocytometer prior to
plating because of the heterogeneous nature of the preparation. Often the pri-
mary culture plating density should be higher than densities that should be used
at later passage because the majority of cells in the initial suspension will not
survive or grow in culture. Medium should be changed 8-16 hr after plating to
remove debris. A significant amount of nonadherent red blood cells may be
present in the initial plating, depending on the nature of the tissue and how the
tissue was prepared in the early steps. Cells in the initial culture may represent
multiple cell types, but the cultures become more homogeneous upon multiple
passage.
C. Multipassage Culture and Cloning

Passaging of suspension cultures may be accomplished simply by dilution or
by centrifugation of cells out of the old culture medium and resuspension into
a larger volume of fresh medium. For routine passaging of adherent cells, remove
the medium, add trypsin/EDTA solution, and incubate the cells until detached.
All cells of primary culture may not detach at the same rate and some may not
detach at all. The percentage of cells that will detach upon routine trypsinization
increases on multiple passage because of the selection for less strongly attached
cells. Add a volume of serum-containing medium equal to the volume of trypsin/
EDTA solution, and centrifuge, resuspend, and replate the cells in fresh medium.
Cloning of primary or early passage adherent cells is best accomplished with

cloning rings rather than by the limiting dilution method. Early passage cells
generally do not tolerate culture at low cell densities well. After removal with
cloning rings, the cells may be placed in small wells (e.g., 24-well plates) in order
to maximize cell density. Suspension cultures may be cloned by limiting dilution,
using conditioned medium if survival at low cell density is a problem.
D. Freezing Cells
Cells should be frozen slowly and thawed quickly for maximal survival. Cells
may be frozen in 10% serum containing 10% dimethyl sulfoxide (DMSO), and
viability upon thawing may vary, depending on the cell type. Greater success
with some cell types can be achieved in a freezing medium of 90% calf or fetal
calf serum and 10% DMSO. After filter sterilization, these solutions may be
stored at -20°C. For freezing, trypsinize, centrifuge, and resuspended cells at a
concentration of 5 X lo5 to 2 X lo6 cells/ml in the freezing medium and aliquot
1 ml into each freezing vial.
Although devices are available for precisely controlled freezing of cells, the
following simple way may be used: refrigerate (4°C) for 30 min, transfer to a
Styrofoam-insulated container, place in a low temperature freezer at -86°C
overnight, and then transfer into liquid nitrogen. A -20°C incubation of a few
hours may also be inserted between the refrigerator and the low temperature
freezer, but may not be essential.
To thaw cells, wearing goggles, remove the vial from the liquid nitrogen and
warm the cells in a 37°C water bath as quickly as possible until ice is completely
gone. Be careful; thawing a vial that explodes because of a rapid expansion of
nitrogen trapped inside can be a deafening, blinding, or otherwise dangerous
experience for you and other that may be around. Transfer the contents to a
flask or plate and change medium in the flask as soon as cells have settled and
stuck to the flask to remove the DMSO. Alternatively, it is possible to centrifuge
the vial contents diluted with culture medium, resuspend the pellet in fresh
medium, and transfer to a flask or plate.
For the long-term storage of primary material, cell suspensions derived from
the initial disaggregation may be frozen in liquid nitrogen in medium with 10%
DMSO and serum, as described earlier. However, the cells must be reasonably
desegregated for good viability upon thawing, as large clumps of cells do not
freeze or thaw evenly, leading to cell death.
Acknowledgments
The author thanks Gordon Sato, Jennie Mather, Hayden Coon, Dick Ham, Rob Hay, Hiroki
Murakami, Wally McKeehan, Penny Roberts, Sam Bradford, Angela Helmrich, Janet Silnutzer
Reing, Deryk Loo, Paul Collodi, Le Sun, Lucy Williams, Sanetaka Shirahata, Masayoshi Iio, Kazuo
Nishiyama, Kate Linberg, Chet Baker, Gram Parsons, Emily Amonett, and numerous others. This
work was supported by NIH Grants ROlES06011 (NIEHS) and ROlRR12063 and is dedicated to
Amber E. Miller.
References
The following is a list of books that investigators exploring cell culture may find helpful.
Barnes, D., Mather, J., and Sato, G. (eds.) (1991). “Methods In Enzymology,” Vol. 198, Part C.
Academic Press, New York 1991.
Barnes, D., and Sirbasku, D. (eds.) (1987). “Methods in Enzymology,” Vol. 146, Part A, and Vol.
147, Part B. Academic Press, New York.
Barnes, D., Sirbasku, D., and Sato, G. (eds.) (1984). “Cell Culture Methods for Molecular and Cell
Biology,” 4 Volumes, Wiley-Liss, New York.
Butler, M. J. (1997). “Animal Cell Culture and Technology.” IRL Press.
Darling, D. C., and Morgan S. J. (1994). “Animal Cells: Culture and Media.” Wiley, New York, 1994.
Darling, D. C., and Morgan, S. J., (1994). “Animal Cell Culture: Introduction to Biotechniques.”
Bios Scientific Publishers Ltd.
Doyle, D., Hay, R., and Kirsop, B. E. (eds.) (1991). “Animal Cells: Living Resources for Biotechnol-
ogy.” Cambridge University Press, Cambridge, UK.
Freshney, R. I. (ed.) (1992). “Animal Cell Culture: A Practical Approach.” IRL Press, Oxford.
Freshney, R. I. (1994). “Culture of Animal Cells: A Manual of Basic Techniques,” 3rd Ed. Wiley-
Liss, New York.
Harrison, M. A,, and Rae, I. F. (1997). “General Techniques of Cell Culture (Handbooks in Practical
Animal Cell Biology),” Cambridge Univ. Press, Cambridge, UK.
Jakoby, W. B., and Pastan, I. H. (1979). “Methods in Enzymology,” Vol. 58. Academic Press,
New York.
Murakami, H., Yamane, I., Hayashi, I., Mather, J., Barnes, D, and Sato, G. (eds.) (1985). “Growth
and Differentiation of Cells in Defined Environments.” Springer-Verlag. New York.
Pollard, J. W., and Walker, J. M. (1990). “Animal Cell Culture: Methods in Molecular Biology,”
Vol. 5. Humana Press, 1990.
Various editors (1989-current). “Proceedings of the Annual Meeting of the Japanese Association
for Animal Cell Technology.” Kluwer Academic Publishers.
Wasley, J. D., and May, J. W. (1971). “Animal Cell Culture Methods.” Lippincott-Raven Publishers.
CHAPTER 2
Malung Informed Choices: Mehum,

Serum, and Serum-Free Medurn
How to Choose the Appropriate Medium and
Culture System for the Model You Wish to Create
Jennie P. Mather
Genentech, Inc.
South San Francisco, California 94080
I. Introduction
11. The Role of Medium
111. pH Control
IV. Selecting the Appropriate Medium
V. Screening Conditioned Medium for Biological Activity
VI. Media Preparation
VII. Serum, Plasma, and Other Undefined Additives
VIII. Testing Media and Components and Quality Control: “It’s in the Water”
IX. Troubleshooting Mehum Problems
X. Altering Commercial Media for Special Uses
XI. Medium Optimization
XII. Choosing the Optimal Medium: The “Quick and Dirty” Method
References
Complex nutrient mixtures, which are usually called “media,” are almost
always supplemented with serum, with another complex biological fluid (e.g.,
milk, embryo extracts, and plasma), or with a defined mixture of hormones and
growth factors. The choice of medium and supplements can have a major impact
on the growth, function, and even phenotypic and genetic stability of cells in
v i m . This choice thus becomes an important part of developing a useful and
meaningful in vitro model system. This chapter defines the various roles that the
WTHODS IN CELL BIOLOGY. VOL. 57
AU righu afrepmducuon in any form reserved
Copynght 0 1998 by Academic Prerr. 19
M)91-679X/98 $25.00
20 Jennie P. Mather
medium plays in supporting cell function and outlines a method for selecting
and optimizing medium in growing the cell of choice.
I. Introduction
Complex nutrient mixtures, which are usually called “media,” are almost
always supplemented with serum, with another complex biological fluid (e.g.,
milk, embryo extracts, and plasma), or with a defined mixture of hormones and
growth factors. The ongoing experimental work of replacing complex mixtures
with defined components, both nutrients and proteins, has been largely responsi-
ble both for our understanding of what the medium does in cell culture and for
our increased technical ability to maintain a broad range of functional cells in
vitro. The choice of medium and supplements can have a major impact on the
growth, function, and even phenotypic and genetic stability of cells in vitro. This
choice thus becomes an important part of developing a useful and meaningful
in vitro model system. The following questions can best be answered after defining
the goals of the research and understanding what the different components of
the medium do: What medium should I use to grow my cells? Should I try to
get the cell to grow as fast as possible? Is it worth the effort to carry my stock
cultures in serum-free medium or to do my experiments in serum-free medium?
Should I attempt to use a chemically defined medium? How much time should
I spend optimizing the medium? What assay(s) should I use for medium optimi-
zation?
11. The Role of Medium

The medium provides essential nutrients that are incorporated into dividing
cells, such as amino acids, fatty acids, sugars, ions, trace elements, vitamins, and
cofactors, and ions and molecules necessary to maintain the proper chemical
environment for the cell. Some components may perform both roles, e.g., sodium
bicarbonate may be used as a carbonate source and may also play an important
role in maintaining the appropriate pH and osmolality. The medium contains
all or part of the buffering system required to maintain a physiological pH (see
Section 111) and should provide the appropriate osmolality for the cells. Nutrients
include amino acids, with the richer medium containing both “essential” and
“nonessential” amino acids. Media also contain lipids; most contain a mixture
of fatty acids, and some contain more complex lipids (e.g., cholesterol). Some
media formulations such as Medium 199 contain detergents (e.g., Tween 80) to
help emulsify the lipids. These detergents can prove toxic to some types of cells,
particularly in serum-free medium. Some media contain macromolecules such
as thymidine, adenosine P, and hypoxanthine, which can be synthesized by cells
2. Choosing the Appropriate Medium and Culture System 21
in vitro. These may nonetheless improve cell growth by maintaining appropriate

pool sizes of precursors inside the cells. Many media contain the common vitamins
such as niacin, folic acid, riboflavin, inositol, and thiamine. Although these vita-
mins are essential to continued cell replication, a detrimental effect may not be
seen until several cell doublings after their removal from the medium. Other
vitamins such as vitamins D (1,25-dihydroxycholecalciferol),C (ascorbic acid),
E (a-tocopherol), and A (retinol, retinoic acid) are not commonly added to
media formulations because they are unstable in solution. However, these may
prove beneficial or even essential for some cell types and, in those cases, should
be added separately (Mather e t d , 1983).They may also be important in maintain-
ing the differentiated state of the cell, in regulating cell function, or acting
as antioxidants.
All media contain some energy source, usually glucose, although the molar
levels can vary widely (0.8-5 g/liter). Amino acids and glucose, as well as ions
such as NaCl, contribute to the osmolality of the medium, as well as having a
nutritional role. In addition to the bicarbonate/C02 buffering system, the medium
may also contain phosphate buffer and perhaps complex organic buffers. The
medium may also contain antioxidants or reducing agents (or these might be
added separately). Most media contain phenol red as a pH indicator.
Most media [e.g., minimal essential medium, Dulbecco’s modified Eagle’s
(DME) medium] were developed specifically for use with serum supplementation
and high-density growth of cells (Dulbecco and Freeman, 1959; Eagle, 1955). In
contrast, Ham’s nutrient mixtures F12 and F10 and the MCDB series of media
were tailored specifically for growing a specific cell type (e.g., CHO, fibroblasts)
at low density with a minimal amount of undefined protein added so as to study
the effects of the nutrient components of the media (Ham, 1965; Ham and
McKeehan, 1979). The F12/DME (1 : 1, v/v) medium was originally devised for
growing cells in defined serum-free conditions (Mather and Sato, 1979) (now
commercially available as a premixed powder). Fl2/DME medium works well
for growing cells at low or high densities and in defined hormone-supplemented
conditions or with serum. Leibovitz L-15 medium (Leibovitz, 1983) is designed
to grow cells in equilibrium with air rather than C02/air and is useful when CO2
incubators are not available (e.g., the teaching laboratory) or when cells are
shipped or handled extensively outside the incubator (e.g., during a long tissue
dissociation protocol).
More recently, vendors are supplying “special use media” to grow a stated
cell line or cell type under special conditions. For example, media have been
specifically formulated for the growth of keratinocytes, human endothelial cells,
or neural cell lines. These sometimes contain undisclosed hormones, growth
factors, or undefined protein components. These media cannot therefore be
considered “defined,” although they may work very well for some applications.
Other such media are supplied with a defined supplement mix that must be
added before use.
22 Jennie P. Mather
111. pH Control
The COz setting on incubators should be chosen to match the medium to be
used. Each medium has been formulated with components designed to work
with a specified COzconcentration (most ranging from 0 to 10%COz/airmixtures)
to give a pH of 7.0-7.4. The mismatch of medium bicarbonate levels and COz
incubator levels will result in the medium pH being out of the optimal range
with resultant growth retardation. If media designed for use with different COz
levels are to be used in the same incubator, the bicarbonate levels should be
adjusted so that they all buffer correctly at the COz level to which the incubator
is set. It should be pointed out that the lowest COz levels (with low bicarbonate)
give a medium with a lower buffering capacity than a high C02/high bicarbon-
ate system.
IV. Selecting the Appropriate Medium

If a new cell line is brought into the laboratory, it is necessary to determine
what medium is recommended for its growth. This information can be obtained
from the same source as the cells. If the recommended medium is incompatible
with the COzsettings on the incubator used for other cells grown in the laboratory,
or is not commonly prepared in the laboratory, it may be best to change the
growth medium. It is best to grow the cells initially in their original medium and
to compare this with the more convenient medium after a passage or two in
each. If the growth rate and morphology of the cells look the same, then a
medium switch can be made. However, when trying to repeat published data, it
must be kept in mind that cells grown in a different medium may respond
differently in some other parameters measured, even when their growth rates
are the same.
If the goal is to grow a primary or established cell type in culture and no
published data describing a preferred medium formulation exist or if the goal
is to grow the cells in a different manner (e.g., with defined supplements rather
than serum), it is best to screen several of the commercially available media
before deciding on the one that is best for that particular use. This can be done
by obtaining 5-10 candidate media powders from a supplier, preparing them all
in the laboratory as described later using the same water and supplementary
components, and doing a direct comparison of cell growth in the different condi-
tions. A sample of commercial media that are available and the cells and condi-
tions they were developed for is given in Table I. Use this table and that given
in Ham and McKeehan (1979) to select several media developed for growing
cell types most similar to those you wish to grow (e.g., fibroblasts, lymphoid
cells) and select media developed for serum-free growth if that is desired. A
“quick and dirty” method for medium optimization is outlined later in this
Table I
Commonly Used Media“
Medium Applicable to Reference
Basal Medium eagle (BME) Growing cells with serum Eagle (1965)
Minimal essential medium Growing cells with dialyzed serum Eagle (1959)
(MEMI
Dulbecco’s modified Eagles’ Many virus-transfected cells, Dulbecco and Freeman (1959)
Medium (DMEM) growth with serum, high-density
growth
Ham’s F10 medium Chick embryo cells, serum Ham (1963)
Ham’s F12 nutrient mixture Chinese hamster ovary cells, low- Ham (1965)
(F12) density, low-serum protein
F12DME (1 : 1) mixture Serum growth, many cells, serum Mather et al. (1979);
free Bottenstein et al. (1979)
William’s medium E Rat liver epithelial cells Williams and Gunn (1974)
RPMI 1630 Mouse leukemia cells, cells in
suspension
RPMI 1640 Human leukemic (and other) cells, Moore and Kitamura (1968)
hybridomas
Leibovitz L-15 medium Buffered for air, human tumors Leibovitz (1963)
Waymouth’s MB 75211 L cells Waymouth (1959)
Fischer’s medium Murine leukemia cells Fischer and Sartorelli (1964)
McCoy’s 5A medium Human lymphocytes McCoy et al. (1959)
MCDB 131 Human endothelial cells Knedler and Ham (1987)
Medium 199 Chick embryo fibroblasts Morgan et al. (1950)
Medium NCTC-109 Hybridomas Evans et al. (1956)
Medium NCTC-135 Serum, serum-free growth Evans et al. (1964)
Neurobasal medium Central nervous system neurons Brewer et al. (1994)
Many of these media are now widely used to grow many different types of cells.
chapter. If end points other than cell growth are important, measure these in
each of the media. Carry the cells in the medium selected for several passages
and freeze them in this medium for future use.
V. Screening Conditioned Medium for Biological Activity

It is important to know the composition of the test medium if conditioned
medium is to be screened for biological activity. As this is often done at 10-50%
conditioned medium, the test medium should minimally use the same bicarbonate
buffer concentration as the assay medium and optimally be identical to the assay
medium except for changes introduced by the conditioning cells. The fewer the
undefined components added to the medium, the easier subsequent purification
of any detected activity should be.
24 Jennie P. Mather
VI. Media Preparation

Media can be purchased as prepared liquid media, made up in the laboratory
from dried powders containing most of the components of the nutrient mixtures,
or prepared in the laboratory from individual stocks of the individual components
or from groups of the components. Purchasing liquid medium, especially for
serum-free culture work, is not recommended. Medium components deteriorate
with time, and do so faster in solution. Some necessary components break down
and are lost, whereas others create toxic breakdown products or oxidize to toxic
components. Although it varies from cell to cell, and with serum-supplemented
or serum-free media, 2 weeks is a safe storage time for serum-free media and
4-8 weeks if serum is added when the medium is prepared. Outdated medium
can be used for washing cells or for preparing tissues for primary culture. Clearly,
this is not adequate time to allow for commercial preparation and storage,
shipping, and further storage of medium in the laboratory. Some prepared liquid
media can be frozen. Those that form a precipitate when thawed should not be
frozen. In any case, it is always safe to store the prepared powdered medium
and make liquid medium in the laboratory on a regular basis.
Powdered nutrient mixtures generally have a shelf life of a year or more if
stored in moisture-proof, airtight containers in the dark. If large volumes of
media are not needed, 1-liter packages are convenient. Preparing medium in the
laboratory from components immediately before use is obviously the best way
to ensure that the medium contains the desired components in the desired form.
This is essential if the investigator wishes to study the role of the nutrients
themselves or to optimize the nutrient portion of the medium, as described
later (Ham and McKeehan, 1979). However, most laboratories will find that the
preparation of medium from commercial powdered nutrient mixtures and a
limited storage of the prepared media in a light tight refrigerator will be adequate
for their needs. This is also less costly than purchasing prepared media, especially
when the cost of filters and so on can be spread over large-volume use.
HEPES, or another organic buffer compatible with cells, provides additional
buffering capacity in the cultures and stabilizes the pH during the time that the
cultures are out of the incubator and at normal atmosphere for observation
and manipulation. Because serum itself has considerable buffering capacity, the
HEPES concentration can be reduced to 10 mM or eliminated if serum (5-15%)
is to be used as a supplement. It is best not to add antibiotics to the medium
for the routine culture of cell lines, as this ensures that a poor sterile technique
will be detected rapidly. Because all antibiotic agents have some toxicity, any
antibiotic to be used should be tested at several concentrations on the cells of
interest. When preparing primary cultures, an antibiotic may be added to the
wash medium during the initial stages of tissue handling. This may be done
by preparing a 1000-fold concentrated stock solution of an antibiotic such as
gentamycin and adding it directly to the wash medium. (A 1 M stock solution
of HEPES buffer is used to dissolve the gentamycin at a final concentration of

1 mg/ml.) Generally, wide-mouthed glass Schott bottles, tissue culture flasks, or
roller bottles are used for storing medium.
VII. Serum, Plasma, and Other Undefined Additives

Serum is frequently spoken of as if it were a defined single substance. This is
very far from the truth. Cell culture media can be supplemented with sera from
any species of animal; bovine (fetal, newborn, or adult), equine, or human sera
are the most frequently used. These are quite different in many ways and can
have very different effects on the properties of cells grown in them. Additionally,
serum varies from animal to animal, with changes in diet, and seasonally. Consid-
erable variability therefore exists from lot to lot of commercially available sera.
To make whole sera, the blood is allowed to clot and the clot removed, or the
blood can be collected with an anticlotting agent and the cellular portion spun
out, resulting in plasma. Serum and plasma, even from the same animal, are
quite different in composition and in their effect on cells. Sera can be treated
before use in one or more ways: filtration, dialysis, diafiltration, heat treatment,
or fractionation. These treatments can act as an added insurance against contami-
nation, remove or inactivate toxic components of the serum, remove or inactivate
growth-promoting or -differentiating components of the serum, and specifically
remove low- or high-molecular-weight components of the serum or particular
serum fractions. Clearly this complex and undefined addition to medium must
be treated with some care to ensure consistent results. The only way to ensure
good results is to thoroughly test several lots of serum for their ability to support
the desired cell characteristics (e.g., growth, differentiation, or lack of differentia-
tion, specific biochemical markers, protein production) and then buy a quantity
of the best lot (store it at -20 to -8OOC) sufficient for the next 1-2 years.
Most commercial sera come sterilely packaged. It is best to purchase serum
that has been sterilely collected as well as an added insurance against viruses or
mycoplasma, which can go through some filters. When adding human serum to
cultures, the entire culture and all waste should be treated as a biohazard. Human
sera should be collected from known donors or blood banks that test for the
most common viruses, such as HIV and hepatitis.
VIII. Testing Media and Components and Quality Control:

“It’s in the Water”
As stated previously, it is best to prepare medium from commercially available
powdered nutrient mixtures. It is important to keep good records and to do
quality control testing of reagents used in making the medium. It is best to keep
26 Jennie P.Mather
one set of glassware exclusively for medium making, which should be rinsed well
with distilled water, but not washed with detergent, between each use. This
avoids any possibility of detergent residue getting into the medium. Bottles of
sodium bicarbonate and any other reagents that are used in medium should be
used only for medium. When weighing out these reagents, a disposable tongue
depressor and weigh boat should be used. This avoids contaminating these re-
agents with other, potentially toxic, chemicals that may be in use in the laboratory.
The major component of the medium is water. Water purity is very important
for good quality medium. Usually, water quality is more critical when cells are
grown in serum-free medium than when the same cells are grown with serum-
supplemented medium. However, some cell types are extremely sensitive to poor
medium quality, even when serum is used. Some sensitive cells (e.g., TR-1, a
capillary endothelial-derived cell line) cannot be grown at all in serum-free
medium made with poor quality water, although they will grow at a decreased
rate if this medium is supplemented with serum. Mather et al. (1986) have
determined that heavy metals and organic compounds can account for some of
the toxicity in poor quality water.
IX. Troubleshooting Medium Problems

