Immune Cell Phenotyping Using Flow UNIT 18.

8
Cytometry
Immunophenotyping is a general term used to describe the use of fluorescently labeled
antibodies to identify and quantify distinct subpopulations of cells within a heteroge-
neous population of cells. Frequently, the term is used more specifically to refer to the
characterization of subsets of cells associated with the immune system (i.e., immune cell
phenotyping). In either case, the antibodies used for immunophenotyping are usually
specific for cell surface proteins that are known to be differentially expressed on discrete
subsets of cells. Immunophenotyping can be carried out using either flow cytometry or
immunohistochemical analysis. However, flow cytometry is often the method of choice,
because it permits the acquisition of nonsubjective data on thousands of cells within sec-
onds. Furthermore, each cell suspension can be labeled with several different antibodies
simultaneously, and the fluorescence associated with each antibody can be collected on
a per-cell basis as correlated data. Modern flow cytometers can routinely measure five
or more different fluorescence emissions simultaneously, providing the means to easily
detect unique subsets within a heterogeneous population of cells. More recently, cell
surface phenotyping with immunofluorescent probes has been performed to ascertain
the functional status (e.g., DNA or cytokine content, mitochondrial membrane integrity,
glutathione status) of cells, making multiparameter flow cytometry a very powerful tool
for characterizing the activation state of a cell. The only drawback of flow cytometry
is that cells must be stained and analyzed in a single-cell suspension, which is easier
to prepare from some tissues than from others. Because cells derived from blood and
lymphoid tissues are readily maintained in suspension, flow cytometry has been widely
used to phenotype these cells.

Immunophenotyping of lymphoid tissues, particularly spleen and thymus, has been used
in the field of immunotoxicology for many years as part of a tiered screening approach
for detecting immunotoxicity following chemical exposure. Since thymic involution fre-
quently occurs after toxicant exposure, changes in thymocyte immunophenotypes are
commonly evaluated. Similarly, just as white blood cell (WBC) counts and differentials
have been used for many years to assess immune status in toxicant-exposed animals,
newer screening approaches such as immunophenotyping of spleen and/or lymph nodes
have been used to quantify frequencies of the major lymphocyte subtypes (B cells, T cells,
and T cell subsets) following chemical exposure. In studies conducted by the National
Toxicology Program, the utility of immune cell phenotyping has been validated using a
large database of environmental toxins and certain pharmaceutical agents. In these studies,
changes in lymphocyte phenotypes in the spleen or thymus following toxicant exposure
was identified as one of the best single correlates with toxicant-induced changes in host
resistance to pathogens or tumors (Luster et al., 1993). However, even though immunophe-
notyping of the spleen and thymus may correlate with toxic chemical exposure, it is not
necessarily a robust predictor of immunotoxicity, particularly when naive animals are
exposed to toxicants. Some highly immunotoxic chemicals, including cyclosporin A and
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), do not alter splenic immunophenotypes in
the absence of antigen challenge (Kerkvliet and Brauner, 1990; Vandebriel et al., 1999).
Likewise, changes in thymic phenotypes can occur indirectly, through stress-induced ef-
fects on the thymus that are independent of direct immunotoxicity. Thus, it is generally
agreed that immunophenotypic analysis of B cells, T cells, and T cell subsets present in
the thymus and/or spleen should be used in conjunction with conventional assessments
of immune function when screening for the immunotoxic effects of drugs and chemicals
(Immunotoxicology Technical Committee, 2001; Food and Drug Administration, 2002).
Immunotoxicology
Contributed by Julie A. Oughton and Nancy I. Kerkvliet 18.8.1
Current Protocols in Toxicology (2005) 18.8.1-18.8.24
Copyright

C 2005 by John Wiley & Sons, Inc.
Supplement 23
 Although immunotoxicology studies have focused primarily on the phenotypic analysis
of B cells and T cell subsets in spleen and/or thymus from naive mice, several extended
applications of immunophenotyping are emerging in the immunotoxicological literature.
One important extension is the analysis of peripheral blood cells from mice and rats
(Oughton et al., 1995; Nygaard and Lovik, 2002; Funatake et al., 2004). The use of
peripheral blood samples from rodents allows more direct comparisons to be made with
toxicity data that may be available from human studies, which nearly always rely on
peripheral blood samples. Analysis of blood also allows evaluation of systemic changes
that may result from toxicant effects in lymphoid tissues, as well as the testing of multiple
samples obtained from the same animal over an extended time.