Even when all these precautions are followed, there will come a time when
problems arise that require “troubleshooting.” This is when meticulous testing
and record keeping pay off. Use the following steps to identlfy and eliminate
the problem.
1. Talk to all persons using the culture facility. Determine whether the problem
is being experienced in many different cell lines or only a few and by all
users or only a few.
2. When did the problem start? Determine the earliest date that anyone
thought they might have a problem.
3. Are there any reagents that are used only with cells having a problem or
by all cells having the problem?
4. Were new lots of any of the medium or supplemental reagents put into use
at, or within 1-2 weeks before, the time the problem started?
5. Were new lots of tissue culture plates, bottles for media preparation or
storage, or a different brand used abost this time? Were new types or lots
of filters used for filtering medium? (It is always a good idea to discard the
first 50 ml of medium put through a filter.)
6. Test all cell lines in the laboratory for mycoplasma and other potential
contaminants. If they are all contaminant free and only one cell line seems
to be having trouble, thaw out a vial of cells from an earlierfreeze of that line.
7. If any of these questions have turned up a suspicious reagent or supply,
test this first. Make up medium using a different lot of medium powder,
serum, and so on (or open another lot of tissue culture dishes) and test by
comparing the newly made medium to the presumptive “bad” lot of me-
dium. It is best to change only one thing at a time.
8. Get water from another source (e.g., a still in another laboratory) and test
medium made with this water.
Although all of these precautions may seem excessive, good quality control
can save days and weeks spent tracking down problems that affect experimental
outcome and can make the difference between success and failure in growing
some types of cells. The author has experienced many problems over the years,
including seasonal variation in distilled water quality, serum lots that will support
the growth of one cell type but not others, serum whose inadequacy to support
growth was only apparent after four to five passages, plasticware to which cells
would not attach, a medium powder lot missing one component, and many more.
Even the best run laboratory will inevitably experience problems. If the problems
experienced in your laboratory are traced to a specific reagent or lot of culture
dishes, notify the manufacturer. They will usually be helpful in correcting the
problem and/or replacing the defective materials.
X. Altering Commercial Media for Special Uses
Sometimes an addition to a commercial medium can improve cell growth. If

cells are being grown at high densities and are very lactogenic (rapidly acidify
the medium), the addition of more glucose to the medium may improve growth
and prolong viability. One may need to add a trace element mix such as those
described by Ham ef al. (Hamilton and Ham, 1977;McKeehan ef al., 1976). Many
of these trace elements are normally provided as trace contaminants that enter
the medium in water or serum. As the medium becomes more defined and the
water more pure, these trace elements need to be purposely added to the medium
formulation. Sometimes an increased concentration of vitamins can be useful.
Vitamin mixtures are commercially available and can be added as such. Some
vitamins, such as vitamin E (or a-tocopherol), A (retinol or retinoic acid), or C ,
are not added to media mixtures because of their instability, but may be important
for some cells to survive or function in vitro. To use vitamin E or A, make a
1000-fold stock solution in absolute ethanol (not benzene distilled) and dilute
in an aliquot of medium immediately before use. The vitamin E solution can be
stored in the dark at -20°C for 3-6 months. Vitamin A solutions should not be
stored longer than 1 week (-20°C, dark), and vitamin C can be made up in an
aqueous solution and discarded after use as it becomes toxic with storage.
XI. Medium Optimization

Many scientists have devoted their careers to understanding the role of nutrient
mixtures in supporting cell growth and survival in vitro. These studies have
28 Jennie P. Mather
resulted in the nutrient mixtures currently published or commercially available

(Ham, 1963,1965;Waymouth, 1959; Eagle, 1959; McCoy et al., 1959; Mather and
Sato, 1979) in Table I. There is, however, still a need for more experimentation to
derive optimal media for other cell types or other culture needs. Optimizing the
medium in which a primary culture or cell line is grown can lead to an increased
growth rate, increased protein secretion, increased viability, increased phenotypic
stability, and better control of differentiation. Optimizing the nutrient mixture
is an important part of this process.
The best way to optimize the nutrient mixture is to sequentially perform
dose-response curves on each component, select the optimal range for each,
and retest each component. This must be done as an iterative process because
the ratios of the components, as well as the absolute levels, are critical in optimiz-
ing the medium. This process should be done using the desired end point to
screen. For example, if a medium is to be optimized to achieve maximal recombi-
nant protein secretion, then the screen should be done using the protein titer as
the end point assayed. If growth is to be optimized, then the cell number is the
end point. Medium optimized for one parameter will not necessarily be best for
others (Mather, 1990; Perez-Infante et al., 1986; Roberts et al., 1990). Cells will
tolerate a broad range of concentrations for some medium components but have
a very narrow optimal concentration range for others.
All of these tests should be done in the presence of the medium supplement
that will be used (e.g., serum, growth factor mix). For optimizing hormone and
growth factor additions for serum-free culture, see Barnes and Sat0 (1980). Many
investigators may not wish to go to the expense and time required to optimize
specifically for their function. In this case, commercial media may be used, but
it is wise to spend a minimum of time determining which of the available options
is best. Steps for this “quick and dirty” optimization technique are outlined in
the following section. It cannot be overemphasized that optimizing medium for
one parameter such as growth may lead to a medium that is suboptimal for the
expression of a given protein, for response to a given growth factor, or for other
physiologic parameters. The medium must therefore be optimized, measuring
the parameter for which optimization is desired.
X I . Choosing the Optimal Medium: The “Quick and

Dirty” Method
1. Obtain and make up medium from several different nutrient mixtures. Be
sure and use the appropriate bicarbonate level for the incubator settings
(see earlier).
2. Supplement media with the required supplements (serum or hormones). If
serum reduction is a desired goal, run a dose-response curve for serum
and choose a serum concentration that gives 50% of the optimal growth,
thus allowing the detection of any “serum-sparing” effects of the media.
280 Melinda Pirity ef al.
The advent of this technology has made the mouse the mammal of choice for
mutagenesis approaches used in the study of embryonic development and disease
conditions. This chapter deals with the maintenance and modification of these
pluripotent cell lines and describes the routes that can be taken for their efficient
introduction to the in vivo environment.
I. Introduction
Since the mid-l980s, embryonic stem cell-mediated alterations of the mouse
genome have revolutionized the genetic approaches that can be employed in
mammalian biology and medical research. Such genome modifications have pro-
vided invaluable information in all fields of the life sciences concerned with
normal and disease biological function.
Historically, the appearance of ES cell lines was the consequence of knowledge
acquired during the 1970s by several investigators working on pluripotent em-
bryonal carcinoma (EC) cells (Martin and Evans, 1974). ES cells are derived
from preimplantation embryos, specifically from the inner cell mass (ICM) of
the blastocyst stage. After establishment these cells retain the potential of one
of the components of the ICM, the primitive endoderm. Thus they can contribute
to all the lineages of the embryo proper when introduced back into an embryonic
environment. Additionally they are restricted to contributing only to the tropho-
blast lineage of the placenta and some extraembryonic membranes, such as
parietal and visceral endoderm (Nagy et al., 1990). Most importantly, ES cells
can differentiate efficiently into germ cells, such that chimeras with germ cell
contributions from ES cells transmit the ES cell genome in vivo to their progeny
(the F1 generation offspring).
One of the most powerful transgenic technologies made possible by ES cells
is gene targeting based on homologous recombination (Capecchi, 1989). This
allows specific mutagenesis of any gene or genomic region of interest if at least
part of the region is characterized. Methods have been developed where this
type of in vivo mutagenesis can be performed to the level of a single base pair
change (site-directed mutagenesis), thus it is now possible to perform not just
null mutations, but specific subtle and conditional changes on a particular gene
(Hasty and Bradley, 1993; Gu et al., 1993,1994; Rossant and Nagy, 1995). These
methods can be categorized as directed genome alterations. The other main
categories of ES cell-mediated genome alterations are the entrapment strategies,
offering a gene expression pattern and/or mutagenesis type screen in ES cells
(Skarnes, 1990; Friedrich and Soriano, 1991;Hicks et al., 1997). Such entrapment
approaches involve random integrations of the vector into the ES cell genome,
such that the identity of the gene or regulatory element under investigation is
initially unknown. A specially designed transgene integrated in the vicinity of a
gene reports its features, such as expression pattern or possible involvement in
functional pathways (e.g., if the reporter responds to morphogens or transcription
16. Embryonic Stem Cells 281
factors) (Forrester et al., 1996). On the basis of this information, the gene or
genomic region can be cloned and its function investigated further. Entrapment
approaches, such as gene, promoter, and enhancer, are nondirected genome
alterations. ES cells can also be used for ‘‘classical’’ transgenesis, where an
exogenous gene expression unit is introduced randomly into the mouse genome
(De Primo et al., 1996).
11. Laboratory Equipment
The technical requirements (instrumental and special skills) for ES cell-

mediated transgenesis have been significantly simplified, thereby making this
technology available to laboratories with limited resources and expertise. There
is no longer a need for specially acquired technical skills such as the efficient
operation of micromanipulators (Wood et al., 1993). The entire procedure, from
establishing new ES cell lines through genetically altering the cells to deriving
transgenic animals, can be done in laboratories equipped for ordinary tissue
culture and having access to an animal facility. Details regarding setting up an
animal facility for preimplantation embryo production and embryo transfer can
be found elsewhere (Hogan et al., 1994). The only major additional equipment
and materials required for ES cell-mediated, transgenesis-related embryo work
are a dissecting microscope equipped with both a transmitted and incident light
source, some basic fine surgical instruments, and obviously a germline-compatible
ES cell line.
111. Culture Conditions

Culture conditions for ES cells and preimplantation stage embryos are well
established. No special medium or solution is needed. The specifications for
culture conditions are listed together with references in Table I.
In order to keep ES cells undifferentiated and at the maximum possible level
of developmental potential, they should be cultured in vitro for as short a time
as possible. The culture protocol should be strictly adhered to whenever possible.
IV. Maintaining Embryonic Stem (ES) Cells

The maintenance of ES cells is very much standardized. They require regular
passaging and splitting in specific ratios onto new tissue culture plates. The only
variation, being the number of days between passages, is due to some differences
in the rates of proliferation between individual cell lines.
There are several good germline-compatible ES cell lines currently available
for investigators starting with the technology. One should be able to choose any
282 Melinda Pirity et al.
Table I
Media and Solutions for ES Cell and Preimplantation Embryo Culture
Use Specification Note Reference
To culture ES cells (ES cell DMEM, high glucose Flow labs No. 430-1600 Manufacturers catalog
medium, referred to as supplemented with:
DMEM+)
+2 mM L-glutamine from lOOX solution of Manufacturer’s catalog
GIBCO NO. 320-5030AG
+lo0 pM fl-mercaptoethanol from 100X solution of Sigma Manufacturer’s catalog
No. 600564AG
+1 mM sodium pyruvate from lOOX solution of Manufacturer’s catalog
GIBCO NO. 320-1360
+0.1 mM nonessential from lOOX solution of Manufacturer’s catalog
amino acids GIBCO NO. 320-1140AG
+15% fetal calf serum Should be ES cell tested for Manufacturer’s catalog
plating efficiency
+lo00 Ulml LIF Necessary if ES cells are Heath et al. (1989)
cultured without feeder
cells, recommended
otherwise
To wash ES cells Phosphate-buffered saline Sambrook et al. (1989)
without Mg and Ca
To dissociate ES cells Trypsin Wurst and Joyner
(1993)
To freeze ES cells 2X freezing medium: VII.3.; Wurst and Joyner
ES cell medium/DMSO/ (1993)
FCS
To manipulate embryos on M2 medium Hogan et al. (1994)
bench
To culture embryos M16 medium Hogan et al. (1994)
To remove zona pellucida Acid Tyrode’s Hogan et al. (1994)
line reported in the literature to have been proven to give germline transmission.
The only factor to consider regarding the choice of ES cells will be addressed
in Section XV.
V. Preparing Tissue Culture Plates for ES Cells

ES cells are usually kept on primary embryonic fibroblast feeders. Protocols
for the preparation of primary embryonic fibroblasts, and feeder plates containing
them, are given in detail elsewhere (Wurst and Joyner, 1993). Some ES cell
lines do not require feeder cells to remain at their full developmental potential
after in vitro culture (Nagy et al., 1993). Such lines require only gelatin coating
of the tissue culture plate, which means rinsing the bottom of the plate with
0.1% (w/v) sterile gelatin (Sigma or BDH) solution prior to plating the cells.
VI. Passaging ES Cells
A properly maintained culture of ES cells should reach 50-60% confluence

prior to passaging. The following procedure is for a 10-cm-diameter tissue culture
plate (Fig. 1). For smaller plates the volume should be changed proportionally.
1. Aspirate ES cell medium from the plate and then wash the plate with
10 ml of phosphate-buffered saline (PBS).
2. Replace PBS with 2 ml of trypsin solution and incubate the plate for 5 min
at 37°C. During this time the clumps should lift up from the bottom of the plate
and the cells become loosely connected.
3. Stop the action of trypsin by adding 4 ml of ES cell medium to the plate
and transfer the cells to a 15-ml centrifuge tube.
4. Spin them down at 1000 g for 5 min.
5. Aspirate supernatant and gently resuspend the pellet in 5-7 ml ES cell
medium.
6. Split the cell suspension in a 1:5 to 1:7 ratio onto prepared tissue culture
plates containing 10 ml of ES medium.
7. Place the plates back into a humidified C 0 2 incubator for culture.
8. Repeat steps 1-7 every other day or every 3 days, depending on the particu-
lar cell line recommendation.
VII. Freezing ES Cells from Confluent 10-cm Tissue

Culture Plates
1. Follow steps 1-4 in the previous protocol for ES cell passaging.
2. After spinning down the cells, aspirate supernatant and gently resuspend
the pellet in 2 ml of ES medium.
Fig. 1 Passaging ES cells.

3. Distribute the cell suspension into four 1.5-ml cryovials and then add
0.5 ml of 2X freezing medium to each.
4. Place the cryovials at -70°C in a Styrofoam box or isopropanol cooler
(Stratagene Cloning Systems, LaJolla, CA) for at least 6 hr and then transfer to
liquid nitrogen for long-term storage.
VIII. Thawing ES Cells from Freezing Vials

1. Prepare a gelatinized or feeder cell containing a 6-cm plate.
2. Thaw vials as quickly as possible in a 37°C water bath.
3. Immediately after the ice has melted, transfer the contents to a centrifuge
tube and slowly add 4 ml of fresh prewarmed medium.
4. Spin cells down at 1000 g for 5 min.
5. Aspirate the supernatant and gently resuspend the pellet in 5-7 ml ES
cell medium.
6. Plate the cell suspension onto the prepared tissue culture plate.
7. Place the plate back into a C 0 2 incubator for culture and then follow the
ES cell maintenance procedure outlined earlier.
IX. Establishing ES Cells

The establishment of new ES cell lines from specific strains or mutant embryos
is increasingly desirable. This task is feasible once some experience has been
gained with maintaining ES cells (Fig. 2).
1. Recover blastocyst-stage embryos from females at 3.5 days postcoitus and
place one embryo in each well on feeders in 4- or 24-well plates containing
ES cell medium.
2. After attachment of the embryo to the feeder layer (usually 2 days), replace
half of the medium daily with prewarmed fresh medium for 3-4 days.
Q
4 ___,
2-3 days 3-4 days
Fig. 2 Establishing ES cell lines.

During this period the original ICM of the blastocyst forms a large colony
of cells (blastocyst outgrowth).
3. Change the medium to PBS. Disaggregate the blastocyst outgrowth by
transferring it into a 96-well plate containing 50 pl of trypsin for 5-10 min
and then transferring the broken up cells to a new feeder-containing 4- or
24-well plate.
4. Replace half of the medium every day with prewarmed fresh medium
for 3-6 days; at this point the appearance of ES cell-like colonies should
be observed.
5. If there are several ES-like colonies of several hundred cells, the cells can
be expanded by regular ES cell maintenance as detailed previously. If
only one or two colonies look promising, they should be picked again and
disaggregated, as was done with the blastocyst outgrowth (step 3).
X. Introducing DNA into and Selecting for Genetically

Altered ES Cells
The most common way of creating genetically altered ES cells is to introduce
exogenous DNA into the cells by electroporation and to subsequently select an
integration into the genome (Fig. 3). Usually the selection is based on a drug
resistance gene implemented into the endogenous DNA, which delivers resis-
tance to the cells that integrate the vector. The most commonly used selectable
markers are neomycin phosphotranferase (neo), puromycin, and hygromycin.
The time required for complete killing of drug-sensitive cells varies among select-
able markers. Of these three, puromycin kills in the shortest time period, 3-4
Fig. 3 Electroporating ES cells.

286 Melinda Pirity et nl.
days, the neo selection requires 7-9 days, and the hygromycin selection takes
about 15-20 days. The design and architecture of the vectors used to introduce
DNA into ES cells vary, depending on the application, and can be found in
specific reviews (Hasty and Bradley, 1993; Gossler and Zachgo, 1993). For the
efficient introduction of a DNA vector into ES cells, the following steps should
be adhered to.
1. Grow up ES cells to the required number, usually 107-108 cells, depending
on the project.
2. Prepare vector DNA, linearize, purify by ethanol precipitation, and adjust
concentration to 1pg.p1 in sterile 10 mMTris, l m MEDTA (TE) or water.
3. Trypsinize cells as for passaging (see earlier), except resuspend the final
pellet in ice-cold PBS.
4. Count cells and adjust their concentration to 7 X 106/mlin PBS.
5. Add 10-40 p1 DNA and 790-760 p1 cell suspension into a 0.8-ml electro-
poration cuvette.
6 . Repeat step 5 according to the number of electroporations planned. Usu-
ally this number is 1 or 2 for ordinary transgenesis and 10-15 for gene
targeting or gene trap experiments.
7. Using an electroporator, such as the Bio-Rad GenePulser, discharge
500 pF at 250 V through the cuvette.
8. Place the cuvettes on ice for 15-20 min.
9. Transfer the cell suspension from the cuvettes into ES cell medium, which
allows plating one cuvette of cells onto two 10-cm tissue culture plates.
The plates could be gelatinized or drug-resistant feeder cell plates.
10. Culture the cells for 1 or 2 days in normal ES cell medium.
11. Start the selection by adding the selective drug to the medium. The concen-
tration of the drug should be the lowest concentration that completely
kills nonresistant cells.
XI. Picking ES Cell Colonies

At the end of the selection period the genetically altered, drug-resistant ES
cells will have formed distant colonies comprising 100-250 cells. These should
be picked and grown up independently for further analysis and studies. Even if
the killing time of the drug used is fast, it is not recommended to pick colonies
earlier than 8-9 days after the electroporation, as the colonies should reach
a certain size (cell number) to efficiently survive the next step: their picking
and expansion.
1. Prepare a V-bottom 96-well plate with 50 pl of trypsin in each well.
2. Prepare a flat-bottom 96-well gelatinized plate with 150 pl of ES medium
in each well for culture of the ES cell colonies.
3. Place a dissecting microscope into a sterile laminar flow hood (this will
be used to view the colonies to be picked).
4. Replace the ES medium with PBS on the selection plate.
5. Using a P20 pipetteman (set to 10 pl), knock off the colonies with the
yellow tip, suck them up, and immediately transfer them into the trypsin-
containing plate, one at a time.
6 . Change the yellow tip and repeat step 5 until the 96-well plate is full or
until no more colonies remain to be picked.
7. Using a multichanel pipette, pipette cells into the trypsin-containing plate
to disaggregate.
8. Transfer the rows of the trypsin-containing plate into the 96-well ES cell
medium-containing plate.
9. Place the plate into the incubator and change the medium after over-
night culture.
10. Expand colonies with regular passaging and create identical sets of 96-
well plates: one for freeze-storage and the others for characterization
of clones.
XII. Detecting Genome Alterations
Usually, clones are screened for the type of integration of the introduced DNA
vector by Southern blotting. Preparing DNA from a 96-well plate is described
in detail elsewhere (Wurst and Joyner, 1993). The only concern is that not
all restriction enzymes cut the 96-well plate-prepared DNA satisfactorily. It is
worthwhile checking in advance whether the chosen diagnostic enzyme(s) works
with DNA extracted using the 96-well plate method. Table I1 shows different
commonly used enzymes. This information should not be taken as fact, but as
experience based on experiments conducted in the authors’ research estab-
lishment.
XIII. Freezing ES Cells in 96-Well Plates
For almost all applications, the freezing of colonies is required shortly after
picking. Fortunately, freezing of cells within the 96-well plate is possible, therefore
large numbers of clones can be stored for short term (up to 2-3 months), while
analyses of the clones are going on for the parallel plate(s).
1. Freshly prepare 2X freezing medium and place it on ice.
2. Using a multichanel pipette, aspirate the ES medium from the wells and
wash the cells with PBS. Add 25 p1 of trypsin to each well and incubate the
plate for 5-10 min at 37°C.
288 Melinda Pirity et el.
Table I1
Ratings of 96-Well Plate ES Cell Genomic
DNA Digestion Enzymes
Enzyme performance
Good Variable Bad
Asp718 EcoRI BspDI

BamHI CIaI
EglII Hind11
EcoRV Not1
Hind111 Sac1
KpnI Sac11
NcoI SalI
PstI SrnaI
PVUII XhoI
ScaI
StUI
3. Stop the action of trypsin by adding 75 pl of ES medium to each well and

then add 100 p1 of 2X freezing medium.
4. Overlay the contents of each well with 30 pl embryo-tested paraffin oil
(Sigma Cat. No. M-6410) and seal the plate with Parafilm.
5. Place the plate in a suitable Styrofoam box and put into a -70°C freezer
for storage.
XIV. Thawing ES Cells in 96-Well Plates

Usually, only a few of the colonies that were originally frozen in the 96-well
plate are needed for further studies. In this case the following protocol should
be used for thawing specific clones.
1. Prepare a gelatinized 24-well plate containing 2 ml of ES medium in
each well.
2. Place the frozen 96-well plate onto the surface of a 37°C water bath on a
properly installed stage.
3. After the ice has melted, carefully aspirate the oil layer and about 100 p1
of medium from the wells of interest. Always aspirate the liquid from the
top of the well.
4. Add 100 pl of medium to the wells of interest and transfer the cells
( 2 0 0 4 total volume) into the wells of the 24-well plate.
5. Culture the 24-well plate overnight and then change the medium daily
until passaging.
6. Expand the cells by regularly passing them into consecutively larger plates
as the cell number increases. When the cells are 70-80% confluent on a
10-cm plate, there are enough for half to be frozen down in order to pro-
duce low passage stocks.
XV. Chimeras
Having established mutant or transgenic ES cell lines, the next step is the in vivo
introduction of the altered genome, which is performed through the production of
germline-transmitting chimeric mice. There are two alternative ways of chimera
production: (1) injection of ES cells into blastocyst-stage mouse embryos using
micromanipulators and (2) aggregating ES cells with eight-cell-stage mouse em-
bryos.
There are pros and cons as to which of the two alternatives should be used
(Nagy, 1997). Generally, for laboratories with limited expertise just starting up
ES cell technology, the latter is recommended, as it does not require expensive
instrumentation or a high skill level. It does, however, require optimum in vitro
embryo culture conditions, as the ES cell<->embryo aggregates need to be
cultured for 24 hr before they are transferred into pseudopregnant recipient
females. A further point to consider is that most ES cell lines are tested for
germline transmission by blastocyst injection. It is, however, possible that not
all the lines that have been shown to be working by blastocyst injection will
work equally well with aggregation. To avoid problems in this phase of the
experiment, it is wise to choose an ES line shown to be germline compatible
with the method of chimera production favored (Wood et al., 1993).
Most of the available ES cell lines are derived from 129 inbred agouti mouse
strains. The favored choice for an embryo donor is usually an outbred strain of
albino mice, as they are the least expensive, give good embryo yields after
superovulation, and the chimerism can be easily detected by the coat color and
eye pigmentation. A method for the production of aggregation chimeras is given;
methods for blastocyst injection are detailed elsewhere (Hogan et al., 1994).
XVI. Preparation of the Aggregation Plate
1. Place M16 microdrops (2-3 mm in diameter) into a 35-mm tissue culture

dish (Fig. 4).
2. Cover the plate with embryo-tested paraffin oil (Sigma Cat. No. M-8410).
3. Using aggregation needles (Biochemical Laboratory Service, 31 Zselyi Ala-
dar utca, Budapest H01165, Hungary), make five to six deep depressions in each
M16 microdrop in the bottom of the plate.
4. Place the plate in the incubator for equilibration with CO2.
290 Melinda Pirity el al.
Fig. 4 Making an aggregation plate.
XVII. Preparation of Embryos

A detailed technical description of recovering and manipulating preimplanta-
tion stage embryos are beyond the scope of this chapter. The reader can find
sufficient information elsewhere (Hogan et al., 1994).
1. Recover eight-cell-stage embryos from females at 2.5 days postcoitum.
2. Remove the zona pellucida by a brief acid Tyrode’s treatment.
3. Place the zona-free embryos in the aggregation plate, one embryo in
each depression.
4. Place the plate in the incubator until the ES cells are ready for aggrega-
tion.
XVIII. Preparation of ES Cells for Aggregation with

Eight-Cell-Stage Mouse Embryos
1. Thaw a vial of ES cells 4 days prior to aggregation on feeder plates.
2. Two days later pass the cells very sparsely (1:25) onto a gelatinized plate.
3. On the day of aggregation (after the embryos are already in the aggregation
plate), trypsinize the plate of ES cells as described previously.
4. Stop the trypsin action by adding the appropriate amount of ES medium
to the plate. (Do not centrifuge cells!)
5. Collect loosely connecting clumps of 10-15 cells from the plate and transfer
them to a plate containing a large amount PBS (to get rid of the ES cell medium).
6. Wash the clumps through one or two M16 drops.
XIX.ES Cell<->Embryo Aggregations

1. Place several clumps of loosely connected ES cells in the M16 drops of the
aggregation plate, where the embryos are already sitting in the bottom of each
depression (Fig. 5).
2. Select an individual clumps of 10-15 cells and place it beside the embryo
in the depression.
3. Make sure that the embryo and the clump are in direct physical contact.
A depression with the proper dimensions should keep the cells and embryo
together, thereby promoting their aggregation.
4. Incubate the plate at 37°C in a humidified C 0 2 incubator for 24 hr and
then transfer the embryos to pseudopregnant recipients (Hogan et al., 1994).
XX. Germline Transmission
Chimeras are usually detected on the basis of coat color. If the host mouse
strain used is albino, then chimeras can be identified in newborn pups on the
basis of eye pigmentation. Male cell lines are used almost exclusively in ES cell-
mediated transgenesis. If male ES cells are aggregated with females embryos,
which happens in 50% of cases, they can take over the sex determination and
render the chimera to be a phenotypic male. Because only male cells can go
through spermiogenesis, a fertile male in such a situation will exclusively transmit
the ES cell compartment to its offspring. Therefore, a distortion in the male/
female ratio among the chimeras, favoring the males, is a promising sign for
germline contribution. The F1 generation fathered by a germline transmitting
chimera should be checked for the presence of the transgene or genome modifi-
cation, as ES cells are usually heterozygous for such a modified allele, only 50%
transmission will be observed within F1 generation.
Fig. 5 Setting up the aggregation.

XXI. Perspectives
The advent of embryonic stem cells has revolutionized genetic approaches
that can be undertaken in mammals, with the analysis of mouse “knockout”
lines having provided a tremendous amount of information concerning a diverse
range of biological phenomena. ES cells provide not only an alternative way of
creating transgenic animals, but have also pioneered the development of a new
set of transgenic technologies exclusively tailored to them. On the virtue of their
high numbers (10-4-10-s), ES cells have allowed the identification of such a rare
event as the recombination between a target vector and its homologous sequence
within the target genome. These new ES cell-based tools were initiated by homol-
ogous recombination-based gene “knockouts”, with the number of genes that
have been “knocked out” to date being close to 2000. As this technology becomes
more commonplace in a larger number of laboratories, the number of generated
knockouts is expected to rise dramatically.
The new generation of tools combines homologous and site-specificrecombina-
tion so efficiently that practically any desired genome alteration, starting with a
simple “directed” point mutation to site-specific chromosomal rearrangements
(translocations and large deletions), is now feasible for creating in ES cells, and
then subsequently introducing into mice. This allows us to produce phenocopies
of any human genetic disease condition, and therefore to create proper animal
disease models.
References
Capecchi, M. R. (1989). Altering the genome by homologous recombination. Science 244,1288-1292.
De Primo, S. E., Stambrook, P. J., and Stringer, J. R. (1996). The human alkaline phosphatase as
a histochemical marker of gene expression in transgenic mice. Transgen. Res. 5(6), 459-466.
Forrester, L. M., Nagy, A., Sam, M., Watt, A., Stevenson, L., Bernstein, A., Joyner, A. L., and
Wurst, W. (1996). An induction gene trap screen in embryonic stem cells: Identification of genes
that respond to retinoic acid in vitro. Proc. Natl. Acad. Sci. U S A 93, 1677-1682.
Friedrich, G., and Soriano, P. (1991). Promoter traps in embryonic stem cells: A genetic screen to
identify and mutate developmental genes in mice. Genes Dev. 5, 1513-1523.
Gossler, A., and Zachgo, J. (1993). Gene and enhancer trap screens in ES cell chimeras. In “Gene
Targeting” (A. L. Joyner), ed.). Oxford Univ. Press, London.
Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994). Deletion of a DNA
polymerase fl gene segment in T cells using cell type-specific gene targeting. Science 265,103-106.
Gu, H., Zou, Y. R., and Rajewsky, K. (1993). Independent control of immunoglobulin switch recombi-
nation at individual switch regions evidenced through Cre-IoxP-mediated gene targeting. Cell
73, 1155-1164.
Hasty, P., and Bradley, A. (1993). Gene targeting vectors for mammalian cells. In “Gene Targeting:
A Practical Approach” (A. Joyner, ed.), pp. 1-31. IRL Press at Oxford Univ. Press, London.
Heath, J. K., Smith, A. G., Wills, A. J., and Edwards, D. R. (1989). Growth and differentiation
factors of embryonic stem cells. In “Cell to Cell Signals in Mammalian Development” (S. W. de
Laat et al., eds.), pp. 247-260. Springer-Verlag, Berlin.
Hicks, G. G., Shi, E. G., L, X. M., Li, C. H., Pawlak, M., and Ruley, H. E. (1997). Functional
genomics in mice by tagged sequence mutagenesis. Nut. Genet. 16(4), 338-344.
Hogan, B., Beddington, R., Costantini I., and Lacy E. (1994). “Manipulating the Mouse Embryo.”
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Martin, G. R., and Evans, M. J. (1974). The morphology and growth of a pluripotent teratocarcinoma
cell line and its derivatives in tissue culture. Cell 2(3), 163-172.
Nagy, A. (1997). Formation of mouse chimeric embryos from ES cells. In “Transgenic Animal
Generation and Use” (L. M. Houdebine, ed.). Hanvood Academic, Amsterdam.
Nagy, A., Gocza, E., Diaz, E. M., Prideaux, V. R., Ivanyi, E., Markkula, M., and Rossant, J. (1990).
Embryonic stem cells alone are able to support fetal development in the mouse. Development
110,815-821.
Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. (1993). Derivation of com-
pletely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci.
USA 90,8424-8428.
Rossant, J., and Nagy, A. (1995). Genome engineering: The new mouse genetics. Nat. Med. 1,592-594.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning: A Laboratory Manual.”
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Skarnes, W. C. (1990). Entrapment vectors: A new tool for mammalian genetics. Biotechnology
8,827-831.
Wood, S . A,, Allen, N. D., Rossant, J., Auerbach, A,, and Nagy, A. (1993). Non-injection methods
for the production of embryonic stem cell-embryo chimeras. Nature 365, 87-89.
Wurst, W., and Joyner, A. L. (1993). “Gene Targeting: A practical Approach” (A. L. Joyner, ed.),
p. 33. Oxford Univ. Press, London.
SECTION IV
Microscopy and Morphology
This section is used to acquaint the reader with the variety of available
techniques on how to look at cells, how these methods can best be adapted
to cultured cells, and how and when to use the individual techniques to
answer specific questions most efficiently. Readers may wish to refer to
Volume 42 in this series for detailed information on flow cytometry meth-
ods that are useful in looking at population distributions in a way that is
difficult or impossible using biochemical methods. The methods discussed
in this section are all designed to see the culture as a collection of individual
cells and to allow one to study the localization of cellular phenomena in
a detailed fashion.
This section starts with a chapter on transmission and scanning electron
microscopy that can be used to obtain detailed information on cultured
cells. This is followed by a chapter (Chapter 18) on indirect immunofluo-
rescence microscopy in cultured cells. This might be expanded by referring
to Volume 29 in this series. Finally, in situ mRNA hybridization histo-
chemistry and in situ ligand binding can be used to visualize cells producing
a specific message or expressing a receptor for a specific ligand.
CHAPTER 17
Electron Microscopy: Use of Transmission

and Scanning Electron Microscopy to
Study Cells in Culture
David M. Phillips
The Population Council
New York, New York 10021
I. Introduction
11. Studying Monolayer Cultures with the Transmission Electron Microscope
A. Glutaraldehyde Fixation
B. Postfixation in Osmium Tetroxide
C. Staining with Uranyl Acetate
D. Dehydration
E. Embedding in Plastic
F. Separating the Epon from the Culture Dish
G. Mounting the Cells
H. Staining
111. Cells Grown on Filters or Matrices
IV. Cells Grown in Suspension
V. Studying Monolayer Cultures with Scanning Electron Microscopy
VI. Cells Grown in Suspension
VII. A Few Hints
References
Standard techniques for electron microscopy were developed for tissues dis-
sected from animals. Optimal methods for electron microscopy of cells in culture
are different. This chapter describes methods for processing cells grown on
plastic or in suspension. Both transmission and scanning electron microscopy
are discussed. The focus is on the procedures for fixation, dehydration, embed-
ding, and staining, which will help the reader to obtain superior electron micro-
graphs of cultured cells.
METHODS IN CELL BIOLOGY, VOL. 57
Copyright Q I998 by Acadenlic Press. AU rightr of reproduction in any form reserved 297
(K)Y1 -67YX/Y8 $25.00
298 David M. Phillips
I. Introduction
This chapter is intended primarily for the investigator who wants to use the
transmission electron microscope (TEM) or scanning electron microscope (SEM)
to study cells grown in culture. Readers should have at least a rudimentary
knowledge of how to process tissues for electron microscopy or have access to
a microscopy facility where they can obtain instruction on the basic techniques
for microscopy. Those who need background information in these techniques
may wish to refer to one of the several textbooks that describe the basics of
electron microscopy. This chapter concentrates on techniques specific for cells
in culture.
11. Studying Monolayer Cultures with the Transmission

Electron Microscope
The procedures for tissue preparation for the TEM that are described in most
textbooks were developed for plant and animal tissue specimens that have been
cut into small pieces. In contrast to cells in culture, which are generally thin
layers on a substrate or individual cells in suspension, pieces of tissue need to
remain immersed in the various solvents for relatively long periods in order to
infiltrate into the tissue. Techniques for embedding in plastic similarly allow time
for the slow infiltration of viscous plastics into tissue. However, with cultured
cells, the intervals between changes of solvents can be far shorter than described
in most electron microscope textbooks, and standard methodology employed for
embedding should be modified.
Most culture work is carried out on monolayer cultures in plastic T flasks or
wells. The standard plasticware is suitable for TEM work. In fact, processing
cells on plastic has a number of advantages over processing cells grown on filters,
glass, or in suspension. After being processed, cells grown on plastic are cut
parallel to the dish. This usually facilitates the clearest appreciation of relation-
ships among neighboring cells (Fig. 1). For example, junctional complexes or
interdigitations between adjacent cells are best observed in this type of section,
which makes it relatively easy to determine the level at which the cells are
transected. This would also be useful in the study of structures at a particular
height in an epithelial cell layer or the area of contact between cells and the
substrate by examining the first sections that are cut from the substrate side of
the specimen (Fig. 2).
The procedure for preparation of cells grown on plastic is as follows.
A. Glutaraldehyde Fixation
A number of different concentrations of glutaraldehyde and different buffers
have been used successfullyfor fixation. It has been found that 3% glutaraldehyde
17. Electron Microscopy 299
Fig. 1 TEM of myofilaments and sarcoplasmic reticulum in a rat cardiac muscle cell in primary
culture. In culture, myofilaments tend to extend parallel to the substrate. Sections cut parallel to
the dish reveal myofilaments in the longitudinal section.
Fig. 2 Section of primary keratinocytes. This is the second or third section and shows microvilli
extending underneath adjacent cells.
(EM grade) in 0.15 M Sorenson’s phosphate buffer at pH 7.4 gives good results
for almost all cultured cells. The culture medium is decanted and replaced with
new medium. This medium is decanted and replaced with fixative. The dish or
T flask is then put in a refrigerator. Cells should remain in fixative for a minimum
of 1 hr, but they can remain in the same fixative in the refrigerator for a few
days before they are processed further. The author generally leaves the cells in
fixative overnight and processes them the next morning.
B. Postfixation in Osmium Tetroxide

The glutaraldehyde fixative should be removed with three or four washes in
0.15 M phosphate buffer (total time 5 min). Be careful that the cells do not dry
out during washing or subsequent processing. Os04 is poisonous, toxic to breath,
and stains skin black on contact. It should be used with caution in a fume hood.
Make a stock solution of 2% Os04 by adding 1 g Os04 to 50 ml of double-
distilled water in a 100-ml screw-top bottle at room temperature. Store the
solution in the refrigerator. The O s 0 4 takes 1-2 days to dissolve. If necessary,
Os04 can be put into solution quickly by heating to 45°C in a water bath while
stirring. The 2% Os04 stock solution will keep in the refrigerator for months.
Fix for 1 to 2 hr at 4°C in a 1:l solution of 2% Os04 stock and phosphate buffer.
C. Staining with Uranyl Acetate

The technique of staining cultured cells in uranyl acetate following fixation
results in membranes that appear crisp and bilaminar and adds additional contrast
to other structures (Fig. 3). The first step in staining is to remove the Os04 by
washing in phosphate buffer three times (total 5 min). If T flasks are used, the
top of the flask needs to be cut off when it is in buffer. Remove a large rectangular
region in the top of the T flask by cutting with a thick scalpel heated red hot in
a Bunsen burner. Remove the buffer by washing in distilled water three times
(total time 5 min) followed by washing three times with 30% methyl alcohol at
room temperature (total time 10 min). Replace the 30% methyl alcohol with a
staining solution of uranyl acetate in 30% methanol. The staining solution of
uranyl acetate can be made from a stock solution of 2% aqueous uranyl acetate.
It takes a while to get uranyl acetate into solution by stirring. Keep the stock
solution in the dark. (Uranyl acetate is moderately radioactive and should be
handled accordingly.)To make the staining solution, add two parts of the aqueous
2% solution to one part 100% methyl alcohol. Cells are stained for 1 to 2 hr in
the dark.
D. Dehydration
Dehydration to absolute alcohol is accomplished by washing at room tempera-
ture in the following series of solutions:
1. 30% methyl alcohol for 10 rnin
2. 50% ethyl alcohol for 10 min
3. 70% ethyl alcohol for 10 rnin
4. 95% ethyl alcohol for 10 min
5. 100% ethyl alcohol for 20 min.
Make four or five changes to ensure that all the 95% alcohol is removed. It
is important that no water remain in the alcohol when the plastic is added. Keep
the dish away from sink or air flow (as in a hood). If the alcohol evaporates
rapidly and cools it may pick up water. When working in a humid room, be
especially careful to assure removal of all water (see Section VII).
Fig. 3 Junction between two (ME 180) cervix-derived epithelial cells. The bilaminar appearance
of membranes is especially obvious when cells are stained with uranyl acetate following fixation.
E. Embedding in Plastic
Acetone and propylene oxide, the solvents usually employed to facilitate
proper infiltration, cannot be used for embedding cells grown on plastic because
they will dissolve the plastic. However, these solvents can be avoided with mono-
layer cultures because only one layer of cells needs to be infiltrated.
Because plastic is messy and difficult to remove from surfaces, all work should
be done with disposable plasticware or glassware. Plastic is made fresh in a
50-ml tripour beaker. Marks on the side of the beaker are accurate enough for
measuring. The formula is 5 ml Dodecenyl Succinic Anhydride (DDSA), 20 ml
Nadic Methyl Anhydride (NMA), 25 ml Epon 812 or equivalent (“Polybed”
from Polysciences Inc.), and 0.8 ml DMP-30. (Because it is viscous, DMP-30
cannot be accurately measured with a pipette. DMP also dissolves some plastics.
The author suggests measuring DMP-30 in a l-ml glass syringe.) Mix the plastic
very well by inverting the beaker for a full 5 min. (It is essential that the plastic
be uniform.)
Replace the alcohol with fresh plastic. Make three changes and rock the culture
dish before pouring out the plastic so that it will mix with the alcohol (15 min
total time). This process can get messy, and quite a bit of plastic may be needed,
but it is important to remove the alcohol. Pour out the plastic, leaving just enough
to thinly coat the bottom of the flask. If too much is left, the polymerized plastic
may be difficult to separate from the cell culture plasticware. However, do not
leave so little that the plastic ends up paper thin. Immediately place in a 60°C
oven. Leave samples in the oven for 48 hr.
F. Separating the Epon from the Culture Dish

Remove the dish from the oven. Break the sides of the well or flask with
pliers and carefully remove the broken pieces of the culture dish around the
polymerized Epon disc. Carefully separate the polymerized plastic from the
bottom of the well. This works better when the dish is warm, so if the process
takes more than a couple of minutes, put the dish back in the 60°C oven for a
while before proceeding. If care is taken, the Epon disc can be separated in one
piece from a 24-, 12-, or 6-well dish or a T flask.
G. Mounting the Cells

The disc will have a higher rim (meniscus) around the edge. Carefully remove
the rim of plastic with a small grinding wheel. The Epon disc can be viewed with
a dissecting microscope. A Magic marker can be used on the side of the disc
away from the cells to distinguish which side the cells are on when they are
mounted. In some cases, selecting areas of the culture or even individual cells
can be done before mounting. To select areas, view the disc in a dissecting
microscope. The lighting may have to be adjusted to see the cells. This allows
you, for example, to find an area that has the density of cells that is desired. The
disc can also be viewed with a phase-contrast microscope with a 40X or even a
lOOX objective. This allows an individual cell to be observed. A circle of various
diameters can be drawn with a diamond tip (Carl Zeiss Cat. No. 662960), which
will etch a circle on the plastic.
Using a small vise to hold the disc, cut out little pieces of the disc with a
jeweler’s saw and mount them on blocks with Crazy glue. It is advisable to make
blank capsules with appropriate labels at the same time the cells are embedded.
The tips of the capsules may need to be flattened with a grinding wheel to provide
a flat surface on which to glue the pieces of Epon disc.
Use a sharp single edge razor blade to trim the blocks. The author prefers
WECK WECPREP razor blades (Electron Microscopy Sciences, Fort Washing-
ton, PA). If care is taken aligning the block when sectioning, the full face of the
block will be gradually cut by the first dozen or so sections. If the region of
contact between the cell and the substrate is of interest, view the first dozen
sections, which contain areas immediately above the substrate (Fig 2).
H. Staining
There are many techniques for staining cells. The author suggests avoiding lead
citrate because it tends to leave the sections grainy or dirty. With the technique of
staining cells in the block with uranyl acetate in methanol, an additional staining
in uranyl acetate alone should result in sufficient contrast without lead staining.
Stain sections with 2% uranyl acetate solution for 30 min at 50°C.
111. Cells Grown on Filters or Matrices
Cutting cells perpendicular to the substrate may be required for making certain
types of observations, e.g., to examine the entrance of a pathogen into a cell or
movement of a cell through an epithelium (Fig. 4). A disadvantage of this
technique is that many fewer cells are seen then when cells grown on plastic are
cut parallel to the substrate. This is because only a single row of cells is seen.
Cells should be cultured on filters that can be infiltrated with plastic. Some insert
filters work better than others. The author has had good success with Costar
insert filters or Millipore HA insert filters (Fig. 5 ) .
The technique of preparing filters involves fixing in glutaraldehyde overnight.
Insert filters are then removed by cutting around the edge of the filter with a
fine dissecting knife or a miniblade scalpel. The filter is then cut into pieces with
sharp microdissecting scissors. Pieces of filter can be processed much like a piece
of tissue, but embedding is done without propylene oxide. Fix, postfix, stain, and
dehydrate as described earlier for cells grown on plasticware, but leave the filters
in the dehydration alcohols a little longer. After dehydrating with 100% alcohol,
mix alcohol with freshly made Epon in a ratio of approximately 1 part alcohol
Fig. 4 Culture of primary human endothelial cells grown on a collagen gel. The section cut
perpendicular to the substrate shows a lymphocyte passing between endothelial cells. For details of
methods, see Muller et al. (1993).
to 5 parts plastic. Replace the alcohol with this blend, mix carefully, and leave
overnight at room temperature. The next morning, make fresh plastic. Place
labels in the cavities of a flat embedding mold and fill the cavities with the freshly
made plastic. Transfer the pieces of filters in a few changes of plastic before
putting in the cavities of the molds. Place the molds in a desiccator. At the end
of the day, place the molds in a 60°C oven.
Cells can also be grown on substrates such as collagen or agarose (Fig. 4),
which can be processed similarly to filters. The author does not recommend
growing cells on glass because techniques for separating glass from plastic can
damage the cells, and cells generally grow better on plastic than on glass.
IV. Cells Grown in Suspension
Cells in suspension can be fixed and centrifuged into a pellet, which can be
processed in the same way as a piece of tissue. Fix the cells in suspension;
centrifugation prior to fixation will distort the cells and the fixative will not
Fig. 5 TEM of a stratified epithelium that was cultured on a transwell membrane insert (Costar).
The layers of the epithelium are best appreciated in sections cut perpendicularly to the plane of the
filter. The structure of the filter is well represented, indicating that this type of filter is especially
suitable for TEM work. For details of methods, see Roberts et al. (1990).
penetrate as quickly if the cells are pelleted. Cells should be fixed only briefly
prior to centrifugation; if they are fixed too well before centrifugation, the fixation
will not cross-link proteins of adjacent cells, and the pellet will disintegrate during
processing (Fig. 6). It is important to have a pellet that is about as high as it is wide.
A very flat pellet may disintegrate. The following technique is recommended.
Fig. 6 TEM of lymphocytes from a suspension culture. Cells were fixed in suspension before
centrifugation and therefore retained their shapes. Because they were fixed only briefly, continued
glutaraldehyde cross-linking occurred after pelleting, causing the adherence of neighboring cells to
maintain the integrity of the pellet.
1. Centrifuge lo6 cells for 2 min at 3000 g in medium in a 1.5-ml Eppendorf

tube.
2. Add 1 ml of fixative (as described earlier) to the pellet of cells and mix
by inversion.
3. One minute after adding the fixative, centrifuge in a microfuge for 2 min
at 3000 g.
4. Place in a refrigerator overnight. The next morning, use a wooden stick
carved to a sharp point with a razor blade to dislodge the pellet. Process
the pellet like a piece of tissue. The cells should be stained in uranyl acetate
in methanol as described earlier.
If fewer than lo6 cells are needed, use a smaller centrifuge tube. Use a
4OO-pl tube with 5 X lo5 cells. If there are only lo5 cells, use a Sarstedt 72.707
tube (Sarstedt, Newton, NC), which has a small nipple at the base.
V. Studying Monolayer Cultures with Scanning

Electron Microscopy
No matter how carefully cells are critical-point dried for the SEM, they shrink.
This is a major problem with cells that are cultured on a solid substrate; as the
cell shrinks, processes that extend to the substrate shorten and frequently break
or flatten (Fig. 7). The way to minimize these artifacts is to find areas where
Fig. 7 Scanning electron micrograph of cultured cervix-derived epithelial cells. ME180 cells are
typically interconnected by processes and junctions. Arrows show where cellular processes are broken,
presumably during critical-point drying.
groups of cells have lifted off the plate and shrunk as a unit or to scratch the
plate so that cells lift off before fixation. The following technique can be used
for processing cells on plastic.
1. Scratch the plate or use a rubber policeman to lift off some cells; wash the
cells two or three times in serum-free medium.
2. Fix cells overnight in phosphate-buffered 3% glutaraldehyde.
3. The next morning, rinse several times in buffer. Use a 1-in. diameter circular
saw blade and a variable-speed Moto-tool (Jensen Tools, Phoenix, AZ) to
cut a piece of plastic that will fit in the critical-point drying apparatus that
is being used. Irrigate the cells with buffer so that they do not dry out when
cutting the plastic.
4. Dehydrate in alcohol series to 100% alcohol and critical-point dry from
100% alcohol.
5. Mount the piece of plastic on a stud and sputter coat with gold.
In some cases, it may be preferable to observe cells from the surface that are
associated with the substrate. This can sometimes be accomplished using double-
sided Scotch tape. Attach the double-sided tape to an aluminum specimen mount.
Apply the mount like a stamp to the critical-point-dried culture (Fig. 8).
Fig. 8 SEM view of the underside of a fibroblast. After drying, the cell was removed from the
substrate by double-sticky tape.
310 David M.Phillips
VI. Cells Grown in Suspension