Another important addition to standard immunophenotyping techniques is the analysis

of activation markers. Most cells in the immune system respond to antigenic stimulation
by altering their expression of a number of different proteins on the cell surface. Many
of these proteins are known to be involved in the functional response of the cell (e.g.,
adhesion, migration, costimulation), while others may reflect the cell’s antigenic history
(as in memory cells). Evaluation of activation markers is likely to provide an enhanced
ability to detect immunotoxic effects. For example, when mice were exposed to low doses
of TCDD for more than a year, no changes in major T- and B cell subsets were observed.
However, analysis of the naive/memory phenotypes of CD4+ T cells revealed that TCDD
exposure had significantly reduced the proportion of memory cells present (Oughton
et al., 1995). Likewise, evaluation of activation markers in the context of an ongoing
immune response may provide novel insight into the selective targets and mechanisms of
action of immunotoxic chemicals. Several laboratories are now using immunophenotyp-
ing to track the effects of toxicants on the activation and fate of antigen-specific T cells
following antigenic challenge, with very exciting results (Shepherd et al., 2000; Mitchell
and Lawrence, 2003; Funatake et al., 2004).

Since the specific antibodies that one might use for immunophenotyping will vary de-
pending on the species tested, the flow cytometer used, and the experimental questions
being asked, the following protocol describes a generic approach to the staining of cells
with fluorescently labeled monoclonal antibodies (MAbs) to assess cell surface protein
expression. Work in the authors’ laboratory has primarily involved mouse cells; however,
all aspects of this unit easily apply to the analysis of cells from rats, dogs, and horses.
The first protocol (Basic Protocol 1) describes the procedure by which cells obtained
from lymphoid tissue are stained with fluorochrome-conjugated MAbs (direct staining
method). A second protocol (Alternate Protocol) describes two indirect methods for the
staining of cells with primary MAbs that are not conjugated to a fluorochrome. Finally,
a third protocol (Basic Protocol 2) describes the staining of peripheral blood cells with
MAbs.

Support protocols are presented for the preparation of lymphoid cells (Support Protocol 1),
the fixation of cells (Support Protocol 2), and discrimination of dead cells (Support
Protocol 3). When freshly stained cells cannot be analyzed immediately, samples must
be fixed so that the cells are stable for storage (Support Protocol 2). Furthermore, because
the inclusion of dead cells in flow cytometric analysis can lead to the misinterpretation
of data and possibly to the rendering of erroneous conclusions, it is desirable to assess
cell viability using DNA dyes (Support Protocol 3).

Finally, a procedure for the determination of optimal antibody concentrations is presented

in Support Protocol 4.
Immune Cell
Phenotyping NOTE: The investigator should consult the manufacturer’s instruction manual for specific
Using Flow information regarding the operation of his or her flow cytometer.
Cytometry

18.8.2
Supplement 23 Current Protocols in Toxicology
DIRECT STAINING OF CELLS PREPARED FROM LYMPHOID TISSUES BASIC
PROTOCOL 1
This is the preferred method for staining cells obtained from lymphoid tissues (e.g., spleen,
thymus, lymph nodes). Basic Protocol 1 is appropriate for immunophenotyping of cells
from either rats or mice. Because this method uses MAbs that are directly conjugated
to fluorochromes, staining is simple and quick and usually produces little background
signal.

Toxicology studies usually involve the assessment of lymphoid tissues from several in-
dividual animals in a number of different treatment groups. For example, a 3-dose-plus-
control experiment that examined spleen and thymus from 5 animals per treatment group
would yield 40 individual cell samples that needed to be stained, usually in more than
one staining configuration. Processing of large numbers of samples can be facilitated by
staining cells in V-bottom 96-well microtiter plates, as opposed to 12 × 75–mm tubes.
These plates can be centrifuged using a microtiter plate carrier, and the contents of the
wells can be mixed easily using a vortex mixer (such as the Vortex-Genie 2, VWR Scien-
tific Products) equipped with a platform head for a microtiter plate. Use of a multichannel
pipettor (such as the 12-channel Pipet-Lite multichannel pipet from Rainin Instrument)
facilitates rapid dispensing of media into the wells, as well as quick transfer of samples
to Titertubes (Bio-Rad) for flow cytometric analysis. Titertubes are small tubes that can
easily be inserted into 12 × 75–mm disposable tubes, which are commonly used to run
samples on a flow cytometer. The abovementioned products greatly increase productivity
and also simplify the processing of large numbers of samples. To help keep track of
stained samples, a template of the microtiter plate can be prepared in which descriptions
of the contents of each well (e.g., animal source, MAbs used) are recorded. In lieu of
microtiter plates, cells can be stained in 12 × 75–mm plastic tubes or microcentrifuge
tubes.

Materials
Tissue of interest (e.g., lymphoid cells, Support Protocol 1)
Fc receptor (FcR)-blocking immunoglobulin (Ig; e.g., normal rat IgG) solution,
200 µg/ml
MAbs or isotype Igs directly conjugated to fluorochromes
PBS supplemented with sodium azide and BSA (PAB; see recipe)
Micropipettor or multichannel pipettor with tips
96-well V-bottom microtiter plates, disposable 12 × 75–mm polystyrene tubes, or
microcentrifuge tubes
Centrifuge, refrigerated and equipped with a microtiter plate carrier
Vortex mixer equipped with a platform head for a microtiter plate
Titertubes (Bio-Rad Laboratories; optional)
Additional reagents and equipment for preparation of lymphocyte suspension to be
analyzed (Support Protocol 1)
NOTE: Several steps need to be performed to minimize antibody cross-linking and the
subsequent internalization of cell surface receptors that may occur when these receptors
are bound to MAbs. This process is often referred to as capping of the antigen-antibody
complex. Some cell surface molecules are less likely to cap than others; however, surface
immunoglobulin on B cells can cap almost instantaneously. The addition of sodium azide
(final concentration, 0.1% w/v) to the staining medium will help to prevent capping. In
addition, all cell preparations and staining procedures, including wash steps, should be
performed at 4◦ C, a condition achieved by incubating the cells on ice. Staining samples
on ice also reduces the off-rate constant associated with antibody binding by a factor of
10 compared with the off-rate constant at 25◦ C.
Immunotoxicology

18.8.3
Current Protocols in Toxicology Supplement 23
 Prepare lymphocyte samples for staining
1. Prepare a single-cell suspension of lymphocytes in 5% HBSS. Lyse red blood cells
(RBCs) if present in large numbers (Support Protocol 1).
2. Using a micropipettor or microchannel pipettor, dispense 1–4 × 106 cells into each
well of a V-bottom 96-well microtiter plate.
Alternatively, dispense cells into 12 × 75–mm tubes or microcentrifuge tubes.

3. Pellet cells by centrifugation for 3 min at 200 to 1500 × g, 4◦ C.

The range of centrifugation speeds quoted in this step reflects the fact that no one speed is
accepted by the entire flow cytometry community. Although 1500 × g may sound excessive,
this is the speed most commonly used by Stewart and Stewart (2001a) in order to minimize
the loss of cells during washing.

4. Remove supernatant by holding the plate over a sink and inverting with a flick of the
wrist or by decanting liquid from tubes. Blot the plate or the lip of each plastic tube
on an absorbent towel to remove excess supernatant.
5. Vortex plate or tubes lightly to resuspend cell pellet.
Label with fluorochrome-conjugated MAbs
6. Add 50 µl FcR-blocking Ig (10 µg) to each cell suspension. Vortex cells lightly and
incubate on ice for 10 min.
A number of cells (especially myeloid cells) have receptors for the Fc portion of an
immunoglobulin and will bind to any MAb. In order to assess the antigen-specific binding
of a MAb, FcR must be blocked using normal IgG from the same animal species as the
selected MAb. For example, for cells stained with rat anti–mouse CD4 MAb, normal rat
IgG should be used to block FcR-mediated binding. As an alternative, a MAb specific for
mouse FcR (CD16/CD32) can be used, although this involves a substantially higher cost.
FcR should not be blocked when assessing FcR expression.