Cells grown in suspension need to be attached to a substrate to be viewed in
the SEM. The method is as follows.
1. Prepare coverslips for attaching cells. The author uses 22 X 22-mm No. 2
glass coverslips because they fit nicely into his critical-point apparatus.
Thinner No. 1 coverslips can also be used but they tend to break. Use a
diamond scribe to write the sample name or number on the coverslip. The
coverslips should be washed carefully. Use a stainless-steel coverslip holder
for washing coverslips and for dehydration. Wash coverslips in an ultrasonic
cleaner in liquid laboratory soap. Rinse several times in distilled water and
air dry. Place coverslips (in a Coplin jar) in a solution of 2 mg/ml of poly-
L-Lysine (MW 50,000, Sigma) overnight to 1 month at 4°C. The coverslip
should be washed in distilled water and shaken dry just before it is used.
2. Wash the cells two or three times in serum-free medium and fix overnight
at 4°C. Transfer cells with a drop of water to the poly-L-lysine-coated
coverslip and allow them to settle and attach. This will take a few minutes.
Do not let the drop of water dry out. The cells will attach more readily if
they are more concentrated. Letting them settle or concentrating them by
slow centrifugation can be done before adding them to the coverslip.
3. The coverslip can now be dehydrated, critical-point dried, and sputter
coated.
VII. A Few Hints

1. Cells should never dry out during processing for electron microscopy. Be
mindful of this whenever solutions are changed during fixing, dehydrating,
and embedding.
2. Use very hard plastic when embedding cells on monolayers. The formulation
described earlier is hard, but Epon tends to be softer in the summer because
it picks up water. To make even harder plastic, use 25 cc Epon, 25 cc NMA,
and 0.8 ml DMP-30.
3. A sharp diamond knife is exceedingly important. Cutting sections from
monolayers is not easy even with a good knife. The author has had the best
success with 45" knives from Diatome.
4. Writing the sample number on the bottom of wells or T flasks with a
diamond scribe will ensure that cultures are not confused if alcohol dissolves
the marker or if the wrong lid is placed on a dish. Use the scribe to write
on polymerized plastic before removing it from the dish.
5. Even a small amount of water in the absolute alcohol or plastic can ruin
the preparation. It is difficult to avoid water in a humid environment because
evaporating solvents cool and cause water to condense. Water condenses

on bottles that are removed from the refrigerator. The more precautions
that are taken to avoid water the better. Keep all plastics in a desiccator
and keep desiccant fresh. Use 100% alcohol at room temperature and do
not use it near a sink or in a fume hood. Cover dishes when 100% alcohol
or plastic is being used.
6. The secret of sputter coating is to coat as thin as possible without charging
and to coat with a very low current for a long time (5-10 min).
References
Muller, W. A., Weigl, S. A., Deng, X., and Phillips, D. M. (1993). PECAM 1 is required for
transendothelial migration of leukocytes. J. Exp. Med. 178,449-460.
Roberts, P., Phillips, D. M., and Mather, J. (1990). A novel epithelial cell from neonatal rat lung:
Isolation and differentiated phenotype. Am. J. Physiol. 259, 415-425.
CHAPTER 18
Indirect Immunofluorescence Microscopy

in Cultured Cells
Sally P. Wheatley and Yu-li Wang
Cell Biology Group
Worcester Foundation for Biomedical Research
Shrewsbury, Massachusetts 01545
I. Introduction
11. Cell Culture
111. Sample Preparation for Immunocytochemistry
IV. Antibodies
A. Handling
B. Titration
C. Primary Antibodies
D. Secondary Antibodies
E. Double Labeling
V. Signal Detection
A. Fluorophores and Filter Sets
B. Green Fluorescent Protein
C. The Microscope
D. Cameras
VI. Miscellaneous Issues
A. Counterstaining
B. Mounting Cells
C. Cell Morphology
D. Focal Plane
VII. Laboratory Protocols for Fixation/Extraction
A. Methanol Protocol
B. Formaldehyde-Triton Protocol
C . Formaldehyde-Acetone Protocol
D. Glutaraldehyde-Triton Protocol
E. Protocol for Antibody Application
F. Counterstaining
G. Protocol for Mounting Cells
References
METHODS IN CELL BIOLOGY, VOL. 57

Copyright 0 1998 by Academic Press. AU n&u of reprodurnan in any form reserved 313
01191-679X/YK525.00
314 Sally P. Wheatley and Yu-li Wang
The technique of fluorescence immunolocalization has evolved steadily since its

first application in the mid-l960s, incorporating innovations in probe chemistry,
microscopy, and image detection. This chapter provides an overview of the
current status of indirect immunofluorescence for those starting to use the
method. It includes both general considerations from cell culture to image detec-
tion and several protocols that should serve as an entry point for this technique.
I. Introduction
Immunolocalization of antigens within cells and tissues has been a routine

procedure in cell biology (see Osborn and Weber, 1982; Wang et d.,1982).
Although methods for preserving and probing cells have not altered dramatically,
the sensitivity and versatility of this technique have improved significantly over
the last decade due to major advances in signal detection, including the commer-
cialization of cooled-charged couple device cameras (CCD), developments in
image analysis, and the availability of a new generation of fluorophores. This
chapter is offered as an introduction to the technique, particularly indirect immu-
nofluorescence microscopy, and is intended to draw the readers’ attention to
ways in which its utility can be maximized. The methods illustrated in this chapter
use microtubule organization in cultured cells as an example. However, the
procedures described are applicable to all immunolocalization studies.
Immunofluorescence localization can be direct or indirect, the principles are
the same. In the direct approach, antibodies are linked to a fluorescent probe
and used directly to localize the antigen in the sample of interest. In the indirect
method, the cell is probed with an unlabeled primary antibody and its location
is in turn reported by a fluorophore-conjugated secondary antibody that recog-
nizes the primary antibody. When working with fixed cells, the indirect method
is most commonly used, as the primary antibody serves to amplify the abundance
of the antigen, enhancing the intensity of the final signal. This method is also
more versatile, allowing a range of fluorophores to be used in combination with
a single primary antibody.
11. Cell Culture

The type of cell that one is working with is often predetermined, e.g., COS
cells are commonly used for transfections and HeLa cells for study of transformed
cells. However, the optical qualities of the cell can be equally important for
obtaining optimal results. The authors have worked extensively with two cell
lines: normal rat kidney epithelial cells (NRK) to monitor cell division and
intracellular organelle dynamics and Swiss 3T3 fibroblasts to study cell locomo-
tion. Both types of cells grow and spread well on glass. NRK cells, which are
18. Indirect Immunofluorescence Microscopy 315
similar to PtK cells have the advantage that they remain spread during cell
division (when most other cells round up), a feature that permits high-resolution
observations of mitosis and cytokinesis. Without such advantage, a confocal
microscope or computer imaging restoration is required to obtain images of
comparable resolution.
Cells are cultured directly on rectangular glass overslips (45 X 50 mm; size 1
or 2 thickness; Fisher Scientific, Pittsburgh, PA), which are passed through a
blue flame immediately before plating the cells. These methods of cell culture
have been described previously (McKenna and Wang, 1989). Briefly, a sterile
coverslip is adhered by vacuum grease to an acrylic block with a circular aperture
(35 mm in diameter) to create a chamber. Cells are passaged directly into the
chamber at the appropriate density 48 hr before experimentation. This arrange-
ment is ideal for viewing and manipulating cells using an inverted microscope.
However, as upright microscopes are more commonly used, most laboratories
culture cells on smaller coverslips,placed in plastic petri dishes or multiwell plates.
Some cells need encouragement to grow on glass. In these cases, coverslips
are incubated for 30 min in a sterile solution of poly-L-lysine (0.1-1 mg/ml) and
washed with sterile water before seeding (Mazia et al., 1995). If cells are grown
in suspension, e.g., HeLa cells or yeast cultures, they need to be concentrated,
then adhered to a poly-L-lysine coated coverslip before observation. Cells can
be attached by gravity or with a “cytospin” before or after processing for immuno-
fluorescence (for details, see Harlow and Lane, 1988). The goal is to obtain an
even distribution of cells, separated by a workable distance, on the coverslip.
For this purpose, immunostaining before mounting may be preferable as a serial
dilution of cells can be made onto a number of coverslips.
111. Sample Preparation for Immunocytochemistry

When choosing or developing an appropriate sample preparation protocol
there are three main concerns: (1) that the true distribution of the antigen
is preserved, (2) that its antigenicity is not compromised by the fixative, and
(3) that the antibody can access the antigen. Ideally, to be certain that the probe
reports the true distribution of the antigen, a variety of fixation protocols should
be tried and the same localization pattern observed with at least two different
procedures. Unfortunately, artifacts introduced from a number of sources can
generate disparate images of the same antigen. For example, organic solvents
dehydrate the specimen and can cause gross distortions in cell structure; certain
buffers, particularly those containing high concentrations of phosphates, can
cause precipitation of proteins and cations, depositing them in nonphysiological
locations. This may be part of the reason why cytoskeleton buffer (CB) (Small
et al., 1982) is better than phosphate-buffered saline (PBS) as the diluent for
fixatives and detergents (see Section VII).
Figure 1demonstrates good versus poor microtubule preservation in two inter-

phase NRK cells. Using glutaraldehyde and mild extraction with Triton X-100,
microtubule ultrastructure is preserved well, revealing a network of continuous
filaments (Fig. 1A). However, microtubule structure is poorly retained with
formaldehyde fixation and they appear as beads along invisible threads (Fig.
1B). All samples are prepared similarly for immunocytochemistry, from yeast
to Drosophila and tissue culture cells, although special procedures are applied
after initial fixation such as removal of the cell wall from yeast (Alfa et al.,
1993;Balasubramanian et al., 1997) and the vitelline membrane from Drosophila
embryos (Theurkauf, 1994) to facilitate the penetration of antibodies. Particularly
yolky specimens such as sea urchin and Drosophila embryos may also need to
be “cleared” after preparation to minimize internal light scattering and autoflu-
orescence (Wright and Scholey, 1993; Theurkauf, 1994).
The authors routinely use three fixatives: glutaraldehyde, formaldehyde, and
methanol. Ethanol and acetone are also commonly used. Glutaraldehyde pro-
vides the highest degree of sample preservation by cross-linking the side chains
of neighboring proteins, locking them in place with little morphological damage.
At the molecular level, however, chemical modifications induced by glutaralde-
hyde can cause severe alterations in the protein structure that result in loss of
antigenicity.Therefore, although glutaraldehyde is preferred for the preservation
of ultrastructure, it is not feasible for many antigens. Formaldehyde also cross-
links proteins. Although poorly understood, its action is known to be unstable
and can be partially reversible in neutral solutions (see Means and Feeney, 1971;
Harlow and Lane, 1988).
Fig. 1 Microtubule preservation in the peripheries of two interphase NRK cells fixed with (A)
glutaraldehyde/Triton and (B) formaldehydelTriton, as detailed in Section VII (extraction in each
case was for 2 min at room temperature with 0.1% Triton). Both cells were probed with a monoclonal
antibody directed against P-tubulin (Amersham; 1/10) and visualized using an FITC-conjugated
antimouse whole IgG secondary antibody (Sigma; 1/50).(A) microtubules are revealed as continuous
filaments forming a network throughout the cell. (B) Staining is discontinuous (arrowheads), giving
the microtubules a broken appearance. Bar: 10 pm.
Methanol preserves protein structure by dehydration and coagulation. It ex-

tracts lipids simultaneously while fixing, causing nonanchored proteins to leak
from the cell. Like other organic solvents, methanol can also cause soluble
proteins to become deposited onto remaining cell structures. Because all these
properties can generate a false pattern of localization, organic solvents tend to
be used when studying antigens that are well anchored.
Knowing certain biochemical properties of the antigen can help in solving the
problem when preservation is suboptimal. In many cases, however, the identity
or biochemistry of all antigens involved may not be known and refinements
and compromises will have to be made over a course of trials. As an exmple,
microtubules are sensitive to cold temperatures and free calcium ions. They retain
their antigenicity unusually well in glutaraldehyde prepared in warm cytoskeleton
buffer. However, if they are to be colocalized with another protein, it is often
necessary to use methanol supplemented with EGTA to optimize the preserva-
tion and antigenicity of both components (see Fig. 2).
All fixation protocols run the risk of destroying antigenic sites, and problems
can arise from overfixation and underfixation. As there are no hard and fast
Fig. 2 Double immunolabeling of P-tubulin (A-C) and the microtubule motor, CHOl (D-F), in
three telophase NRK cells fixed with glutaraldehyde (A, D), formaldehyde (B, E), or methanol (C,
F), according to the protocols in Section VII. Using the glutaraldehyde protocol, the microtubule
structure is well preserved (A), but CHOl distribution appears to resemble that of the microtubules
(D). With formaldehyde the microtubules appear broken (B), yet CHOl is located in its true position
at the developing midbody (E). Methanol fixation preserves the microtubule structure better than
formaldehyde (compare C and B) and reports a similar CHOl distribution as did formaldehyde
fixation (compare F and E). Methanol fixation is therefore chosen in this colocalization study. Bar:
10 pm.
318 Sally P. Wheatley and Yu-li W a n g
rules regarding the reactivity or sensitivity of antigens to different fixatives, most

laboratories have a favorite set of basic protocols that can be modified when
necessary (see Section VII).
To facilitate antibody penetration, cells are “extracted” or “permeabilized”
with nonionic detergents or organic solvents during or after fixation. The most
commonly used permeants are nonionic detergents, acetone, and methanol. De-
tergents can also be added to the initial fixative solution to assist its penetration
within the cell. This is particularly helpful when dealing with thick or yolky
specimens such as Drosophila or sea urchin embryos (Wright and Scholey, 1993).
If the antigen in question is anchored within the cell, extraction generally
does not cause problems, as further extraction may even reduce nonspecific
background staining. However, if the antigen is soluble, extraction can remove it
completely (see Melan and Sluder, 1992).Extraction with commercially available,
nonionic detergents such as Triton X-100, Tween 20, Brij 35 and Nonidet P-40 is
more gentle and can be adjusted to give the best results by varying concentration,
temperature (although not when fixing and extracting tissue culture cells simulta-
neously), or duration of incubation.
Another problem encountered is “epitope shielding” where access of the
antibody to the antigen of interest is obstructed by other proteins or material.
This is classically represented by tubulin immuolocalization at the midbody of
a dividing mammalian cell. In a living NRK cell injected with labeled tubulin
the midbody appears as the brightest band of fluorescence within the cell (Fig.
3A, arrows; see also Wheatley and Wang, 1996). In contrast, in a fixed cell probed
with an antibody against P-tubulin the midbody is manifested as a dark zone
(Fig. 3B, arrowhead). Tricks to unveil masked epitopes include treatment with
Fig. 3 Comparison between midbody appearance in a living NRK cell microinjected with
rhodamine-labeled tubulin (A) and a glutaraldehyde-fixed NRK cell probed with anti-p-tubulin (B),
as described in Fig. 1. Microinjected rhodamine-tubulin incorporates readily into the midbody.
Because of the overlap of microtubules in this region, the central part appears most intensely
fluorescent (A, arrow). In contrast, in the fixed preparation, antibodies have difficulty accessing
midbody microtubules, hence the region appears as a dark zone (B, arrowhead). Bar: 10 pm.
protease, trypsin, and inclusion of the denaturant sodium dodecyl sulfate (SDS)
in the fixative (see Harlow and Lane, 1988; Ding et al., 1995).
IV.Antibodies
This section briefly outlines antibody handling and selection. For a more
complete account and for details on antibody structure and production, readers
are referred to the laboratory manuals of Harlow and Lane (1988) and Celis
(1994).
A. Handling
Antibodies are robust proteins; however, repeated freeze-thaw cycles should
be avoided. Undiluted stocks should be aliquoted into appropriate working
volumes and stored at -20 or -80°C. Secondary antibodies can be maintained
undiluted at 4°C for up to 6 months. Those conjugated to a fluorochrome should
be stored in a light-tight container. For immunocytochemistry, all antibodies
should be routinely diluted immediately before use in PBS containing 1%bovine
serum albumin (BSA) and 0.1%sodium azide. BSA blankets nonspecific epitopes
that may interact with the antibody when it is presented. Other commonly used
concealants include dry milk powder (up to lo%), fetal calf serum (up to lo%),
or higher concentrations of BSA. Azide is included to deter the growth of
microorganisms (note that azide is extremely toxic!). Before use the diluted
antibody is spun at top speed in a microfuge (at room temperature) for 30 min
to remove debris such as unconjugated dye and aggregated antibodies.
B. Titration
A new primary antibody should always be tested with a known secondary
antibody and vice versa. New antibodies should be tested in serial dilution, e.g.,
1/10, 1/100,1/500,and 1/1000, paying close attention to the range suggested by
the manufacturer (if applicable). Occasionally, when using ascites fluid, dilution
may have to be increased to 1/10,000. However, if monoclonal antibodies are
supplied as tissue culture supernatants, they are often used undiluted. When
using purified antibodies it is common practice to express the concentration of
the antibody in micrograms per ml, particularly if these are noncommercial
stocks. For commercially available antibodies it suffices to indicate the dilution
factor used for each antibody.
C. Primary Antibodies
Antibodies recognize and bind to specific “epitopes” or “antigenic determi-
nants.” They can be either monoclonal, recognizing a single epitope, or poly-
clonal, a collective of antibodies recognizing different antigenic determinants of

the same molecule. Most commonly,monoclonal antibodies derive from mice and
polyclonals from rabbits. The specificity of monoclonal antibodies has obvious
advantages. For example, after assembling into microtubules, tubulin subunits
undergo a number of posttranslational modifications, including detyrosination.
Therefore, ID5, a monoclonal antibody developed against the detyrosinated end
domain of a-tubulin (Wehland and Weber, 1987), can recognize a specific subset
of microtubules and give a markedly different localization pattern to that of total
P-tubulin (Fig. 4).Although sometimes desirable, this specificity can be confusing
with monoclonal antibodies reacting against different parts of the same antigen
reporting different distributions (see Oka et a l , 1994). As a consequence of their
specificity, monoclonal antibodies tend to be more susceptible to preparation
artifacts than polyclonal antibodies (see later). Nevertheless, they tend to cause
fewer problems associated with nonspecific binding. When combining immunocy-
tology with genetic manipulations, it is important to know which part of the
molecule is recognized by the antibody. In such studies, polyclonal antibodies
may be preferable as they react with multiple parts of the molecule and are less
likely to be completely impeded by alterations in the protein. A compromise
between monoclonal and polyclonal antibodies is the use of pooled monoclonal
antibodies as these are not limited to one antigenic determinant, yet minimize
nonspecific staining.
In addition to purified antibodies, whole immune serum is sometimes used.
Rigorous controls should be performed when using sera as additional antibodies
are present that can give false staining patterns. The most pertinent control is
staining with preimmune serum from the same animal, if possible. If this is
unavailable, serum from a control animal can be used as a minimal requirement.
Autoimmune serum from humans has led to the identification of many interest-
ing antigens. Unfortunately, as such serum tends to be obtained from patients
with severe illnesses, their supply is highly unpredictable (although this can be
circumvented using molecular techniques). Because the mortality of hosts can
lead to batch variation, it is wise to reassess new batches of antibodies on arrival
both for the appropriate titration and to ensure that the staining pattern has not
deviated. Monoclonal antibodies have the distinct advantage that, if hybridoma
cell lines are maintained properly, a supply should be available ad infiniturn.
The quality and specificity of a primary antibody are assessed by standard
Western blotting. The antibody should recognize only target antigens. Often it
is necessary to perform affinity purification using either chromatographic tech-
niques or on a microscale (Hammerback and Vallee, 1990) to remove nonspecific
reactivity. After affinity purification, antibodies should be reexamined by West-
ern blotting. For details on Western blotting and affinity purification, readers
are referred to Harlow and Lane (1988).
D. Secondary Antibodies
Immunologically, the primary antibody used dictates the secondary antibody
required. The secondary must be targeted against the appropriate class of immu-
18. Indirect Immunofluorescence Microscopy 32 1
noglobulin (or light chains) of the species in which the primary was made and
must be manufactured in a distinct species. Most secondaries are polyclonal and
are generated in a wide range of species. It is necessary to perform controls
on unknown secondary antibodies. In particular, incubation with a secondary
antibody alone will report nonspecific interactions between cellular components
and the secondary antibody or its conjugate. If primary antibodies are applied
as antiserum, cells should be tested by incubation with preimmune serum then
with secondary antibodies.
E. Double Labeling
When immunolocalizing more than one protein within a sample, primary
antibodies should be from different species and fluorochromes must contrast.
There are combinational and sequential options to the application of antibodies
when labeling multiple antigens, e.g., for double labeling: (1) primary x then
secondary x then primary y then secondary y; (2) primary x then primary y then
secondary x then secondary y; (3) primaries x and y, then secondaries x and y;
or (4)primary x then primary y then secondaries x and y . Despite being more
laborious, the authors tend to use the first sequence punctuated with extended
incubations in blocking solution (PBS/BSA/azide) to minimize cross-reactions
between antibodies.
Although up to five different components have been labeled simultaneously
within a single cell, rarely is immunostaining performed with more than two
probes as, in addition to confusion arising from combining antibodies from
different species, the availability of fluorochromes becomes limiting. Although
triple labeling can be achieved using immunological probes entirely (Herzog et
al., 1994), it is commonly achieved by double immunolabeling and counterstaining
once (see Sections VI1,F) or by immunolocalizing a single antigen and then
counterstaining with two fluorescent probes.
V. Signal Detection
A. Fluorophores and Filter Sets

Fluorophores are molecules that absorb light of one wavelength and emit light
at a longer wavelength. They differ in their absorption and emission spectra,
extinction coefficient, quantum efficiency, and stability. Most fluorescence micro-
scopes are fitted with fluorescein isothiocyanate (FITC) and tetramethylrhoda-
mine isothiocyanate (TRITC) filter sets as these two fluorochromes have been
available for many years. However, secondary antibodies conjugated to alterna-
tive fluorophores are now available and may be preferable. Useful information
regarding fluorophores can be found in relevant manufacturers’ catalogs, such
as Molecular Probes (Eugene, OR) and Jackson ImmunoResearch Laboratories
Inc. (West Grove, PA).
322 Sally P. Wheatley and Yu-Li Wang
Three commonly used red fluorophores are TRITC, Texas red, and indocarbo-
cyanine 3 (Cy3). There are advantages and disadvantages to each. As TRITC is
the traditional choice, most microscopes have a filter set designed for its maximum
absorption and emission. Cy3 has similar excitation and emission spectra (see
Table I), but is brighter and more photostable. Therefore, one might select Cy3
over TRITC when an antigen is present in low abundance or the signal with
TRITC is weak.
When double labeling a sample, it is common to use FITC and a red fluoro-
phore. Cy3 and FITC or TRITC and FITC, although often used, are less favorable
as their excitation peaks are close enough to cause considerable overlap or “bleed
through” between channels. These pairs should be used only with carefully tested
band-pass filters. A better combination is FITC and Texas red as their spectra
are well separated. Using a TRITC filter set, Texas red absorbs and emits light
at margins of its spectra but can still be detected (particularly by a CCD camera).
To maximize the signal from Texas red, filters that allow its peak excitation and
emission light to pass can be obtained. (One caveat with Texas red, however, is
its nonspecific affinity for proteins, which may cause high background staining.)
An alternative approach is to find a substitute for FITC, such as indodicarbocya-
nine (Cyz) or a probe of a different wavelength, e.g., aminomethylcoumarin
(AMCA or coumarin) or a short wavelength BODIPY.
B. Green Fluorescent Protein