7. Without removing the FcR-blocking reagent, add the appropriate fluorochrome-

labeled MAbs (see Support Protocol 4 for determination of optimal antibody con-
centrations) or isotype Igs to each well or tube. Vortex plate or tubes and incubate
cells on ice for 10 min, protected from light.
Cells can be stained with a number of MAbs simultaneously as long as there are no
interactions among these MAbs. For large studies, a cocktail of all the MAbs of interest
can be used to stain each sample. For example, if cells are to be stained for CD3, CD4,
and CD8, a single cocktail including all three MAbs can be prepared in a total volume of
10 µl. Cocktail staining greatly expedites sample preparation and ensures that all samples
are stained uniformly.
Over time, MAbs in solution tend to form aggregates. Cells that express Fc receptors can
bind these antibody aggregates with far greater avidity than they can antibody monomers.
Aggregates should be removed by microcentrifugation of the antibody solution for 5 min
at 12,000 to 13,000 rpm, 4◦ C, prior to staining.
Fluorochromes [especially the phycoerythrin (PE)–Cy5 and PE-Cy7 tandem conjugates]
are particularly sensitive to photodegradation. Therefore, all staining procedures should
be conducted in subdued light.

8. Add 150 µl PAB to each well or tube and centrifuge for 3 min at 200 to 1500 × g,
4◦ C. Remove supernatant as in step 4.

Immune Cell
Phenotyping
Using Flow
Cytometry

18.8.4
Supplement 23 Current Protocols in Toxicology
 9. To remove unbound MAb, which can contribute to background fluorescence, wash
cells once by adding PAB (total volume in microtiter plate not to exceed 200 µl)
to each well or tube and centrifuging for 3 min at 200 to 1500 × g, 4◦ C. Remove
supernatant as in step 4.
10. Resuspend cells in 500 µl PAB with gentle vortexing.
For samples prepared in microtiter plates, resuspend each cell pellet in 200 µl PAB. Next,
using a multichannel pipettor, transfer the contents of each well to a Titertube and add an
additional 300 µl PAB, for a total volume of 500 µl. These Titertubes can then be inserted
into 12 × 75–mm plastic tubes for flow cytometric analysis.

11. Keep samples on ice until analyzed using a flow cytometer.

If stained cells cannot be analyzed on the day of preparation, they must be fixed for
longer-term storage (Support Protocol 2).

INDIRECT STAINING OF CELLS PREPARED FROM LYMPHOID CELLS ALTERNATE

PROTOCOL
In general, it is best to stain lymphoid cells with a MAb that is directly conjugated to a
fluorochrome (i.e., using the direct method). It is relatively easy to obtain MAbs specific
for mouse or rat markers in a number of conjugated forms from commercial sources.
However, there are very few mouse or rat MAbs that are conjugated to PE–Texas Red.
This will impact studies requiring four or five colors for immunophenotypic analysis,
since one MAb may require a second-step staining reagent. This concern is even more
relevant when dealing with species other than mice, rats, or humans, as it is very difficult
to find commercial sources of fluorochrome-conjugated MAbs for such species. In those
situations, it may be necessary to resort to one of two indirect staining methods.

In one method, cells are incubated first with an unconjugated primary MAb and then with a
fluorochrome-conjugated antibody that is specific for the primary antibody. For instance,
if cells are stained with an unconjugated rat IgG2a MAb that is specific for mouse CD4,
the secondary antibody would be a fluorochrome-conjugated antibody that is specific
for rat IgG. In most cases, the secondary antibody will be a polyclonal anti-Ig antibody,
although Ig isotype–specific MAbs can sometimes be obtained from commercial sources.
In this example, one can limit cross-reactivity of the secondary antibody with all other IgG
isotypes by restricting the specificity of the secondary antibody to the rat IgG2a isotype,
if available. To minimize FcR-mediated binding of the secondary anti-Ig antibody, it is
preferable to use the F(ab )2 fragment rather than the whole immunoglobulin.

An alternative indirect staining method relies on the use of a biotin-conjugated primary

antibody, which is then detected with fluorochrome-conjugated streptavidin (SA). This
biotin-SA method is the preferred indirect staining approach, because reagent interactions,
which are often associated with the use of anti-Ig antibodies, are less of a concern. In
addition, there are commercially available SA conjugates to a variety of fluorochromes,
making SA useful in multicolor staining protocols. However, as with directly conjugated
MAbs, biotin-conjugated MAbs are not always available.

The use of secondary anti-Ig antibodies can be circumvented by labeling purified MAbs
with specific fluorochromes in the laboratory. Detailed methods for MAb conjuga-
tion are available on the Internet (Roederer, 1997), and conjugation kits can be ob-
tained from commercial vendors. The authors have had success using the Zenon Alexa
Fluor 488 Mouse IgG Labeling Kit (Molecular Probes, 2004a) to label unconjugated
MAbs. Using this antibody labeling kit, small quantities (≥0.4 µg) of primary MAbs
can easily be conjugated to fluorescent labels. Currently, the Zenon system can only
be used with mouse IgG1 , mouse IgG2a , mouse IgG2b , rabbit IgG, and human IgG
antibodies. Immunotoxicology

18.8.5
Current Protocols in Toxicology Supplement 23
 Additional Materials (see also Basic Protocol 1)
Unconjugated primary MAb or biotin-labeled primary MAb
Fluorochrome-conjugated anti-Ig F(ab )2 fragment (for use with unconjugated
primary MAb) or fluorochrome-conjugated streptavidin (for use with
biotin-labeled primary MAb)
1. Stain cells with primary MAbs according to steps 1 through 9 of Basic Protocol 1.
If necessary, a combination of several fluorochrome-conjugated MAbs plus one non-
fluorochrome-conjugated MAb (preferably a biotinylated MAb) can be used in a single
MAb cocktail.

2. Resuspend cells in 50 µl PAB with gentle vortexing.

3. Add fluorochrome-labeled streptavidin or an appropriate secondary antibody. Vortex
lightly and incubate on ice for 20 min.
4. Add 150 to 200 µl PAB and centrifuge cells for 3 min at 200 to 1500 × g, 4◦ C.
Remove supernatant as in Basic Protocol 1, step 4.
5. Wash cells once in PAB as in Basic Protocol 1, step 9.
6. Resuspend cells in 500 µl PAB with gentle vortexing.
Alternatively, for samples prepared in microtiter plates, resuspend each cell pellet in
200 µl PAB. Next, using a multichannel pipettor, transfer the contents of each well to a
Titertube and add an additional 300 µl PAB, for a total volume of 500 µl. These Titertubes
can then be inserted into 12 × 75–mm plastic tubes for flow cytometric analysis.

7. Keep samples on ice until analyzed using a flow cytometer.

If stained cells cannot be analyzed on the day of preparation, they must be fixed for
longer-term storage (Support Protocol 2).

SUPPORT PREPARATION OF CELLS FROM LYMPHOID ORGANS

PROTOCOL 1
The processing of mouse lymphoid organs into single-cell suspensions is a fairly simple
procedure.

Materials
Lymphoid organ of interest
HBSS supplemented with 5% (v/v) FBS (5% HBSS; see recipe)
Endotoxin-screened distilled water for cell culture (Gibco)
10× HBSS (Sigma)
ACK lysis buffer (optional; see recipe)
60 × 15–mm untreated culture dish
25 × 75 × 1–mm frosted glass microscope slides
15-ml conical centrifuge tubes
12 × 75–mm disposable cell culture tubes
Coulter counter or hemacytometer
1. Transfer a freshly dissected lymphoid organ to a 60 × 15–mm culture dish containing
4 ml of 5% HBSS.
2. When ready for processing, place the lymphoid organ between the frosted ends of
two glass microscope slides. Disrupt the organ by gently pressing the frosted ends in
Immune Cell a circular motion until only the empty capsule remains.
Phenotyping
Using Flow
Cytometry

18.8.6
Supplement 23 Current Protocols in Toxicology
 3. Transfer the resulting cell suspension to a 15-ml conical centrifuge tube. Rinse the
culture dish and capsule with a combined total of 3 ml of 5% HBSS and then add
this liquid to the cell suspension.
4. Centrifuge the cell suspension for 10 min at 200 × g, 4◦ C. Discard supernatant.