With the introduction of green fluorescent protein (GFP) as a reporter mole-
cule, it is now possible to visualize directly a fluorescently tagged protein within
a living cell using transfection (see Stearns, 1995). Although in viva application
is its major strength, GFP also has distinct advantages in fixed preparations
(although avoid using methanol as it denatures GFP). First, as GFP is manufac-
tured by the cell itself, the problem of target accessibility is eliminated and
extraction becomes unnecessary. Second, GFP can be excited using the regular
FITC channel; although this is not its peak absorption, when excited in this way
GFP is relatively photostable, making it preferable to FITC as a fluorophore
(Stearns, 1995). Third, mutations in GFP are now available that offer a range
Table I
Absorption and Emission Peaks of
Commonly Used Fluorophores
Fluorochrome Absorption (nm) Emission (nm)
FITC 492 520
CY3 550 570
TRITC 550 570
Texas red 596 620
of absorption and emission spectra, providing a greater scope in fluorophore

selection (Heim and Tsien, 1996).
C. The Microscope
A number of adjustments can be made to the microscope to maximize observa-
tions.
1. Illumination
Fluorescence microscopes are usually fitted with a mercury arc lamp. The
intensity of emitted light can be altered by interposing neutral density filters
between the light source and the sample; alternatively, the light source can
be adjusted through an electronic controller available from Carl Zeiss. Also
commonly used are xenon arc lamps, which give a more uniform excitation across
the visible and UV light spectrum, and quartz halogen lamps, which are dimmer
and more suited to fluorescence imaging in live cells.
2. Objective Lenses
In addition to selecting an appropriate magnification, there are several other
considerations in choosing an objective lens for immuofluorescence: degree of
correction, material, number of lens elements, and its numerical aperture. Al-
though lens correction is desirable to prevent spherical aberration and color
splitting, it requires additional lens elements, which lead to loss of signal. In
addition, some glass material is noticeably less autofluorescent than others. The
most critical parameter, however, is the numerical aperture (NA). In epifluo-
rescence microscopy, because the objective lens serves as both the condenser
and the detector, signal intensity is proportional to the fourth power of the NA.
3. Field I n s
Microscopes are equipped with an adjustable field iris. Although observations
are generally made with the field iris fully open, it is often advantageous to close
the iris around a particular region to minimize light scattering and remove out-
of-focus haze. It can also be used to mask areas of high signal (e.g., densely
crowded areas) so that less brightly labeled regions can be viewed.
D. Cameras
As mentioned in the Section I, major advances have been made in the instru-
mentation for recording microscope images. Although it has been common prac-
tice to use 35-mm cameras fitted with high-sensitivity film (>400 ASA), these
are being superseded with cooled charge-coupled device cameras, which can
detect very low levels of light. The high sensitivity of CCDs translates as a
decrease in exposure time and also makes dimmer fluorophores a viable option.
This decrease can be substantial-an order of magnitude over a regular 35-mm
camera. CCD cameras also have the advantage that they are operated via a
personal computer, hence images can be viewed, exposure times and image
quality optimized, and data filed easily and rapidly. However, without additional
coating, CCD chips do not respond well to ultraviolet (UV) light. Even with
UV-sensitive coating, CCDs are more sensitive to long wavelength dyes such as
Texas red, TRITC, and Cy3 than to Hoechst 33258, DAPI, or fluorescein. On
the other hand, our eyes are extremely inefficient in detecting red light so without
a CCD camera signals often appear deceptively weak at long wavelengths.
Although color CCD cameras are available, black and white models are more
sensitive as signals of virtually all wavelengths are accumulated into one image.
When using black and white CCD cameras, color images can be generated
through the application of pseudocolor to images of different channels and
merging with computer programs such as Adobe Photoshop 4.0 and Metamorph
(Universal Imaging, West Chester, PA). Alternatively, the use of 35-mm high-
speed color film remains as a simple option for recording color fluorescence
images.
VI. Miscellaneous Issues

A. Counterstaining
When presenting immunofluorescence data it is often necessary to familiarize
the audience with the general appearance of the cell before indicating the specific
points of interest. One commonly encountered problem is that the fixation proto-
col that preserves the antigen may leave the cell looking ravished in phase contrast
or bright field. An alternative way to orientate the reader is to counterstain cells
with a fluorescent marker such as Hoechst 33258 or DAPI to show the position
of the chromatin or fluorescently tagged phalloidin to localize filamentous actin
(see Sections VI1,F). In addition, because fungi have the advantage that the cell
wall and septa stain with the laundry brightner Calcofluor, dividing cells can be
identified easily in fixed preparations (Alfa et al., 1993; Balasubramanian et
al., 1997). Counterstaining is a quick, easy, and reliable way of comparing the
localization of the antigen with other structures in the cell. However, as with
double immunostaining, fluorochrome selection becomes more constrained.
B. Mounting Cells
When viewing cells with fluorescence they should be mounted in an appropriate
medium. This should contain an antibleaching compound such as n-propyl gallate
or p-phenylenediamine, should be viscous, and should be in the pH range 7-8
(FITC prefers pH 8). The authors make their own stock (see Section VII,H),
but many mounting media are available commercially. To prevent oxidation the
coverslip should be sealed to the slide with nail varnish. If the sample is going
to be kept for a long time (months-years), a self-hardening mounting medium
such as Gelvatol or Mowiol is preferable (see Harlow and Lane, 1988).
C. Cell Morphology
It is important to become acquainted with the morphology of control cells
and their substructures before studying experimental cells. It is also important to
consider the dynamics of structures within living cells. For example, microtubule
organization alters rapidly, particularly during mitosis. Likewise, many motility
events, both intracellular and locomotory, take place as cells explore and respond
to environmental cues. Thus cells can look considerably different from one
moment to the next. Using immunocytochemistry, only static images of the cell
are obtained. Nevertheless, some information regarding the dynamic nature of
the cell or its constituents can be inferred through cell cycle staging and compari-
son of structures between individual cells. When dealing with unfamiliar struc-
tures, it may be advantageous to take snap shots at low magnification first, as
these can be used later for unbiased statistical analyses.
D. Focal Plane
Structures of interest are often found at different focal planes. For example,
in kidney cells the primary cilium usually sits above the interphase nucleus,
protruding into the external medium (Wheatley et al., 1994). Unless one focuses
above the cell, the primary cilium is undetectable (see Fig. 4). Focusing through
the specimen is a good twitch to develop, but better still is the use of an optical
sectioning device, which operates by the same principle as in a confocal micro-
scope, but is a simple attachment to the conventional light microscope (Wang,
1997).
VII. Laboratory Protocols for Fixation/Extraction

The basic protocols are listed from the quickest (methanol) to the most in-
volved (glutaraldehyde). Empty coverslip boxes are ideal as “fixation contain-
ers.” When using smaller coverslips, disposable multiwell plates or petri dishes
may be more appropriate. Prevent coverslips from colliding, and be aware which
side has cells attached: the cell side will appear matt rather than glossy. If
preparing cells in suspension, it is important to ensure that they are exposed
uniformly to solutions. This is best achieved by performing incubations in micro-
fuge tubes fitted to a cell rotator. It is often convenient to place these tubes in
empty black 35-mm film canisters to contain any leaks and to protect the sample
Fig. 4 Deconvolved and reconstructed images (90" perspective) comparing the distribution of
detyrosinated a-tubulin (A) and total P-tubulin (B) in two interphase NRK cells. (A) The cell was
fixed using the formaldehyde protocol (Section VII,B), probed with undiluted monoclonal antibody
(ID5; a tissue culture supernatant), and visualized using FITC-conjugated antimouse IgG (Sigma;
1/50). Detyrosinated a-tubulin specifically labels the primary cilia that project from the top surface
of the cell. (B) The cell was fixed using the glutaraldehyde method and probed with anti-P-tubulin
as detailed in Fig. 1. The entire interphase microtubule network can be seen, including a primary
cilium (arrow) Bar: 10 pm.
from light where necessary. When using chilled organic solvents, place coverslip
sideways into a 100-ml glass beaker. The large volume and small surface area
help prevent heat exchange. Fixatives should be made up in disposable containers
or in bottles designated for fixatives only. Buffer pH is adjusted at ambient
temperature (22°C); the solution is then warmed or chilled as appropriate. Buffers
are stored at 4°C. Other reagents are stored as detailed below. Unless stated,
reagents are supplied by Sigma Chemical Co.
Buflers
1. Phosphate-buffered saline, pH 7.4.
2. Cytoskeleton buffer
a. 1X CB: 137 mM NaCl, 5 mM KC1,l.l mM Na2HP04,0.4 mM KH2P04,
2 mM MgC12, 2 mM EGTA, 5 mM PIPES, and 5.5 mM glucose, pH 6.1.
b. 1.3X CB: 182 mM NaCl, 6.6 mM KCI, 1.46 mM Na2HP04, 0.5 mM
KH2P04, 2.7 mM MgC12, 2.7 mM EGTA, 6.6 mM PIPES, and 7.3 mM
glucose, pH 6.1.
3. EGTA: Store stock solution (100 mM, pH 6.0) in a plastic, not glass con-
tainer.
Fixatives and Permeabilizing Reagents

1. Acetone: store at room tempeature.
2. Formaldehyde: 16% stock (methanol free), supplied in 10-ml ampoules
(Electron Microscopy Sciences, Fort Washington, PA) and stored at room
temperature. (Ampoules should be discarded if opened for more than 1
week. Avoid prepared solutions of formaldehyde that contain methanol
such as formalin. Some researchers prefer to make a fresh solution of
formaldehyde from paraformaldehyde powder.)
3. Glutaraldehyde: 70% stock in water, EM grade, supplied in 2-ml ampoules
(Polysciences Inc., Warrington, PA) and stored at 4°C.
4. Methanol: store at room temperature.
5. Triton X-100.
Other Reagents
1. Sodium borohydride: For glutaraldehyde protocol only. Sodium borohy-
dride reduces aldehyde groups that interfere with the final signal by autoflu-
orescing. Store light tight at room temperature as an anhydrous powder,
and prepare working solution in CB immediately prior to use (within 1min).
2. PBS/BSA/azide: PBS containing 1% BSA (Fraction V, Boehringer-
Mannheim Corp., Indianapolis, IN) and 0.1% sodium azide.
A. Methanol Protocol
This protocol is very quick and ideal for a “look see” preparation. Some
researchers use straight methanol, whereas others do not chill it. As trace calcium
ions may be present in the methanol that could affect microtubules, the authors
add 5 mM EGTA, hence the methanol concentration is diluted to 95%.
1. Prepare and chill a solution of 95% methanol with 5 mM EGTA to -20°C.
2. Transfer this solution to a chilled beaker or suitable container.
3. Drain medium from cells and wash thoroughly in prewarmed PBS.
4. Remove the coverslip from the chamber and plunge it into methanol. Incu-
bate for 10 min without agitation.
5. Remove coverslip and submerge it immediately in PBS. Change PBS twice.
Proceed with antibody application (see Section VILE).
B. Formaldehyde-Triton Protocol
1. Within 2-3 hr of use, prepare a 4% formaldehyde solution by mixing 1
part 16% formaldehyde stock with 3 parts 1.3X CB, cap tightly.
2. Warm PBS, formaldehyde solution, and 1X CB buffer to 37°C.

3. Prepare 0.1-0.5% Triton X-100 solution in prewarmed 1 X CB buffer.
Note: As Triton is viscous, cut the end of the disposable pipette tip to assist
flow. To ensure accurate measuring, wipe off excess Triton on the outside of the
tip before transferring it to the CB. Prewarming the CB will facilitate detergent
dissolution and its exit from the tip.
4. Transfer the formaldehyde solution to a clean fixation container.
5. Drain medium from cells and rinse twice with warm PBS.
6. Remove coverslip from chamber dish and place it cell side down on the
surface of the formaldehyde solution. Incubate with gentle rocking for 10 min
at room temperature. Ensure that the coverslip remains floating. If the coverslip
does not float, turn it cell side up and submerge it fully.
7. Transfer warm Triton solution to a clean fixation container.
8. Remove coverslip and rinse two to three times with PBS.
9. Place the coverslip on top of the Triton solution (or submerge as before).
Incubate with gentle agitation for 2 min at room temperature. (Triton concentra-
tion, temperature, or incubation time can be varied to optimize extraction.)
10. Remove coverslip and rinse thoroughly with PBS. Proceed with antibody
application (see Section VI1,E).
C. Formaldehyde-Acetone Protocol
Because the dehydrating properties of acetone make cells particularly suscepti-
ble to damage, ensure that the coverslip surface is wet at all times.
1. Chill an appropriate volume of 100% acetone to -20°C (100 ml for a 45 X
50-mm coverslip).
2. Follow the protocol for formaldehyde-Triton to the end of Step 6, (skip
step 3).
3. Transfer chilled acetone to a 100-ml glass beaker or appropriate container.
4. Remove the coverslip from fixative and rinse thoroughly with PBS.
5. Plunge the coverslip into the cold acetone, rock gently at first, and then
incubate without agitation for 5 min at -20°C.
6. Remove the coverslip from the acetone and plunge it immediately into a
beaker of PBS.
7. Change PBS twice and proceed with antibody application (see Section VI1,E).
D. Glutaraldehyde-Triton Protocol
1. Warm PBS and 1 X CB to 37°C.
2. Within 2-3 hr of use, prepare and warm the following solutions. Solution
1: 0.5% glutaraldehyde, 0.1-0.2% Triton X-100 (see Note in Section VI1,B) in
1 X CB. Solution 2: 1%glutaraldehyde in 1 X CB.
3. Transfer solution 1 to a fixation container.

4. Drain medium from cells and gently rinse twice with PBS.
5. Remove coverslip from chamber dish and float it on the surface of solution
1 for 1 min, rocking gently at room temperature (or submerge cell side up
completely; if the coverslip does not float, turn it cell side up and submerge
it fully).
6. Remove coverslip and rinse twice with 1 X CB.
7. In a clean fixation container, float the coverslip cells side down on the
surface of solution 2. Incubate with gentle rocking for 15 min at room temper-
ature.
8. After 13-14 min, prepare a OS-mg/ml solution of sodium borohydride in
I X CB. Transfer to a clean fixation container.
9. Rinse coverslip quickly as before with 1 X CB.
10. Submerge the coverslip cell side up in the sodium borohydride solution.
Incubate for 5 min at room temperature with periodic agitation. Check regularly
that the coverslip has not floated to the top and that the solution is in contact
with the entire surface of the coverslip. (The solution should be visibly effervescing
when the coverslip is introduced.)
11. Rinse thoroughly with PBS and proceed with antibody application (see
Section VI1,E).
E. Protocol for Antibody Application

Wash the specimen thoroughly between antibody incubations. Keep the sample
wet during incubations to ensure that the antibody is applied uniformly. To
prevent the further dilution of antibodies, dab off excess washing solutions by
tilting the coverslip and touching its corner with filter paper. Antibody incuba-
tions can be performed at 4°C overnight, at room temperature for 2-4 hr, or at
37°C for 45-60 min. Although longer incubations at lower temperature may
decrease nonspecific background staining, there is usually no detectable differ-
ence among these conditions. To conserve antibodies and minimize drying out,
which can lead to nonspecific interactions, present antibody solutions to the
sample on Parafilm in a petri dish with risers (tooth picks) to prevent the Parafilm
from contacting the bottom surface of the petri dish and place this in a humidifying
chamber (soaked paper toweling can provide humidity). Before application,
dilute antibodies in PBS/BSA/azide and clarify by centrifugation as detailed
earlier.
1. Incubate the prepared coverslips on a shaker for a minimum of 30 min at
room temperature in PBS/BSA/azide. Prewarming the solution to 37°C can im-
prove this “blocking” step.
2. Cut a piece of Parafilm the same size as the coverslip and dispense the
primary antibody solution in its center. [Volume required will depend on the
size of the coverslip. For an area 35 mm in diameter (the chamber dish aperture),
use between 100 and 200 4 . 1
3. Invert the coverslip and touch its surface to the antibody droplet. Capillary
action will lift the Parafilm, allowing the antibody solution to coat the coverslip
surface uniformly. (For small coverslips, place the antibody droplet on a large
piece of Parafilm in a petri dish and invert the coverslip onto the droplet.)
5. Incubate coverslips in the inverted position at the desired temperature as
stated earlier.
6. To remove the Parafilm, turn the coverslip cell side up (i.e., Parafilm up)
and immerse in PBS. The Parafilm should float to the surface. (Small coverslips
are separated from the Parafilm by flooding the petri dish with PBS/BSA/azide.)
7. Drain and reimmerse in fresh PBS/BSA/azide.
8. Repeat steps 1 to 6 using the secondary antibody, keeping the sample
protected from light.
9. Wash in two to three changes of PBS.
10. The sample is ready for mounting or counterstaining.
F. Counterstaining
1. Staining with Fluorescent Phalloidin
Phalloidin is a fungal toxin that binds specifically to filamentous actin. It is
extremely poisonous and should be handled with care. Because methanol destroys
its binding site on F-actin, methanol fixation should be avoided when counter-
, staining with fluorescently labeled phalloidin. As stock solutions are usually
stored in methanol (-20°C) it is necessary to dry the stock prior to use.
1. Dry the appropriate volume of phalloidin using nitrogen gas or a Speed Vac.
2. Dissolve phalloidin in PBS to a final concentration of 200 nM by vigor-
ous vortexing.
3. Using the Parafilm method just described for antibody application, apply
phalloidin to the center of the Parafilm and incubate the sample inverted for
30 min at 37°C or for 45 min at room temperature.
4. Wash twice with PBS before mountinglviewing.
2. Staining with Hoechst 33258

1. Prepare a lO-pg/ml solution of Hoechst 33258 i PBS from lO-mg/ml
frozen stock in dimethyl sulfoxide.
2. As described earlier, apply Hoechst 33258 solution to Parafilm and invert
a coverslip onto its surface.
3. Incubate for 5-15 min at room temperature.
4. Wash with PBS and then mounthiew.
G. Protocol for Mounting Cells

1. Prepare a stock solution of 1% (wh) p-phenylenediamine (PPD) in H 2 0
(dissolves at pH 9.9, and store light tight in 1-ml aliquots at -20°C.
2. Prepare a mounting medium (MM) solution: 50% glycerol, 1 X PBS, and
0.1% PPD. Adjust pH to 7.0 or pH 8.0 for fluorescein.
3. Store excess in 0.5- or 1-ml aliquots, in the dark, at -20°C.
4. Keep protected from light. Flood the chamber dish with 1ml of MM or, if
using an upright microscope, place a small drop (50-100 pl) of MM on a clean
glass slide and invert a coverslip over the droplet.
5. Cells are ready to view. If the sample is to be stored for future observation
(up to 6 months), seal the edges of the coverslip with nail polish.
Acknowledgments
We thank Dr. Ryoko Kuriyama, for supplying anti-CHO 1 antibody and Dr. Jurgen Wehland
for supplying antidetyrosinated a-tubulin antibody (IDS).
References
Alfa, C. E., Gallagher, I. M., and Hyams, J. S. (1993). Antigen localization in fission yeast. Meth.
Cell Biol. 37, 201-222.
Balasubramanian, M. K., McCollum, D., and Gould, K. L. (1997). Cytokinesis in the fission yeast
Schizosaccharomyces pombe. Meth. Enzymol. 283,494-506.
Celis, J. E. (1997). “Cell Biology.” A Laboratory Handbook.” Academic Press, New York.
Ding, M., Robinson, J. M., Behrens, B. C., and Vandre, D. D. (1995). The microtubule cytoskeleton
in human phagocytic leukocytes is a highly dynamic structure. Eur. J. Cell Biol. 66, 234-245.
Hammerback, J. A., and Vallee, R. B. (1990). Antibody exchange immunochemistry. J. Biol. Chem.
265, 12763-12766,
Harlow, E., and Lane, D. (1988). “Antibodies: A Laboratory Manual.” Cold Spring Harbor Labora-
tory Press, Cold Spring Harbor, NY.
Heim, R., and Tsien, R. Y. (1996). Engineering green fluorescent protein for improved brightness,
longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178-182.
Herzog, M., Draeger, A,, Ehler, E., and Small, J. V. (1994). Immunofluorescence microscopy of the
cytoskeleton: Double and triple immunofluorescence. In “Cell Biology: A Laboratory Manual.”
(J. E. Celis, ed.), pp. 355-360. Academic Press, New York.
McKenna, N. M., and Wang, Y. L. (1989). Culturing cells on the microscope stage. Meth. Cell Biol.
29, 195-205.
Mazia, D., Schatten, G., and Sale, W. (1975). Adhesion of cells to surfaces coated with polylysine.
J. Cell Biol. 66, 198-200.
Means, G. E., and Feeney, R. E. (1971). Chemical Modification of Proteins.” Holden-Day, Inc.
Melan, M. A,, and Sluder, G. (1992). Redistribution and differential extraction of soluble proteins
in permeabilized cultured cells: Implications for immunofluorescence microscopy. J. Cell Sci.
101,731-743.
Oka, M. T., Arai, T., and Hamaguchi, Y. (1994). Different reactivity with monoclonal antitubulin
antibodies between native and fixed mitotic microtubules in sea urchin eggs. Cell Motility Cytoskel.
29, 241-249.
Osborn, M., and Weber, K. (1982). Immunofluorescence and immunocytochemical procedures with
affinity purified antibodies: Tubulin-containing structures. Meth. Cell Biol. 24, 97-132.
Small, J. V., Rinnerthaler, G., and Hinssen, H. (1982). Organization of the actin meshworks in
cultured cells: The leading edge. Cold Spring Harbor Symp. Quant. Biol. 46,599-611.
Stearns, T. (1995). The green revolution. Curr. Biol. 5,262-264.
Theurkauf, W. E. (1994). Imrnunofluorescence analysis of the cytoskeleton during oogenesis and
early embryogenesis. Meth. Cell Biol. 44,489-505.
Wang, K.. Feramisco, J. R., and Ash, J. F. (1982). Fluorescent localization of contractile proteins in
tissue culture cells. Meth. Cell Biol. 85, 514-561.
Wang, Y.-L. (1998). Digital deconvolution of fluorescent images for biologists. Meth. Cell Biol.,
in press.
Wehland, J., and Weber, K. (1987). Turnover of the carboxy-terminal tyrosine of a-tubulin and
means of reaching elevated levels of detyrosination in living cells. J. Cell Sci. 88, 185-203.
Wheatley, D. N., Feilen, E., Yin, Z., and Wheatley, S. P. (1994). Primary cilia in cultured mammalian
cells: Detection with an antibody against detyrosinated a-tubulin (ID5) and by electron microscopy.
J. Submicroscop. Cytol. Pathol. 26,91-102.
Wheatley, S . P., and Wang, Y.-L. (1996). Midzone microtubule bundles are continuously required
for cytokinesis in cultured epithelial cells. J. Cell Biol. 135, 981-989.
Wright, B. D., and Scholey, J. M. (1993). Nonfluorescent irnmunolocalization of antigens in mitotic
sea urchin blastomeres. Meth. Cell Biol. 37, 223-240.
CHAPTER 19
Cellular Locahzation of mRNA and

Protein: In Situ Hybridization
Histochemistry and in Situ Ligand Binding
Teresa K. Woodruff
Northwestern University
Departments of Memcine and Neurobiology and Physiology
Chicago, Illinois 60611
I. Introduction
11. Method: In Situ Hybridization
A. Tissue Preparation and Sectioning
B. Prehybridization of Cells or Tissue Sections on Slides
C. Probes
D. Hybridization
E. Posthybridization
F. Autoradiography
111. Protocol: In Situ Hybridization
A. Reagent and Buffer Preparation
B. Tissue Sectioning
C. Prehybridization Conditions
D. Synthesis of Radiolabeled Antisense Riboprobe
E. Hybridization
F. Posthybridization Treatment
G. Exposure to X-Ray Film and Emulsion
IV. Method: In Situ Ligand Binding
A. Cell and Tissue Processing
B. Probe
C. Binding
D. Washes
E. Autoradiography
V. Protocol: In Situ Ligand Binding
METHODS IN CELL BIOLOGY, VOL. S7

Copynght 8 1998 by Academic Press. All rights of reproduction I" any form reserved 333
oo91-679xm s2s.00
334 Teresa K. Woodruff
C. Synthesis of Radiolabeled Proteins