For spleen cell preparations

5a. Lyse RBCs by adding 4.5 ml cold distilled water (for cell culture) to the cell pellet
and resuspending with gentle vortexing for 10 sec. After vortexing, immediately add
0.5 ml of 10× HBSS to restore isotonicity, followed by an additional 5 ml of 5%
HBSS. Centrifuge cells as in step 4, discard supernatant, and resuspend the cell pellet
in 10 ml of 5% HBSS with gentle vortexing.
This procedure also lyses dead cells. If it is necessary to preserve dead cells (e.g., for
apoptosis studies), RBCs can be removed by (1) resuspending the cell pellet in 5 ml of a
standard ammonium chloride lysing solution (ACK lysing buffer; see recipe) and vortexing
briefly; (2) incubating the resuspended pellet for 5 min at room temperature; (3) adding
5 ml of 5% HBSS (inverting the tube several times to mix); and then (4) centrifuging the
cells for 10 min at 200 × g, 4◦ C, and discarding the supernatant.

In general, the viability of spleen cell suspensions will be substantially higher when the
hypotonic lysis method (>95%), as opposed to the ammonium chloride lysis method (85%),
is used.

6a. Let cell debris settle for 10 min on ice, and then remove the cell-rich supernatant and
transfer to a clean tube.

For thymus and lymph node cell preparations

5b. Resuspend the cell pellet in 10 ml of 5% HBSS with gentle vortexing.
6b. Let cell debris and clumps settle for 8 to 10 min on ice, and then remove the cell-rich
supernatant and transfer to a clean tube.
7. Determine cell concentration using a Coulter counter or hemacytometer (APPENDIX 3B).
Use 5% HBSS to adjust the final concentration of the cell suspension to 10–40 ×
106 cells/ml.

CELL FIXATION SUPPORT

PROTOCOL 2
If it is not possible to analyze freshly stained cells on the day of preparation, the cells must
be fixed in electron microscopy (EM)–grade formaldehyde (such as Ultrapure formalde-
hyde, Polysciences) prior to storage at 4◦ C. Fixation not only cross-links proteins to make
cells stable for storage but also inactivates the infectious activity of any viruses present.
Hence, cell fixation is highly advisable when handling biological specimens capable of
transmitting infection, and especially when handling human-derived cells.

Fixed samples should be analyzed within 5 days of fixation, since prolonged storage can
result in increased autofluorescence. This is usually not a problem when antigens that are
expressed at high levels are being analyzed. However, increased autofluorescence may
make it more difficult to resolve negative cells from dimly stained cells. Cell fixation
often leads to increased cell aggregation; therefore, it may be prudent to filter samples
through a 40-µm nylon mesh prior to running them on the flow cytometer.

Additional Materials (see also Basic Protocol 1)

1% formaldehyde: 10% (v/v) Ultrapure EM-grade formaldehyde (Polysciences)
diluted 1:10 in PAB (see recipe)
40-µm nylon mesh (Small Parts, Inc.; optional) Immunotoxicology

18.8.7
Current Protocols in Toxicology Supplement 23
 1. Stain cells with MAbs according to steps 1 through 9 of Basic Protocol 1 or steps 1
through 5 of the Alternate Protocol.
2. Resuspend cell pellet in residual supernatant by gently vortexing.
Cells must be thoroughly resuspended prior to fixation. If gentle vortexing does not com-
pletely resuspend the cell pellets in the microtiter plate, use a multichannel pipettor
(equipped with plastic tips) to break up the pellets.

3. Add 200 µl 1% formaldehyde to each well and store cells on ice, protected from
light, for 30 min.
If necessary, cells can be stored overnight in 1% (v/v) formaldehyde at 4◦ C.