D. Prehybridization
E. Hybridization
F. Posthybridization
VI. Analysis of Slides
VII. Combination of in Situ Hybridization with in Situ Ligand Binding,
Immunohistochemistry, or Metabolic Studies
References
Powerful methods for the detection of mRNA and proteins in cells and tissue
sections have been developed since the mid-1980s. This chapter discusses the
applications of in situ hybridization histochemistry and in situ ligand binding to
cells in culture and tissue sections. In situ hybridization takes advantage of
paired nucleotide interactions between a labeled probe (antisense strand) and
the endogenous mRNA (sense strand). Following processing, the mRNA is
localized through detection of the disintegration pattern of the radiolabeled
probe. Protein-protein interaction is detected in a similar fashion. Proteins are
radiolabeled and incubated with tissues that contain target-binding proteins or
receptors. On processing, the interaction sites are localized through detection of
the radiolabeled probe. The methods are rapid, sensitive, specific, and provide
important information regarding the sites of mRNA synthesis, abundance of
protein, and the ability of the ligand to interact with the receptor in restricted
cellular populations. Application of these techniques to cells in culture allows
in vitro manipulation of endogenous mRNA or protein with various hormones
or growth factors and a method to detect the results.
I. Introduction
The cellular localization of mRNA and protein in individual cells of a tissue
or in a mixed cell population provides important data about neuronal structure,
hormonal control of mRNA accumulation, hormone/growth factor site of synthe-
sis, and the ontogeny of developmentally relevant mRNAs and proteins (Funa-
bashi et al., 1995;Levin et al., 1995;Mercer et al., 1996;Norris et al., 1995; Roberts
et al., 1994;Woodruff et al., 1988). In principle, in situ hybridization is the process
whereby a labeled “antisense” RNA strand is hybridized to a “sense” RNA
strand present in cells from thin tissue sections or monolayers of cells plated
onto glass microscope slides. Hybridization is achieved by the hydrogen bonding
of complementary base pairs found in the single-stranded probe and tissue mes-
senger RNA. Detection of the complex is based on imaging radioactive or biotin-
ylated nucleic acid (usually UTP) incorporated into the antisense probe. The
mRNA of interest is thus localized to a specific cell or group of cells expressing
19. Cellular Localization of mRNA and Protein 335
a mRNA of interest. Comparison of mRNA localization or abundance in tissues

or cells collected under variant experimental conditions can provide important
molecular information about genes of interest.
In situ ligand binding detects protein-protein interactions between a labeled
ligand and cells in a thin tissue section or monolayer culture attached to micro-
scope slides. Receptor-ligand and binding protein-ligand interactions are the
most commonly examined associations. In this application, the receptor or bind-
ing protein and labeled ligand interact and binding occurs as it would in vivo,
via affinity interactions. Similar to in situ hybridization, in situ ligand binding
provides data relative to the cellular site of protein synthesis.
The power of each method is in its specificity for individual cells of a heteroge-
neous tissue or cell population. When applied appropriately, the methods can
also be semiquantitative. The challenges of both methods are (1) selection of
appropriate probes, (2) preparation of tissues or cells, (3) optimization of hybrid-
ization conditions, and (4) evaluation of results (either by analysis of X-ray films
or following liquid film emulsion autoradiography). Each of these points must
be systematically optimized for each probe and tissue or cell preparation. This
chapter will describe details of the in situ hybridization and in situ ligand-binding
protocols and provide investigators with starting points for experimental design.
11. Method: Ifi Situ Hybridization
Numerous methods are described in the literature for the detection of mRNA
in tissue sections (Bloch, 1993; Koji and Nakane, 1996; Mitchell et al., 1992;
Raval, 1994; Strotmann et al., 1996; Szakacs and Livingston, 1994; Wilcox, 1993)
(Fig. 1). The method described in this chapter deals with mRNA detection in
sections obtained from frozen tissues or cells plated as monolayers on microscope
slides. The method of tissue sectioning will depend on the cryostat available.
Cells that are grown on chamber slides can also be processed according to the
following method. Chamber slides can be purchased from Lab-Tek (Chicago,
IL). The sensitivity of in situ hybridization depends on the preservation of mRNA
integrity in the tissue or cell preparations. Therefore, throughout the procedure,
steps should be taken to minimize contact between ungloved hands and RNase-
free equipment and reagents. Dedicated RNase-free spatulas, stir bars, pH
probes, glassware, and pipettes should be utilized where possible. A treatment
of glassware and equipment with 0.1% H202followed by a methanol rinse can
be used on surfaces that cannot otherwise be sterilized (e.g., pipetteman). Solu-
tions can be treated with 0.1% diethylpyrocarbonate (DEPC), an inhibitor of
RNase, although this step is not necessary if all reagents are autoclaved and kept
separate from sources of contaminating RNase.
A. Tissue Preparation and Sectioning

Cells can be grown on chamber slides under standard conditions. Chamber
slides can be purchased with one-, two-, four-, or eight-well compartments. The
Fig. 1 I n situ hybridization of a 32P-labeled antisense probe generated against a hormone/growth

factor (inhibin a-subunit) hybridized to a tissue section taken from the ovary of a cycling female
rat. Sections were processed as described in the text and then exposed to X-ray film for 12 hr (A)
followed by autoradiographic emulsion -12 days (B, lOOX magnification; C, 5OOX magnification).
(A) The dark circular is clearly defined on X-ray film.A qualitative assessment of hybridization
intensity can be analyzed using this type of data output. Following exposure of the slide to X-ray
film, the slide is dipped in liquid emulsion, exposed, and then developed at a time to maximize the
intensity of the signal and minimize nonspecific background. (B) The developed slide has black
silver grains deposited (precipitated in situ by the D19 developer) over the cells in which radiolabeled
molecules are hybridized to endogenous mRNA. At higher magnification (C) the individual grains
can be seen over specific cell populations. The grains can be counted per cell or per square area to
give a quantitative measure of grain density. The slides were photographed using bright-field optics
so the grains appear black over the field of stained cells.
advantage of the multicompartment slides is that several treatment groups can

be compared on a slide with a given probe. For example, cells could be plated
in an eight-well chamber slide, incubated with a range of doses of a particular
ligand, and then processed for mRNA detection using one probe against a
particular target gene product. This technology allows tremendous precision in
comparing groups of cells for expression of any molecule. Some cells do not
adhere to glass well. Options for improving cell adhesion include coating the
wells with whole serum for 30 min, followed by rinsing with warm media prior
to plating the cells. Alternatively, attachment factors such as vimentin, fibronec-
tin, or poly-L-lysine can be used to coat the wells to improve cell attachment.
The chambers should be removed from the slide prior to proceeding through
the remainder of the protocol.
If tissues are used, they should be collected rapidly (<3 min), and the smallest
representative tissue chunk (3-4 cm2) should be immersed in a freezing block
(either fabricated out of foil or purchased from Baxter) and filled with a mounting
medium (OCT, Miles Laboratories). The tissue face to be sectioned should lie
flat and free of air bubbles against the base of the mold. OCT mounting medium
should be added to fill the block and the block lowered into an isopentene bath
(with dry ice; -1SOC) or set on foil above dry ice. Liquid nitrogen should not
be used at this step because it will cause the tissue block to fracture. Once frozen,
the tissue blocks should be stored in zip-lock bags with little or no air and stored
at -80°C until sectioned. It is preferable to use the tissue blocks once; however,
sectioned tissue blocks can be resealed with OCT, remounted into molds, and
stored again at -80°C. Some investigators section tissue blocks and store the
sections on slides at -80°C. This method can succeed when mRNA is in high
abundance. However, results from tissues stored in this manner can vary due
to poor (or variant) tissue and mRNA integrity. For best results, use freshly
sectioned tissues.
Tissue sections are usually 4-12 pm in thickness. Sections are mounted onto
slides that have been treated to maximize tissue adherence during the numerous
processing steps. Precoated slides can be purchased from a number of vendors
(Baxter, Promega, Fisher). Alternatively, uncoated slides can be coated with a
reagent known by its trade name, Vectabond (Vector Laboratories, Burlingame,
CA). These two sources of treated slides provide reliably good tissue adherence
and minimal background to the slide.
B. Prehybridization of Cells or Tissue Sections on Slides

A series of prehybridization treatments should be done within 20 min of
sectioning. The cells or sections are first fixed. Several fixative reagents can be
used, including glutaraldehyde (3%) and paraformaldehyde (5%). Fixation must
satisfy two, at times conflicting, requirements. Tissue morphology must be main-
tained and the mRNA anchored in position, but proteins must not be so highly
cross-linked that probe penetration is obstructed. Paraformaldehyde is the most
economical choice of fixative. Rinse the slides in 2X SSC (5 min) and dip briefly
in water followed by triethanolamine (two dips) and incubate for 10 min in
triethanolamine and acetic anhydride to remove residual fixative and reduce
nonspecific probe binding. Following a rinse in 2X SSC, dehydrate the slides
through a graded ethanol series (50,70,95,100%) and store under vacuum until
hybridized (<8 hr).
C . Probes
Three types of probes can be used for in situ hybridization studies: complemen-
tary DNA probes, antisense RNA probes, and synthetic oligonucleotides. Control
probes are constructed to represent the “sense” configuration of the endogenous
mRNA (i.e., identical to mRNA). The control probe is used to delineate nonspe-
cific binding to labeled nucleic acid in tissue sections. The most important aspect
of planning a probe is designing specificity. Regions of discreet identity should
be used. Each prove type requires different conditions for labeling, hybridization,
and washing stringency. Riboprobes will be considered in this chapter.
Single-strand riboprobes are derived from antisense cRNA synthesized under
the control of bacterial promoters in specially designed vectors. Typically, cDNA
representing the gene of interest is cloned into a vector containing T3, T7,or
Sp6 bacterial promoters. The vector is linearized at a convenient restriction site
250-450 bp 5‘ relative to the promoter start site. The linearized plasmid is then
used as an antisense template [sequence generated will be complementary (3’-5’)
relative to the mRNA strand expressed by the cells (5’-3’)]. To generate a
suitable sense control probe, the vector should be linearized 250-450 bp 3’ to a
promoter start site. Probes can be radiolabeled with 35S-UTP,32P-UTP,or 33P-
UTP. Alternatively, nonradioactive probes can be generated using biotinylated
UTP. Incorporation of the labeled nucleotide is done during the synthesis phase
of cRNA from DNA template.
D. Hybridization
Hybridization between a labeled nucleic acid sequence and the sequence held
in situ within a sectioned cell is defined by hydrogen bonding and hydrophobic
interactions in equilibrium. The mathematical calculation for this phenomenon
is represented by the mean thermal stability of hybrids ( T,), or the temperature
at which 50% of target sequences are dissociated. This number depends on the
number of mismatches between target and probe, the length of the probe, the
G + C content of the probe, and the salt and formamide concentration in the
hybridization buffer. Salt concentrations (generally sodium ions) neutralize
the negative or repulsive nature of the nucleic acid strand net negative charge
(due to the negatively charged phosphate groups which make up the backbone
of nucleic acid chains). Formamide disrupts hydrogen bonds and destablizes
duplexes. Mathematically, the calculation of T, is defined as
T, = 16.6 log [Na] + (0.41 X %GC base pairs) + 81.5 - 1”C/1% mismatch
- [300 + (2000 X [Na]/probe length)] - % formamide/2.
Practically, the temperature of hybridization ( Th)for in situ is 20°C below the T,:
Th = T, - 20°C.
Hybridization buffers contain reagents to maximize nucleic acid duplex, inhibit
nonspecific binding of probe to tissue, and reduce the hybridization volume to
a small amount in order to conserve probe. The hybridization buffer base is a
10: 1 molar ratio of sodium chloride and sodium citrate. Formamide is used at
a percentage to lower the optimal hybridization temperatures to minimize tissue
damage and encourage specific probe binding. Formamide is also a nuclease
inhibitor and allows the salt concentration to be adjusted to allow physiological
osmolality and pH. Dextran sulfate is included in the buffer as a high molecular
weight “volume excluding” macromolecule, in effect concentrating the probe
into a smaller physical space. Detergents such as sodium dodecyl sulfate and
heterologous nucleic acids inhibit background due to charge or nonspecific inter-
action with nucleic acid. The probe concentration should be in excess of mRNA
contained in tissue section resulting in a “probe-driver” condition. Hybridization
takes place on a coverslipped slide in a mineral oil bath or in a humidified
chamber for 18 hr.
E. Posthybridization
Following an overnight incubation, carefully remove the coverslips using 2X
SSC. If an oil bath has been used, remove the oil with chloroform. A 30-min
incubation of slides with RNase A (37°C) removes the majority of nonspecific
hybridization. Four 30-min washes in decreasing salt solution at 42°C further
eliminate nonspecific hybrids. Steps should be taken to ensure the containment
of RNase-contaminated slide racks, slide vessels, and pipettes away from future
in situ hybridization equipment and reagents.
F. Autoradiography
X-ray film or a liquid emulsion specific for /3 emitters (Kodak NTB-2) can be
used to image the radionucleotide. X-ray film can only be used for 32P-or 33P-
UTP-labeled probes. The procedure is simple, rapid, and allows gross assessment
of hybridization intensity in all sections simultaneously. If precise cellular localiza-
tion is required, the slides should be exposed to a hardened emulsion that has
been coated directly onto the slide. NTB-2 emulsion can be solubilized at 42”C,
diluted 1 : 1 with water in a darkroom, and then coated onto slides by dipping
and removing slides with a slow even movement. Allow the slides to dry while
standing in a vertical position. Place dried slides in a light-tight box with disiccant
and store at 4°C until developed. Several sets of emulsion-dipped slides should
be generated. Slides should be developed on a weekly basis in order to capture

the output at a time of maximal signal to least noise. Develop exposed slides
with Kodak 19 developer, water, and Kodak fixer. Wash the slides in water for
another 30 min. At this point the slides can be counterstained and coverslipped
for analysis.
111. Protocol: In Situ Hybridization

1. Vectabond slides: Set up all reagents in glass containers with metal racks).
Uncoated 3” X 1”slides (Baxter, 48300-240).
a. 30 min 1 M HCl.
b. 5 min water.
c. 5 min water.
d. 5 min acetone.
e. 5 min Vectabond (Vector Laboratories, SP-1800,7 ml reagent/350 ml
acetone).
f. 5 min water.
g. 5 min 100% EtOH. Change buffers after 500 slides. Do not create
bubbles. Dry slides in dust-free area.
2. 5% paraformaldehyde: 12.5 g paraformaldehyde (Sigma P-6148), 1 bead
NaOH, and 250 ml PBS. Heat solution until paraformaldehyde is solubilized.
Allow solution to cool and pH to 7.8. Filter through a 0.45-pm filter.
3. 0.1 M triethanolamine (TEA): 4.63 g TEA (Sigma T 9534) and 250 ml PBS;
pH to 8.0. Filter through a 0.45-pm filter.
4. 0.1 M TEA + 0.2% acetic anhydride: 250 ml 0.1 M TEA and 0.5 ml acetic
anhydride (Sigma A-6404).
5. RNase A solution: 25 ml 5 M NaCl, 25 ml 1 M Tris, pH 8.0, 0.5 ml 0.5 M
EDTA, and 0.5 ml10 mg/ml RNase A (Sigma R 6513). Solution should be
made up 30 min in advance and heated to 37°C.
6. 20X SSC: 175.3 g NaCl, 88.2 g sodium citrate, and 1 liter water; autoclave.
7. 50X Denhardt’s solution (Sigma D-8906): 100 mg Ficoll (MW 400,000)
(Sigma F2637), 100 mg polyvinylpyrrolidone acid (Sigma P-5288), 100 mg
BSA (Intergen 3160-80), and 1 ml water. Filter through a 0.45-pm filter
into autoclaved Eppendorf tubes. Store at -20°C.
1. Mount tissue onto OCT covered microtome block (OCT Miles Labs Cat
#4583; mounting molds-Baxter M7307-7 X 7 mm; 15 X 15 mm; 24 X 24 mm).
2. Allow tissue to equilibrate to sectioning temperature.

3. Section tissue (5-12pm).
4. Thaw mount tissue onto Vectabond treated slide.
(Note: cells cultured on chamber slides do not need to be processed prior to the
prehybridization steps.)
C. Prehybridization Conditions
1. Fix tissue sections mounted onto slides in 5% freshly prepared paraformalde-
hyde for 5 min.
2. Wash in 2X SSC for 5 min.
3. Dip four times in water.
4. Dip in 0.1 M TEA.
5. Incubate for 10 min in 0.1 M TEA/AA.
6. Dip in 2X SSC.
7. Dehydrate in 50, 70, 95, 100% ethanol for 3 min each.
8. Store under vacuum until ready to hybridize.
D. Synthesis of Radiolabeled Antisense Riboprobe

Promega Biotech offers a kit for riboprobe transcription (P1420, P1430, P1440)
1. Add in order at room temperature: 4 pl 5X transcription buffer, 2 pl 100
mM DTT, 1 p1 RNasin, 4 pl 2.5 mM rib0 A,G,C, 2.4 pl 100 p M UTP, 1 pg
linearized template DNA, 5 pl 32P-UTP(800 Ci/mmol), and 1 p1 SP6, T7,T3.
2. Incubate at 37°C for 1 hr.
3. Ethanol precipitate twice.
4. Count probe.
Note: The concentration of all four nucleotide triphosphates must exceed the
K, of the RNA polymerase used (i.e., 10-60 p M ) for effective transcription.
Clean, completely linearized templates are required for the production of pure
riboprobe. Probe can be monitored on 6% acrylamide/urea gel chromatography.
E. Hybridization
1. Prepare hybridization cocktail: 500 p1 formamide (Fluka 47671), 60 p15 M
NaC1, 10 pl 1 M Tris 8.0, 4 p1 0.25 M EDTA, 10 p1 lOOX Denhart's, and 200
pl 50% dextran sulfate (Sigma D 8906).
2. Prepare probe mix: 50 pl yeast tRNA (10 mg/ml) (Sigma R 8759), 50 p1
poly(A)RNA (10 mg/ml) (Boeringer-Mannheim 109-495),and lo8 cpdprobe in
138 p1 water.
3. Mix and heat probe mix to 65°C for 5 min.

4. Combine cocktail and probe mix, vortex, and heat to 65°C for 5 min.
5. Spot 75 pl onto slide, coverslip (Fisher No. 1coverslips 1254414), carefully
minimizing air bubbles.
6. Incubate at 42°C for 18hr in a heavy mineral oil chamber (mineral oil, Sigma
400-5) (a five slide mailer works well as hybridization chambers) (Baxter M6271).
F. Posthybridization Treatment
1. Dip slides in chloroform for 15 min.
2. Dip slides in 4X SSC for 20 min.
3. Remove coverslips.
4. Incubate slides in RNase A solution at 37°C for 30 min.
5. 4 X SSC; 42"C, 30 min.
6. 2X SSC; 42"C, 30 min.
7. 1 X SSC; 42"C, 30 min.
8. 0.5X SSC; 42"C, 30 min.
9. Dehydrate through an ethanol series (50, 70, 95, 100%) for 3 min each.
10. Dry under vacuum.

1. Expose dry slides to X-ray film.
2. Dip slides in NTB-2 emulsion (diluted 1: 1 with water and melted for 5 min
at 42°C) (Kodak 1654433).
3. Allow to dry.
4. Place in light-tight box (foil wrapped) for 9-12 days with desiccant.
5. Develop with D19 for 1 rnin (Sigma P-5670).
6. Wash with water for 5 min.
7. Fix with Kodak fix for 5 rnin (Sigma P-5670).
8. Rinse with water, counterstain, and coverslip.
IV. Method: In Situ Ligand Binding

Studies of ligand binding to receptors (or to other macromolecules) in situ
have increased in number and sophistication in a number of scientific disciplines
(Jakeman et al., 1992, 1993; Lynn et al., 1996; Roberts et af., 1996; Schwartz et
af., 1996; Wilkin et al., 1981; Woodruff et af., 1993). Identification of cellular sites
of protein-protein interactions is important in delineating sites of growth factor,
cytokine, or hormone action (Fig. 2). Although in situ ligand binding has been
used to determine saturation kinetics and binding constants, most notably in
brain nuclei, it is most suited as a method of localization.
A. Cell and Tissue Processing

Unlike in situ hybridization, the cells or tissues used for in situ ligand binding
are not fixed prior to the addition of labeled ligand. Fixation causes the cross-
linking of proteins and usually blocks the ability of ligand to bind the target site.
(Indeed, protein cross-linking prior to ligand binding is sometimes used as a
negative control.) The cells are grown on chamber slides or sections are mounted
onto pretreated slides and the slides are directly incubated with a radiolabeled
ligand.
B. Probe
Probes are generally proteins that are iodinated. Iodine isotopes, which are
y-emitters, have a short half-life and can be chemically or enzymatically coupled
to most proteins. Attachment of iodine to tyrosine (through phenolic anion
attack at positions ortho to hydroxyl groups) is the most common modification.
Because some proteins or peptides contain insufficient tyrosines or tyrosine
modification bioinactivates the protein, reagents that modify histidine or lysine
molecules should also be tested.
Iodination of tyrosine residues can be accomplished chemically using the oxi-
dating reagent chloramine-T (Sigma C-9887). The use of chloramine-T (because
it is strongly oxidizing and because inactivation of the reaction requires a reducing
agent) many times causes loss of biological activity. Iodo-Beads (Pierce 28665)
is a solid-phase chloramine-T reagent that is less cumbersome to use than the
liquid reagent. The beads can also be physically removed, eliminating the terminal
reduction step. Additionally, iodination with Iodo-Beads at pH 7-8.2 causes the
modification of histidine residues. Enzymatic lactoperoxidase (Sigma L-2130)
modifies tyrosine and is a mild and reliable alternative to chloramine-T. The
enzymatic reaction is inhibited by high salt or azide.
If tyrosines are not available, are contained in active sites, or are under-
represented (leading to a low specific activity of probe), a Bolton-Hunter
reagent can be used. Bolton-Hunter modifies primary amines (lysines) and is a
N-hydroxysuccinimide (NHS) ester of 3-(4-hydroxyphenyl)propionic acid.
The decision to use any of the methods of protein modification should be
done in concert with a careful assessment of biological and specific activity. The
target-specific activity for an iodinated probe is 60-100 pCi/pg.
C. Binding
Binding of ligand to tissue section is done in a simple buffer with protease
inhibitors. The buffer includes F12 :DMEM to maintain normal osmolarity
Fig. 2 In situ ligand binding of iodinated activin A to potential receptors in rat embryos. Sections
were processed as described in the text and then exposed to X-ray film (-14 days) (A) and then
autoradiographic emulsion (-14 days) (B and C). The specificity of in siru ligand binding of an
iodinated probe to a variety of structures in the developing embryo is illustrated. (A) The labeled
ligand is '251-inhibinA. Areas of positive hybridization have been delineated by capital letters. D,
dermis; B, brain; TG, trigeminal; L, liver; SG, spinal ganglia; SC, spinal cord. X-ray film results are
important in this type of gross anatomical assessment of binding sites. The emulsion autoradiographic
and pH, cytochrome c and bovine serum albumin (BSA) as inhibitor of

nonspecific protein binding, and a cocktail of protease inhibitors, including
phenylmethylsulfonyl fluoride (PMSF) (inhibits serine proteases: chymotrypsin,
trypsin, thrombin, cysteine protease papain) and leupeptin (inhibits serine and
cysteine proteases, including cathepsin B). Binding should be done over a
range of molar concentrations, time, and temperature in order to establish
optimal conditions for binding while minimizing nonspecific association of
proteins. Once concentration, time, and temperature have been determined,
competition experiments in which homologous unlabeled ligands are coincu-
bated over a range of concentrations (up to 1000-fold excess) should be
included in the experiment.
D. Washes
Following incubation of slides with ligand, fix the slides in a mixture of para-
formaldehyde and glutaraldehyde, rinse in a low salt buffer, and dehydrate
through an ethanol series (50,70,95,100%).Dry the slides and ready for autoradi-
ography .
E. Autoradiography
Autoradiography for in situ ligand-binding slides incubated with iodinated
ligands can be first imaged on X-ray film. Following gross analysis on film, dip
the slides in a liquid emulsion specific for y emitters (NTB-3, Kodak) and allow
to expose embedded silver grains over time. Several sets of slides should be
generated so that the optimal exposure time can be established for each experi-
mental ligand.
V. Protocol: Irt Situ Ligand Binding