4. Prior to flow cytometric analysis, centrifuge cells 3 min at 1500 × g, 4◦ C. Remove

supernatant as in Basic Protocol 1, step 4, and resuspend the cell pellet in 500 µl PAB.
After resuspension in PAB, it may be desirable to filter samples through a nylon mesh to
remove cell aggregates, as this will prevent clogging of the flow cell nozzle.

BASIC STAINING OF PERIPHERAL BLOOD LEUKOCYTES

PROTOCOL 2
This protocol details the immunophenotyping of peripheral blood leukocytes, a process
often referred to as a “whole-blood” technique, as RBC lysis takes place after the sample
has been stained with specific MAbs. It is appropriate for the immunophenotyping of
blood cells from a number of animal species, including mouse, rat, horse, and dog.

Materials
Animals of interest
Sodium heparin, 250 IU per ml in PBS (APPENDIX 2A)
PBS supplemented with sodium azide and BSA (PAB; see recipe)
FcR-blocking Ig solution, 2 mg/ml (note that concentration differs from that used
in Basic Protocol 1)
Appropriate primary MAbs and (if necessary) secondary antibodies
FACS Lysing Solution (BD Biosciences), diluted 1:10 in distilled water according
to the manufacturer’s instructions
1-cc syringe equipped with 22-G needle
Coulter counter
Disposable 12 × 75–mm polystyrene tubes
NOTE: Staining conditions are similar to those noted in Basic Protocol 1. However, all
staining procedures should be performed in 12 × 75–mm polystyrene tubes, not microtiter
plates.

NOTE: Be sure to protect sample from light during incubation with fluorochrome-
conjugated MAbs.

Prepare peripheral blood leukocytes for staining

1. Using aseptic technique (APPENDIX 3B), collect blood (by cardiac puncture for mice)
in a 1-cc syringe containing sodium heparin.
A sufficient amount of heparin can be drawn into the syringe barrel by simply pulling the
plunger back to the 0.05-cc mark.
In order to reduce animal-to-animal variation, the same volume of blood should be col-
lected from each animal (for example, ∼500 µl from each mouse).
Immune Cell 2. Determine WBC concentration of blood sample using a Coulter counter.
Phenotyping
Using Flow
Cytometry 3. Add 50 to 100 µl blood to a 12 × 75–mm polystyrene tube.

18.8.8
Supplement 23 Current Protocols in Toxicology
 4. Wash cells twice by adding 2 to 3 ml PAB to tube, centrifuging for 5 min at 1500 ×
g, 4◦ C, and removing supernatant.
This wash step minimizes artifactual staining of serum Igs with MAbs. Washing is per-
formed by adding 2 ml PAB and centrifuging cells for 5 min at 1500 × g, 4◦ C. This
centrifugation speed is required to produce a soft pellet of blood cells.
The supernatant is removed by carefully decanting it and then touching the lip of the tube
to an absorbent towel. Alternatively, the supernatant can be aspirated, taking care not to
disturb the soft cell pellet.

5. Resuspend blood cells in residual medium (∼100 µl) by gently vortexing.

Label with fluorochrome-conjugated MAbs
6. To block FcR-mediated binding of Igs, add 5 µl FcR-blocking Ig (10 µg) to the cell
suspension. Vortex cells lightly and incubate on ice for 10 min (Basic Protocol 1,
step 6).
7. Without removing the FcR-blocking reagent, add the appropriate MAbs (see
Support Protocol 4 for determination of optimal antibody concentrations) to the
cell suspension, vortex gently, and incubate on ice for 10 min.
All MAbs can be added concurrently in this step (Basic Protocol 1, step 7). It is highly ad-
visable to include a MAb to CD45 to help define the leukocyte gate during data acquisition
and analysis.

8. If all MAbs are fluorochrome-conjugated, proceed to step 11.

9. Add 2 ml PAB to sample and centrifuge for 5 min at 1500 × g, 4◦ C. Remove
supernatant and wash cells once with PAB as in step 4.
10. Add secondary antibodies to sample, vortex gently, and incubate on ice for 20 min.
11. Without removing or washing away the MAbs, add 2.0 ml FACS Lysing Solution,
1:10 dilution.
FACS Lysing Solution does not lyse nucleated erythrocytes, which are present in certain
animal species.