1. Vectabond slides: Treated as described earlier.
2. Binding buffer
results provide detailed information about specific cell populations. (B) A lOOOX magnification of
cells in a spinal ganglia bundle. The labeled ligand is '2sI-activin A. Specific hybridization is assessed
by incubating an adjacent section (C) in binding buffer, labeled ligand, plus 1000-fold excess unlabeled,
competitive, activin A. The binding sites in the spinal ganglion are bound by competitor and therefore
these cells are binding the ligand specifically. The apparent binding to the rib is nonspecific because
the excess unlabeled ligand is unable to compete for the binding in this tissue. Slide photography
was done with dark-field optics, making the grains appear white over a black background.
346 Teresa I(. Woodruff
Reagent and final concentration Source and catalog number Stock concentration Per 500 ml
DMEM :F12 GIBCO 1:l 490 ml
20 mM HEPES 1M 10 ml
0.05% cytochrome c Boehringer 103 888 0.25 g
0.3% BSA Intergen 3160-80 1.5 g
0.01 mg/ml PMSF Boehringer 236 608 1 m g / d in EtOH 50 p1
0.4 pg/ml leupeptin Boehringer 101 7101 0.2 g
3. Aldehyde fixative
Reagent and final concentration Source and catalog number Stock concentration Per 500 ml
3.7% formalin Fluka 47629 37% 50 ml
2% glutaraldehyde Kodak 8648 50% 20 ml
Phosphate buffered saline (PBS) 430 ml
1. Mount tissue onto OCT-covered microtome block.
2. Allow tissue to equilibrate to sectioning temperature.
3. Section tissue (5-12 pm).
4. Thaw mount tissue onto Vectabond-treated slide.
(Note: Cells cultured on chamber slides do not need to be processed prior to
the incubation of cells with labeled ligand.)
C. Synthesis of Radiolabeled Proteins

Optimal conditions for iodinating specific proteins should be developed for
each ligand. The following method is a modified lactoperoxidase method and
iodinates proteins at tyrosine residues.
1. Pipette 0.5 nmol protein into 1.5-ml microfuge tube.
2. Add 40 pl 0.4 M sodium acetate, pH 5.6.
3. Add Na 12’1 to final concentration of 0.5 nmol/mCi on calibration data).
4. Add lactoperoxidase to a final concentration of 5 mIU.
5. Add H202to a final concentration of 0.25 nmol.
6. Incubate for 5 min at room temperature with intermittent vortexing.
7. Quench reaction by adding 500 p1 column buffer.
8. Separate labeled and unlabeled protein on a Sephadex G-25M column
(Pharmacia 17-0851-07). Column chromatography choice will have to be made
carefully based on the size of the expected product. HPLC purification may be
necessary in some cases.
9. Collect column fractions of 0.5 ml.
10. Determine specific activity of peak fraction.
19. Cellular Localization of mFWA and Protein 347
D. Prehybridization
1. Add protease inhibitors to binding buffer immediately before use.
2. Distribute slides into slide mailers in binding buffer and incubate at room
temperature for 3 hr.
E. Hybridization
1. Prepare labeled ligand and labeled ligand plus cold competitor in binding
buffer.
2. Filter hot hybridization buffer (0.45 pM).
3. Incubate slides in buffer at temperature and for period of time determined
experimentally (recommended starting temperature range 4-37°C).
F. Posthybridization
1. Rinse in binding buffer (without labeled ligand).
2. 2 x 10 rnin PBS.
3. Fix in aldehyde for 10 min.
4. Rinse in water.
5. Dehydrate through an ethanol series (50, 70, 95, 100%) for 3 min each.
6. Dry slides under vacuum.

1. Expose dry slides to X-ray film.
2. Dip slides in NTB-3 emulsion (diluted 1:1with water and melted for 5 min
at 42°C) (Kodak 1654441).
3. Allow to dry.
4. Place in light-tight box (foil wrapped) for 9-12 days with desiccant.
5. Develop with D19 for 1 min.
6. Wash in water for 5 min.
7. Fix with Kodak fix for 5 min.
8. Rinse with water, counterstain, and coverslip.
VI. Analysis of Slides

Four identical slides (if tissues are to be analyzed, this means adjacent sections)
should be prepared for each probe (either riboprobe or iodinated protein). Each
set of slides is then exposed to emulsion for different lengths of time (1-6 weeks).
In this manner, the best exposure for low and high density binding sites can
be assessed. Slides or films should be examined by two or three independent

investigators. Evidence of binding can be done in a qualitative manner using a
densitometer able to count grains in particular areas of the slide. Instrumentation
and computer programs are available from a vareity of sources, including Nikon
and Carl Zeiss. Qualitative scores can also be used. For example, the abundance
of ligand binding or mRNA expression can be estimated based on the relative
abundance of silver grains over specific structures. One type of graded scale is
as follows: -, silver grains are not present above background; +, silver grains
are sparse in number but form a discrete hybridization pattern; + +, silver grains
are abundant but do not cover the whole area; +++, silver grains are very
abundant and appear to fuse together; ++++, silver grains are so abundant
that they form a continuous structure without any interruptions (Meunier et
al., 1988).
Representative sections with silver grains can be photographed using a variety
of microscopes equipped with epiluminescent (grains appear white on dark tissue
background) bright-field (grains appear black on stained background), or dark-
field optics (white grains appear with no tissue background).
VII. Combination of in Situ Hybridization with in Situ Ligand

Binding, Immunohistochemistry, or Metabolic Studies
The power of in situ localization methods can be amplified by the coordinate
localization of mRNA and protein in the same cell or tissue (Fig. 3). Valuable
information about the mRNA site of synthesis and receptor localization can be
obtained from studies in which cells are treated with various hormones, growth
factors, or cytokines or in which adjacent tissue sections are mounted onto
separate slides and processed to localize mRNA on one slide and protein on
another (Altar et al., 1994;Jakeman et al., 1993; Woodruff et al., 1993). Addition-
ally, immunohistochemical detection of proteins using specific antibodies can be
used in tandem with either in situ hybridization or in situ ligand binding to
colocalize two independent gene products to a particular cell or to identify the
specificcell type in which a particular protein is expressed. Immunohistochemistry
should be done immediately after processing the slides for in situ hybridization.
If it is preferable to do the immunolocalization first, then all reagents must be
kept RNase free. Because the emulsion coat does not allow penetration of
antibodies, immunohistochemistry following emulsion autoradiography is not
an option.
Colocalization of expressed mRNA and protein can be valuable in delineating
targets cells for hormone action, potential sites of growth factor activity, or
changes in abundance of cellular proteins under different experimental condi-
tions. For example, colocalization of the mRNA for vascular endothelial growth
factor (VEGF) (in situ hybridization) with the VEGF receptor (in situ ligand
binding) suggests sites of interaction of ligand with receptor in vascular tissue
Fig. 3 Colocalization activin receptor subunit mRNA (Act RIIB) (in situ hybridization) and follicle
stimulation hormone (FSH) containing pituitary gonadotrope cells (immunohistochernistry). This
figure exemplifies the added information that can be gained by using cellular localization methods
in tandem. Pituitary sections were processed for in situ hybridization (antisense probe against activin
receptor) and then incubated with antibodies against FSH. Once stained the sections were dipped
in autoradiographic emulsion and developed -12 days later. Activin receptor mRNA is depicted
as silver grains and gonadotropes (FSH-containing cells) are localized by brown staining. Double-
labeled cells are shown with arrows.
and predicts potential roles for the ligand in angiogenesis (Jakeman et al., 1993).
Another example of combination methodology is in metabolic studies. For exam-
ple, labeled proteins (such as nerve growth factor) can be injected into the brain
in vivo and the distribution of the label can be localized to specific target cell
populations. The analysis can be extended using in situ hybridization to colocalize
the mRNA for specific receptor targets (in this example, TrkA and TrkB). In
so doing, the specific receptor isotype regulated by the exogenous hormone can
be mapped and studied (Anderson et al,, 1995).
In conclusion, in situ hybridization and in situ ligand binding are powerful and
precise methods used to localize mRNA and protein to specific cell populations.
Acknowledgments
The author thanks Vickie Roberts, University of California, San Diego, for the photomicrographs
in Fig. 2 and Robert J. Handa and Melinda Wilson, Loyola University, Chicago, for Fig. 3.
References
Altar, C. A., Siuciak, J. A,, Wright, P., Ip, N. Y., Lindsay, R. M., and Wiegand, S. J. (1994). In situ
hybridization of trkB and trkC receptor mRNA in rat forebrain and association with high-affinity
binding of [lZ5I]BDNF,[12'I]NT-4/5 and [1251]NT-3.Eur. J. Neurosci. 6(9), 1389-1405.
Anderson, K. D., Alderson, R. F., Altar, C. A., DiStefano, P. S., and Corcoran, T. L. (1995).
Differential distribution of exogenous BDNF, NGF, and NT-3 in the brain corresponds to the
relative abundance and distribution of high-affinity and low-affinity neurotrophin receptors.
J. Comp. Neurol. 357(2), 296-317.
Bloch, B. (1993). Biotinylated probes for in situ hybridization histochemistry: Use for mRNA detec-
tion. J. Histochem. Cytochem. 41(12), 1751-1754.
Casey, J., and Davidson, N. (1977). Rates of formation and thermal stab es of RNA :DNA and
DNA :DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 4(5), 1539-1552.
Funabashi, T., Brooks, P. J., Kleopoulos, S. P., Grandison, L., Mobbs, C. V., and Pfaff, D. W. (1995).
Changes in preproenkephalin messenger RNA level in the rat ventromedial hypothalamus during
the estrous cycle. Brain Res. 28(1), 129-134.
Jakeman, L., Mather, J., and Woodruff, T. (1992). In vitro ligand binding of '251-recombinanthuman
activin A to the female rat brain. Endocrinology l31(6), 3117-3119.
Jakeman, L. B., Armanini, M., Phillips, H. S., and Ferrara, N. (1993). Developmental expression of
binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role
for this protein in vasculogenesis and angiogenesis. Endocrinology 133(2), 848-859.
Koji, T., and Nakane, P. K. (1996). Recent advances in molecular histochemical techniques: In situ
hybridization and southwestern histochemistry. J. Electron Microsc. 45(2), 119-127.
Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M., and Tabin, C. (1995). A molecular pathway
determining left-right asymmetry in chick embryogenesis. Cell 82(5), 803-814.
Lynn, R. B., Cao, G . Y., Considine, R. V., Hyde, T. M., and Caro, J. F. (1996). Autoradiographic
localization of leptin binding in the choroid plexus of oblob and db/db mice. Biochem. Biophys.
Res. Commun. 219(3), 884-889.
Meinkoth, J., and Wahl, G. (1984). Hybridization of nucleic acids immobilized on solid supports.
Anal. Biochem. 138(2), 267-284.
Mercer, J. G., Hoggard, N., Williams, L. M., Lawrence, C. B., Hannah, L. T., Morgan, P. J., and
Trayhurn, P. (1996). Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate
nucleus of mouse hypothalamus. J. Neuroendocrinol. 8( lo), 733-735.
Meunier, H., Cajander, S. B., Roberts, V. J., Rivier, C., Sawchenko, P. E., Hsueh, A. J., and Vale,
W. (1988). Rapid changes in the expression of inhibin alpha, beta A-, and beta B-subunits in
ovarian cell types during the rat estrous cycle. Mol. Endocrinol. 2(12), 1352-1366.
Mitchell, B. S., Dhami, D., and Schumacher, U. (1992). In situ hybridisation: A review of methodolo-
gies and applications in the biomedical sciences. Med. Lab. Sci. 49(2), 107-118.
Norris, P. J., Charles, I. G., Scorer, C. A., and Emson, P. C. (1995). Studies on the localization and
expression of nitric oxide synthase using histochemical techniques. Histochem. J. 27(10), 745-756.
Raval, P. (1994). Qualitative and quantitative determination of mRNA. J. Pharmacol. Toxicol. Meth.
32(3), 125-127.
Roberts, V. J., Barth, S., el-Roeiy, A., and Yen, S. S. (1994). Expression of inhibidactivin system
messenger ribonucleic acids and proteins in ovarian follicles from women with polycystic ovarian
syndrome. J. Clin. Endocrinol. Metabol. 75(5), 1434-1439.
Roberts, V. J., Bentley, C. A., Guo, Q., Matzuk, M. M., and Woodruff, T. K. (1996). Tissue-specific
binding of radiolabeled activin A by activin receptors and follistatin in postimplantation rat and
mouse embryos. Endocrinology 137(10), 4201-4209.
Schwartz, M. W., Seeley, R. J., Campfield, L. A., Burn, P., and Baskin, D. G. (1996). Identification
of targets of leptin action in rat hypothalamus. J. Clin.Invest. 98(5), 1101-1106.
Strotmann, J., Konzelmann, S., and Breer, H. (1996). Laminar segregation of odorant receptor
expression in the olfactory epithelium. Cell Tissue Rex 284(3), 347-354.
Szakacs, J. G., and Livingston, S. K. (1994). mRNA in situ hybridization using biotinylated oligonucleo-
tide probes: Implications for the diagnostic laboratory. Ann. Clin. Lab. Sci. 24(4), 324-338.
Thomas, C. A., Zimm, B. H., and Dancis, B. M. (1973). Ring theory. J. Mol. Bid. 77(1), 85-99.
Wilcox, J. N. (1993). Fundamental principles of in situ hybridization. J. Histochem. Cytochem.
41(12), 1725-1733.
Wilkin, G. P., Hudson, A. L., Hill, D. R., and Bowery, N. G . (1981). Autoradiographic localization
of GABAB receptors in rat cerebellum. Nature 294(5841), 584-587.
Woodruff, T. K., D’Agostino, J., Schwartz, N. B., and Mayo, K. E. (1988). Dynamic changes in inhibin
messenger RNAs in rat ovarian follicles during the reproductive cycle. Science 239, 1296-1299.
Woodruff, T. K., Krummen, L., McCray, G., and Mather, J. P. (1993). In situ ligand binding of
recombinant human [1251] activin-A and recombinant human [1251]inhibin-Ato the adult rat ovary.
Endocrinology 133(6), 2998-3006.
INDEX
A principle, 267-268
Adenovirus, immortalization of cells, 72-73 staining of cells, 272-273
American Type Culture Collection Antibody
authentication of cell lines, 41-43 humanization, 112-113, 127
categorization of cell lines, 34-35 immunofluorescence microscopy
development of cell lines, 38-40 double labeling, 321
fees, 35 handling, 319
functions, 32 incubation protocol, 329-330
patent culture repository, 36, 38 primary antibodies, 319-320
representative cell lines, 33, 35-37 secondary antibodies, 320-321
Anaplastic astrocytoma, see Glioma titration, 319
monoclonal antibody, see Hybridoma
Animal cell culture, see also Glioma; Schwann
Apoptosis
cell; Testicular cell
DNA laddering analysis, 259-261
cloning, 16
enzyme-linked immunosorbent assay of
contamination, see Contamination, cell
histone-associated DNA fragments, 261
culture
flow cytometry assays
equipment
annexin Vkell cycle analysis
autoclave, 8, 53-54
filters, 273
cell counter, 8-9
performance of assay, 273-274
centrifuges, 9
preparation of cells, 272
freezer, 9
principle, 267-268
hoods, 4-6 staining of cells, 272-273
incubators, 6-7, 52 propidium iodidelsurface staining, 256-257
liquid nitrogen storage tank, 9 TUNELkell cycle analysis
medium filtration, 8 filters, 269
microscopes, 7-8 fixation of cells, 268
water baths, 9 labeled nucleotides, 275-276
water purification, 8, 53 performance of assay, 269-272
freezing, 9, 16, 40-41 principle, 259, 267
glassware, 13, 54-55 staining, 268-269, 271-272
immortalization, see Immortalization, cell TUNELhrface staining, 257-259
lines ISEL assay, 255-256
laboratory design, 10-11, 52-53 lactate dehydrogenase assay, 261
large-scale culture, see Scaleup, cell culture mechanism, 266
passaging of cultures, 15 morphology of cells
plasticware, 13, 54-55 characteristics, 251-252, 266
primary culture establishment, 14-15 electron microscopy, 253
reagents, medium, and serum, 11-13 light microscopy, 253
sterile technique, 13-14, 51-52 M l T assay, 261-262
Annexin V, apoptosiskell cycle analysis with nuclear staining assays, 254-255
flow cytometry TUNEL assay, principle and applications,
filters, 273 255-256
performance of assay, 273-274 vital dye staining assays, 253-254
preparation of cells, 272 XTT assay, 262
353
354 Index
ATCC, see American Type Culture Collection performance of assay, 273-274

Authentication, cell lines preparation of cells, 272
cell bank protocol, 41-43 principle, 267-268
chromosome analysis, 209-211 staining of cells, 272-273
DNA fingerprinting, 211-214 TUNEL/cell cycle analysis
history, 204-206 filters, 269
importance in various applications, 204-205 fixation of cells, 268
invertebrate cell culture, 198 labeled nucleotides, 275-276
isoenzyme phenotyping, 207-209 performance of assay, 269-272
species-specific immunofluorescence, principle, 259, 267
206-207 staining, 268-269, 271-272
Autoclave, animal cell culture, 8, 53-54 stages and transitions, 230
synchronization, see Synchronization, cell
cycle
B Cell hybridization, see Hybridization, cells;
Hybridoma
Block, cell cycle, see Synchronization, cell cycle
Cell line, see also Animal cell culture; Glioma;
Bovine corneal endothelial cell basement
Testicular cell
membrane, glioma culture
authentication, see Authentication, cell lines
biological effects, 161-162
commercial utility, 37-38
cell attachment to culture dishes, 151, 161
development by American Type Culture
Bovine serum albumin, gradient preparation,
Collection, 38-40
172-173 embryonic stem cell line establishment,
284-285
government regulation, 35
C
immortalization, see Immortalization, cell
Camera, immunofluorescence microscopy, 314, lines
323-324 nomenclature, 36-37
Cell counter repositories, 32-36, 44-45
Coulter counter, 8 source species selection, 32-33
maintenance, 8-9 Centrifugal elutriation, cell cycle
Cell culture, see Animal cell culture; synchronization, 244-246
Invertebrate cell culture Centrifuge, animal cell culture, 9
Cell cycle Chromosome analysis
difficulty in defining phases, 247-248 fluorescence in situ hybridization, 209, 211
phase duration estimation Giemsa staining, 209
flow cytometry, 237 quinacrine mustard staining, 209, 211
importance in synchronization studies, trypsin-Giemsa banding, 209-210
231-232 Cloning, animal cell culture, 16, 98-99
labeling index determination, 234-235 Colcemid, cell cycle synchronization, 239-240
mean generation time, 232 Contamination, cell culture
mitotic index determination, 233, 235-237 chemical contamination, 53-55
proliferative fraction determination, cross-contamination, 41,43
232-233 mycoplasma, 41-43,55-63
pulse-chase labeling, 235-237 physical contamination, 52-53
regulation prevention, 50-52
cyclins and kinases, 230-231 types, 49-50
growth-dependent versus regulatory Coulter counter, see Cell counter
events, 247-248 Cryopreservation, see Freezing
simultaneous analysis with apoptosis using
flow cytometry D
annexin V/cell cycle analysis Disaggregation, tissue for primary cell culture,
filters, 273 14-15, 148-150, 175-177,180
Index 355
DNA fingerprinting F
databases, 214
Feeding layer, invertebrate cell culture,
embryonic stem cells, 287
193- 194
polymerase chain reaction amplification of
Fermentor, cell culture scaleup, 226-227
polymorphisms, 212-213
FISH, see Fluorescence in sifu hybridization
principle in cell line authentication, 211
Flow cytometry
restriction fragment length polymorphism
apoptosis assays
analysis, 212
annexin V/cell cycle analysis
DNA laddering, apoptosis analysis, 259-261
filters, 273
performance of assay, 273-274
E preparation of cells, 272
E l A , immortalization of cells, 72-73 principle, 267-268
E7, immortalization of cells, 72 staining of cells, 272-273
EBV, see Epstein-Barr virus TUNEL/cell cycle analysis
Electrofusion, cell hybridization, 116, 136-138 filters, 269
Electron microscopy fixation of cells, 268
apoptosis assay, 253 labeled nucleotides, 275-276
scanning electron microscopy, see Scanning performance of assay, 269-272
electron microscopy principle, 259, 267
transmission electron microscopy, see staining, 268-269, 271-272
Transmission electron microscopy TUNEL/surface staining, 257-259
Electroporation, T-cell immortalization, 77, cell cycle phase duration determination, 237
80-81 characterization of immortalized
ELISA, see Enzyme-linked immunosorbent mononuclear cells, 81
assay testicular germ cell, analysis of DNA,
Embryonic stem cell, see also Transgenic 106-107
animal Fluorescence in sifu hybridization,
applications in transgenic technology, chromosome analysis, 209, 211
280-281,292 Formaldehyde, fixation for
cell line establishment, 284-285 immunofluorescence microscopy, 316-317,
characteristics of cell lines, 280 327-329
culture conditions, 281-282 Freezing
feeder plate preparation, 282 animal cell culture guidelines, 16,40-41
freezing embryonic stem cells, 283-284, 287-288
plates, 287-288 freezers, 9
vials, 283-284 lymphocytes, 129
genome alteration detection, 287
maintenance, 281-282
passaging, 283 G
picking colonies, 286-287 Gene delivery, immortalization of cells, 77-78,
thawing 80-81, 95-98, 105-106
plates, 288-289 Germ cell, see Testicular germ cell
vials, 284 GFP, see Green fluorescence protein
Enzyme-linked immunosorbent assay Giemsa staining, chromosome analysis, 209
histone-associated DNA fragments in Glassware, animal cell culture, 13, 54-55
apoptosis, 261 Glioma
hybridoma screening cell isolation from tumors
human, 131-132, 138-139 enzymatic digestion, 148
mouse, 125-126 mechanical dispersion, 149-150
Ependymoma, see Glioma sampling site, 148
Epstein-Barr virus, immortalization of cells, 73 cell line establishment
ES cell, see Embryonic stem cell anaplastic astrocytoma, 159
356 Index
efficiency, 151-152 humanization of antibodies, 112-113, 127

ependymoma, 159 polyethylene glycol, 112
heterogeneity of cell lines, 162 hybridomas,.see Hybridoma
maintenance, 152-154 invertebrate cells, 197
medulloblastoma, 160-161 principles of techniques
nestin-positive cell line, 160 electrofusion, 116
oligodendroglioma, 159 hemagglutinating virus of Japan, 114-115
progenitor cells, 161 polyethylene glycol, 115-116, 124
stabilization, 152 selection of hybrid cells, 116
characterization of cell lines 1Hybridoma
immunostaining markers for lineage and applications of monoclonal antibodies
phenotype, 155-158 human
neoplasticity assays cancer diagnosis, 140-141
genetic analysis, 158 light chain modification, 141-142
xenotransplantation, 158-159 multifunctional antibodies, 141
heterogeneity of disease, 147, 162 mouse, 126-127
primary culture definition, 118
bovine corneal endothelial cell basement human-human hybridoma preparation
membrane cell fusion
biological effects, 161-162 A4HI2cells, 136
cell attachment to culture dishes, 151, efficiency, 132, 134-135
161 electrofusion, 136-138
goals, 150 HO-323 cells, 135
Glutaraldehyde, cell fixation NAT-30 cells, 135
immunofluorescence microscopy, 316-317, RF-Sl cells, 136
328-329 immunization of lymphocytes in vitro
transmission electron microscopy, 298, 300 cancer cell antigens, 130-132
Green fluorescence protein, rationale, 129-130
immunofluorescence microscopy, 322-323 soluble antigens, 132-134
immunization of lymphocytes using SCID
H mice, 134
Hemagglutinating virus of Japan, cell lymphocyte preparation
hybridization, 112,114-115 cryopreservation, 129
Herpesvirus saimiri, immortalization of cells, lymph node, 128-129
74 peripheral blood, 128
Histone, associated DNA fragments in screening with enzyme-linked
apoptosis, 261 immunosorbent assay, 131-132,
Hollow fiber, cell culture scaleup, 223-224 138-139
Hood, animal cell culture mouse-mouse hybridoma preparation
horizontal flow hoods, 4-5 apparatus, 119-120
laminar flow hoods, 5-6 fusion of lymphocytes and myeloma cells,
placement in laboratory, 10 124-125
sterilizing, 6, 13-14 immunization of mouse
HPV, see Papillomavirus emulsion preparation, 121-123
HTLV-1, see Human T-cell leukemia virus-1 injection, 123
Human papillomavirus, see Papillomavirus overview, 121
Human T-cell leukemia virus-1, lymphocyte preparation, 123-124
immortalization of cells, 73-74 myeloma cell preparation, 124
Hybridization, cells parent cell lines, 119
applications, 117-118 reagents and medium, 120-121
history of techniques screening with enzyme-linked
hemagglutinating virus of Japan, 112 immunosorbent assay, 125-126
Index
357
Hydroxyurea, cell cycle synchronization, filter sets, 321-322

240-241 green fluorescence protein, 322-323
focal planes, 325
1 microscope adjustments, 323
Immortalization, cell lines morphology of cells, 325
gene delivery, 77-78 mounting cells, 324-325, 331
genes for transfection sample preparation
adenovirus, 72-73 antigen preservation, 315-317
Epstein-Barr virus, 73 epitope shielding, 318-319
herpesvirus saimiri, 74 fixation
human T-cell leukemia virus-1, 73-74 buffers, 326
oncogenes, 74-76 fixative selection, 316-318
p53,76-77 formaldehyde, 316-317, 327-328
papillomavirus, 71-72 glutaraldehyde, 316-317,328-329
simian virus 40, 70-71 methanol, 316-317, 327
invertebrate cells, transgenesis, 198-199 stock solutions, 327
spontaneous immortalization, 70 permeabilization of cells, 318, 327-329
T-cell immortalization by oncogene simian virus 40 large T antigen, 99-100
transfection Incubator, animal cell culture, 6-7, 52
bone marrow extract preparation, 79 In situ hybridization, see also Fluorescence in
characterization of cells, 81-83 situ hybridization
efficiency, 81 applications, 334-335
electroporation, 80-81 autoradiography, 339-340, 342
oncogene expression plasmid combining with in situ ligand binding,
construction, 79 348-349
peripheral blood mononuclear cell hybridization conditions, 338-339, 341-342
preparation, 79-80 posthybridization treatment, 339, 342
yolk lipoprotein preparation, 79 prehybridization of cells, 337-338, 341
telomere length in immortalized cells, 84 principle, 334
testicular cells, see Testicular cell probes, types and labeling, 338, 341
tumorigenicity, 84 reagent and buffer preparation, 340
Immunization, hybridoma production, see RNase inhibition, 335
Hybridoma tissue preparation and sectioning, 335, 337,
Immunofluorescence microscopy 340-341
antibodies In situ ligand binding
double labeling, 321 applications, 335, 342-343
handling, 319 autoradiography, 345, 347
incubation protocol, 329-330 combining with in situ hybridization,
primary antibodies, 319-320 348-349
secondary antibodies, 320-321 hybridization conditions, 343, 345, 347
titration, 319 interpretation of results, 347-348
cameras, 314, 323-324 posthybridization, 345, 347
cell culture, 314-315 prehybridization, 347
cell line authentication principle, 334-335
glioma markers for lineage and probe labeling, 343, 346
phenotype, 155-158 reagent and buffer preparation, 345-346
species-specific immunofluorescence, sample processing, 343
206-207 tissue sectioning, 346
counterstaining, 324, 330 . Invertebrate cell culture
direct versus indirect approach, 314 buffering system, 194
fluorophores feeding layers, 193-194
commonly used probes, 321-322 feeding schedules, 195
358 Index
gas phase, 194 transfection

hybridization of cells, 197 calcium phosphate transfection with
importance, 188-189 simian virus 40 large T antigen gene,
intact organisms, 197 97-98
media principles, 95-96
additives, 191-192, 196-198 Light microscopy
selection, 190-191 apoptosis assay, 253
nonadherent cell and fragment rejection, 193 microscopes for animal cell culture, 7-8
passaging Liquid nitrogen storage tank, animal cell
cell harvest, 196-197 culture. 9
passaging without splitting, 197
proliferation encouragement, 196 M
scoring of culture conditions, 195
single-cell versus tissue fragment MAG, see Myelin-associated glycoprotein
preparation, 193 Mean generation time, determination, 232
species Media
confirmation of origin, 198 buffers, 24-25
selection, 190 chemical contamination, 54
success by species, 188-189 components, 20-21
sterile culture cleanup, 192 filtration, 8
temperature optimization, 194 invertebrate cell culture
transgenesis for immortalization, 198-199 additives, 191-192, 196-198
trypsinization in culture maintenance, 197 selection, 190-191
ISEL, apoptosis assay, 255-256 optimization, 27-29
ISH, see In situ hybridization pH control, 22
Isoenzyme phenotyping, cell lines, 207-209 preparation, 24-26
screening for biological activity, 23
selection, 22-23
storage of liquid medium, 12,24
J
supplementation, 12-13, 20-21, 27
Jun, immortalization of cells, 74-75 troubleshooting, 26-27
types, 21, 23
water purification, 8, 25-26
L Medulloblastoma, see Glioma
Labeling index, cell cycle phase duration Melting temperature, calculation for
determination, 234-235 oligonucleotide probes, 338-339
Laboratory design, animal cell culture, 10-11, Messenger RNA localization, see In situ
52-53 hybridization
Lactate dehydrogenase, apoptosis assay, 261 Methanol, fixation for immunofluorescence
Leydig cell, establishment of cell lines microscopy, 316-317, 327
cell preparation Microcarrier
fractionation over discontinuous Percoll cell growth, 222-223
gradient, 96-97 types, 221-222
isolation, 96 Microscopy, see Electron microscopy;
clone selection, 98-99 Immunofluorescence microscopy; Light
histology of immortalized cells, 100-101 microscopy
immunodetection of simian virus 40 large T Mitotic detachment, cell cycle synchronization,
antigen, 99-100 241-242, 244
medium for immortalized cell culture, 99 Mitotic index, cell cycle phase duration
overview, 94-95 determination, 233, 235-237
passaging, 99 Monoclonal antibody, see Hybridoma
seminiferous cord formation in vitro with M l T , apoptosis assay, 261-262
other testicular cells, 101-103, 105 Myc, immortalization of cells, 74-76
Index 359
Mycoplasma, culture contamination transfection

containment and elimination, 63 calcium phosphate transfection with
effects and consequences, 57-60 simian virus 40 large T antigen gene,
prevention and control, 58, 60, 63-64 97-98
sources, 56-57 principles, 95-96
testing, 41-43, 55, 60-63, 153-154 pH, control of media, 22
types of contaminants, 56 Plasticware, animal cell culture, 13, 54-55
Myelin-associated glycoprotein, myelination Polyethylene glycol, cell hybridization, 112,
marker, 185 115-116, 124
Polymerase chain reaction, polymorphism
amplification in cell line authentication,
N 212-213
Primary culture
Nitrous oxide, cell cycle synchronization, 240
animal cell culture, 14-15
Nocodazole, cell cycle synchronization,
glioma, 150-151
239-240
Schwann cells, 177, 180
Programmed cell death, see Apoptosis
Proliferative fraction, cell cycle phase duration
0
determination, 232-233
Oligodendroglioma, see Glioma Pulse-chase labeling, cell cycle phase duration
Oncogenes, immortalization of cells, 74-76, determination, 235-237
79-81
Osmium tetroxide, cell postfixation for
transmission electron microscopy, 300-301 Q
Quinacrine mustard staining, chromosome
analysis, 209, 211
P
p53, immortalization of cells by mutant genes,
R
76-77, 105-106
Papillomavirus, immortalization of cells, 71-72 Ras, immortalization of cells, 74-76
Passaging Restriction fragment length polymorphism
animal cell culture, 15, 99, 177-180 analysis, cell line authentication, 212
embryonic stem cells, 283 RFLP analysis, see Restriction fragment length
invertebrate cell culture polymorphism analysis
cell harvest, 196-197 Roller bottle
passaging without splitting, 197 cell harvesting, 221
PCR, see Polymerase chain reaction medium harvesting, 221
PEG, see Polyethylene glycol plating, 220-221
Peritubular cell, establishment of cell lines types, 220
cell preparation
fractionation over discontinuous Percoll
gradient, 96-97 S
isolation, 96 Scaleup, cell culture
clone selection, 98-99 applications of large-scale culture, 219-220
histology of immortalized cells, 100-101 fermentors, 226-227
immunodetection of simian virus 40 large T hollow fibers, 223-224
antigen, 99-100 large plates, 220
medium for immortalized cell culture, 99 microcarriers
overview, 94-95 cell growth, 222-223
passaging, 99 types, 221-222
seminiferous cord formation in vitro with roller bottles
other testicular cells, 101-103, 105 cell harvesting, 221
360 Index
medium harvesting, 221 clone selection, 98-99

plating, 220-221 histology of immortalized cells, 100-101
types, 220 immunodetection of simian virus 40 large T
suspension adaptation antigen, 99-100
approaches, 224 medium for immortalized cell culture, 99
culture, 225-226 overview, 94-95
medium, 224 passaging, 99
scaleup, 226 seminiferous cord formation in vitro with
spinners, 224-225 other testicular cells, 101-103, 105
Scanning electron microscopy transfection
cell processing hints, 310-311 calcium phosphate transfection with
monolayer cultures, 308-309 simian virus 40 large T antigen gene,
sputter coating, 310-311 97-98
suspension cultures, 310 principles, 95-96
Schwann cell Serum, cell culture additive
conduction, 168-169 handling, 12, 25
culture from human peripheral nerve trunks heterogeneity, 25
dissociation of cells, 180
preparation, 25
nerve harvesting, 179
supplementation, 12-13
passaging, 180
toxicity testing, 54
primary culture, 180
Simian virus 40
culture from rat dorsal root ganglia
immortalization of cells with large T antigen
adult
gene, 70-71,77-78,97-98,105-106
dissociation of cells, 177
immunodetection of large T antigen, 99-100
dorsal root ganglia dissection, 175
embryo Sperm, see Testicular germ cell
dissociation of cells, 175-177 Sterile technique, animal cell culture, 13-14,
dorsal root ganglia dissection, 173-175 51-52
passaging, 178-179 Suspension adaptation
primary culture, 177 approaches, 224
development, 169 culture, 225-226
growth factors, 169 medium, 224
materials for culture scaleup, 226
bovine serum albumin gradient spinners, 224-225
preparation, 172-173 SV40, see Simian virus 40
serum-free medium -~ preparation and Synchronization, cell cycle
growth factor supplementation, centrifugal elutriation, 244-246
170-171 GO arrest, release, 237, 239
stock solution preparation, 171-172 growth-dependence versus cell cycle
substrate coating of culture vessels, 173 regulation in measured events, 247-248
surgical instruments, 170 heterogeneity of synchronized populations,
morphology of cultured cells, 181 246-247
myelination mitotic detachment, 241-242,244
remyelination in culture, 182-183, 185 M phase block, release, 239-240
status, 168 S phase block, release, 240-241
proliferation markers, immunocytochemistry,
182
T
SEM, see Scanning electron microscopy
Seminiferous cord, formation in vitro with Tax, immortalization of cells, 73-74
testicular cells, 101-103, 105 T-cell, immortalization by oncogene
Sertoli cell, establishment of cell lines transfection
cell preparation bone marrow extract preparation, 79
fractionation over discontinuous Percoll characterization of cells, 81-83
gradient, 96-97 efficiency, 81
isolation, 96 electroporation, 80-81
Index 361
oncogene expression plasmid Transmission electron microscopy

construction, 79 cells grown on filters or matrices, 304-305
peripheral blood mononuclear cell monolayer cultures
preparation, 79-80 dehydration, 301
yolk lipoprotein preparation, 79 embedding in plastic, 303
Telomere, length in immortalized cells, 84 Epon separation from culture dish, 303
TEM, see Transmission electron microscopy glutaraldehyde fixation, 298, 300
Terminal deoxynucleotidyl transferase mounting of cells, 303-304
apoptosis assay, see TUNEL osmium tetroxide postfixation, 300-301
Testicular cell, see Leydig cell: Peritubular cell; substrates for growth, 298, 304-305
Sertoli cell; Testicular germ cell uranyl acetate staining, 301, 304
Testicular germ cell suspension cultures
establishment of cell lines fixation, 305-307
cell preparation harvesting, 305, 307-308
fractionation over discontinuous Percoll staining, 307
gradient, 96-97 Trypsin-Giemsa banding, chromosome
isolation, 96 analysis, 209-210
characterization of cotransfected TUNEL
immortalized cells, 107-108 flow cytometry assays
clone selection, 98-99 TUNELkell cycle analysis
flow cytometry analysis of DNA, 106-107 filters, 269
histology of immortalized cells, 100-101, fixation of cells, 268
108 labeled nucleotides, 275-276
immunodetection of simian virus 40 large performance of assay, 269-272
T antigen, 99-100 principle, 259, 267
medium for immortalized cell culture, 99 staining, 268-269, 271-272
overview, 94-95 TUNELhrface staining, 257-259
passaging, 99 principle and applications, 255-256, 257-259
transfection with simian virus 40 large T
antigen gene
calcium phosphate transfection, 97-98 U
cotransfection with temperature- Uranyl acetate, cell staining for transmission
sensitive p53 mutant, 105-106 electron microscopy, 301, 304
principles, 95-96
seminiferous cord formation in vitro with
other testicular cells, 101-103, 105
V
spermatogenesis, 94
tumor lines, 94 Vitamins, media supplementation, 27
Thymidine, cell cycle synchronization, 240-241
Transfection, see Gene delivery
Transgenic animal, see nlso Embryonic stem W
cell Water bath, animal cell culture, 9
aggregation Water purification, animal cell culture, 8,
embryo, preparation, 290 26, 53
embryonic stem cell-embryo aggregation,
291
embryonic stem cell preparation, 290 X
plate preparation, 289
chimera production, overview, 289
XlT,apoptosis assay, 262
embryonic stem cell applications in
transgenic technology, 280-281, 292
Y
germline transmission, 291
laboratory requirements for production, 281 Yolk lipoprotein, preparation, 79
VOLUMES IN SERIES
Founding Series Editor

DAVID M. PRESCOTT
Volume 1 (1964)
Methods in Cell Physiology
Edited by David M. Prescott
Volume 2 (1 966)
Edited by David M. Prescotl
Volume 3 (1968)
Volume 4 (1970)
Volume 5 (1972)
Volume 6 (1973)
Volume 7 (1973)
Edited by David M . Prescott
Volume 8 (1974)
Edited by David M. Prescoti
Volume 9 (1975)
Volume 10 (1975)
363
364 Volumes in Series
Volume 11 (1975)
Yeast Cells
Volume 12 (1975)
Yeast Cells
Volume 13 (1976)
Volume 14 (1976)
Volume 15 (1977)
Volume 16 (1977)
Chromatin and Chromosomal Protein Research I
Edited by Gary Stein, Janet Stein, and Lewis J. Kleinsmith
Volume 17 (1978)
Chromatin and Chromosomal Protein Research Il
Volume 18 (1978)
Chromatin and Chromosomal Protein Research III
Volume 19 (1978)
Chromatin and Chromosomal Protein Research IV
Volume 20 (1978)
Advisory Board Chairman

KEITH R. PORTER
Volume 21A (1980)

Normal Human Tissue and Cell Culture, Part A Respiratory, Cardiovascular,
and Integumentary Systems
Edited by Curtis C. Harris, Benjamin F. Trump, and Gary D. Stoner
Volumes in Series 365
Volume 21B (1980)

Normal Human Tissue and Cell Culture, Part B: Endocrine, Urogenital, and
Gastrointestinal Systems
Edited by Curtis C. Harris, Benjamin F. Trump, and Gary D. Stoner
Volume 22 (1981)
Three-Dimensional Ultrastructure in Biology
Edited by James N. Turner
Volume 23 (1981)
Basic Mechanisms of Cellular Secretion
Edited by Arthur R. Hand and Constance Oliver
Volume 24 (1982)
The Cytoskeleton, Part A Cytoskeletal Proteins, Isolation and
Characterization
Edited by Leslie Wilson
Volume 25 (1982)
The Cytoskeleton, Part B: Biological Systems and in Vitro Models
Edited by Leslie Wilson
Volume 26 (1982)
Prenatal Diagnosis: Cell Biological Approaches
Edited by Samuel A. Latt and Gretchen J. Darlington
Series Editor
LESLIE WILSON
Volume 27 (1986)
Echinoderm Gametes and Embryos
Edited by Thomas E. Schroeder
Volume 28 (1987)
Dictyostelium discoideum: Molecular Approaches to Cell Biology
Edited by James A. Spudich
Volume 29 (1989)
Fluorescence Microscopy of Living Cells in Culture, Part A Fluorescent
Analogs, Labeling Cells, and Basic Microscopy
Edited by Yu-Li Wang and D. Lansing Taylor
Volume 30 (1989)
Fluorescence Microscopy of Living Cells in Culture, Part B: Quantitative
Fluorescence Microscopy-Imaging and Spectroscopy
Edited by D. Lansing Taylor and Yu-Li Wang
Volume 31 (1989)
Vesicular Transport, Part A
Edited by Alan M. Tartakoff
Volume 32 (1989)
Vesicular Transport, Part B
Volume 33 (1990)
Flow Cytometry
Edited by Zbigniew Darzynkiewicz and Harry A. Crissman
Volume 34 (199 1)
Vectorial Transport of Proteins into and across Membranes
Selected from Volumes 31, 32, and 34 (1991)
Laboratory Methods for Vesicular and Vectorial Transport
Volume 35 (1991)
Functional Organization of the Nucleus: A Laboratory Guide
Edited by Barbara A. Hamkalo and Sarah C. R. Elgin
Volume 36 (199 1)
Xenopus Zuevis: Practical Uses in Cell and Molecular Biology
Edited by Brian K. Kay and H. Benjamin Peng
Series Editors
LESLIE WILSON AND PAUL MATSUDAIRA
Volume 37 (1993)
Antibodies in Cell Biology
Edited by David J. Asai
Volume 38 (1993)
Cell Biological Applications of Confocal Microscopy
Edited by Brian Matsumoto
Volume 39 (1993)
Motility Assays for Motor Proteins
Edited by Jonathan M. Scholey
Volume 40 (1994)
A Practical Guide to the Study of Calcium in Living Cells
Edited by Richard Nuccitelli
Volumes in Series 367
Volume 41 (1994)
Flow Cytometry, Second Edition, Part A
Edited by Zbigniew Darzynkiewicz, J. Paul Robinson,
and Harry A. Crissman
Volume 42 (1994)
Flow Cytometry, Second Edition, Part B
Edited by Zbigniew Darzynkiewicz, J. Paul Robinson,
and Harry A. Crissman
Volume 43 (1994)
Protein Expression in Animal Cells
Edited by Michael G. Roth
Volume 44 (1994)
Drosophilu melunoguster: Practical Uses in Cell and Molecular Biology
Edited by Lawrence S. B. Goldstein, and Eric A. Fyrberg
Volume 45 (1994)
Microbes as Tools for Cell Biology
Edited by David G. Russell
Volume 46 (1995)
Cell Death
Edited by Lawrence M. Schwartz, and Barbara A. Osborne
Volume 47 (1995)
Cilia and Flagella
Edited by William Dentler, and George Witman
Volume 48 (1995)
Cuenorhubditis eleguns: Modern Biological Analysis of an Organism
Edited by Henry F. Epstein, and Diane C. Shakes
Volume 49 (1995)
Methods in Plant Cell Biology, Part A
Edited by David W. Galbraith, Hans J. Bohnert, and Don P. Bourque
Volume 50 (1995)
Methods in Plant Cell Biology, Part B
Edited by David W. Galbraith, Don P. Bourque, and Hans J. Bohnert
Volume 51 (1996)
Methods in Avian Embryology
Edited by Marianne Bronner-Fraser
Volume 52 (1997)
Methods in Muscle Biology
Edited by Charles P. Emerson, Jr. and H. Lee Sweeney
Volume 53 (1997)
Nuclear Structure and Function
Edited by Miguel Berrios
Volume 54 (1997)
Cumulative Index
Volume 55 (1997)
Laser Tweezers in Cell Biology
Edited by Michael P. Sheez
Volume 56 (1998)
Video Microscopy
Edited by Greenfield Sluder and David E. Wolf
Volume 57 (1998)
Animal Cell Culture Methods
Edited by Jennie P. Mather and David Barnes

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This book is printed on acid-free paper. @

Copyright Q 1998 by ACADEMIC PRESS

All Rights Reserved.

Academic Press Limited

International Standard Book Number: 0-12-544159-2 (hb)

PRINTED IN THE UNITEDSTATES OF AMERICA

Shahabuddin Alam (69), Laboratory of Cellular Regulation Technology, Gradu-

Hildegard Meissner (147), Department of Neurosurgery, Laboratory for Brain

In this volume we provide a broad introduction to animal cell culture tech-

Principles of Cell Culture

Animal Cell Culture Equipment

dents, mistakes, and misunderstandings encountered since the mid-1970s in cell

material is considered by some to represent a risk uniformly. Even under pre-

Fans in incubators are useful when precise temperature control is required,

E. Water Purification Equipment and Medium Filtration Devices

G. Liquid Nitrogen Storage Tanks

H. Water Baths, Centrifuges, Freezers, and Refrigerators

111. Laboratory Design

and consistency of the product. Unusual medium formulations can be obtained

B. Cell Culture Plasticware and Glassware

V. Cell Culture Methods

C. Multipassage Culture and Cloning

Cloning of primary or early passage adherent cells is best accomplished with

Malung Informed Choices: Mehum,

11. The Role of Medium

in vitro. These may nonetheless improve cell growth by maintaining appropriate

IV. Selecting the Appropriate Medium

Medium Applicable to Reference

V. Screening Conditioned Medium for Biological Activity

VI. Media Preparation

of HEPES buffer is used to dissolve the gentamycin at a final concentration of

VII. Serum, Plasma, and Other Undefined Additives

VIII. Testing Media and Components and Quality Control:

IX. Troubleshooting Medium Problems

X. Altering Commercial Media for Special Uses

Sometimes an addition to a commercial medium can improve cell growth. If

XI. Medium Optimization

resulted in the nutrient mixtures currently published or commercially available

X I . Choosing the Optimal Medium: The “Quick and

11. Laboratory Equipment

The technical requirements (instrumental and special skills) for ES cell-

111. Culture Conditions

IV. Maintaining Embryonic Stem (ES) Cells

V. Preparing Tissue Culture Plates for ES Cells

VI. Passaging ES Cells

A properly maintained culture of ES cells should reach 50-60% confluence

VII. Freezing ES Cells from Confluent 10-cm Tissue

Fig. 1 Passaging ES cells.

VIII. Thawing ES Cells from Freezing Vials

IX. Establishing ES Cells

Fig. 2 Establishing ES cell lines.

X. Introducing DNA into and Selecting for Genetically

Fig. 3 Electroporating ES cells.

XI. Picking ES Cell Colonies

XII. Detecting Genome Alterations

XIII. Freezing ES Cells in 96-Well Plates

Good Variable Bad

Asp718 EcoRI BspDI

3. Stop the action of trypsin by adding 75 pl of ES medium to each well and

XIV. Thawing ES Cells in 96-Well Plates

XVI. Preparation of the Aggregation Plate

1. Place M16 microdrops (2-3 mm in diameter) into a 35-mm tissue culture