12. Vortex cells thoroughly and incubate for 10 min at room temperature.
13. Centrifuge sample for 5 min at 1500 × g, 4◦ C. Remove supernatant and wash cells
once with PAB as in step 4.
14. Resuspend cells in 500 µl PAB with gentle vortexing.
15. Keep samples on ice until analyzed using a flow cytometer.
The FACS Lysing Solution contains formaldehyde, so no further cell fixation is required
for longer-term storage.

ASSESSMENT OF CELL VIABILITY SUPPORT

PROTOCOL 3
Cell viability should routinely be assessed in all flow cytometric analyses. Because the
membrane integrity of dead cells has been compromised, most antibodies will freely
pass through such membranes and stain dead cells nonspecifically. This nonspecific
staining can make data difficult, if not impossible, to interpret and ultimately lead to
erroneous conclusions.

In general, cell viability is high (>95%) when lymphoid cells are taken directly from
animal tissues, unless the mechanical process used to create single-cell suspensions has
resulted in large numbers of dead cells. In contrast, the viability of cultured cells can be
Immunotoxicology
quite low, especially when these cells are chemically treated. In either case, to minimize
18.8.9
Current Protocols in Toxicology Supplement 23
 cell death during sample processing, all cell preparations and staining procedures (in-
cluding wash steps) should be performed at 4◦ C or on ice and in the presence of BSA or
heat-inactivated FBS. The elimination of dead cells prior to staining is not recommended,
since any manipulation of the cell sample could lead to the inadvertent loss of viable cells.
Cells should be stained promptly after they are harvested and, if not fixed, analyzed in a
flow cytometer as soon as possible.

To assess cell viability, a sample of unstained cells is incubated with a DNA dye, such as
propidium iodide (PI) or 7-aminoactinomycin D (7-AAD); dead cells will stain positively
for either of these two nuclear dyes. Next, a data acquisition region is placed around the
positively stained cells. By color-eventing or backgating on the PI+ or 7-AAD+ cells
present, one can easily discern that most, but not all, dead cells exhibit lower forward
scatter (FS) and higher side scatter (SS) than do viable cells. A gating region is then
established around a cluster of viable cells (PI-negative) on the basis of their light scatter
profile. This gating region can be used for all subsequent samples, even if these samples
do not include a viability indicator. However, the best method for excluding dead cells
from data analysis is to use a vital DNA dye in all samples. Some of the more common
vital dyes used in multicolor analyses are PI, 7-AAD, TO-PRO-3 (Molecular Probes),
and pyronin Y(G) [PY(G)].
Materials
Nuclear staining compound dissolved in PBS (APPENDIX 2A): 200 µg/ml PI,
250 µg/ml 7-AAD, 250 µg /ml TO-PRO-3 (Molecular Probes), or 200 µg/ml
PY(G)
12 × 75–mm polystyrene tubes
Flow cytometer
1. Add 500 µl cells (1–2 × 106 ; unfixed and unstained) to a 12 × 75–mm polystyrene
tube.
2. Add 5 µl PI to tube.
Alternatively, add 4 µl 7-AAD, 4 µl TO-PRO-3, or 5 µl PY(G).
3. Incubate cells on ice for at least 5 min.
4. Analyze cells by flow cytometry.

SUPPORT TITRATION TO DETERMINE OPTIMUM ANTIBODY CONCENTRATION

PROTOCOL 4
The single most important factor in selecting an appropriate antibody is antibody quality.
Good antibodies are characterized by their high specificities and high binding affinities,
which allow specific staining to be distinguished from nonspecific staining. However,
even with high-quality antibodies, it is important to determine the optimal staining con-
centration (or titer) for each MAb. This titer will ensure that cells are stained under
saturating conditions. Under such conditions, specific fluorescence will not be readily
influenced by cell number or incubation time (Kantor and Roederer, 1997). The method
for titrating MAbs has previously been described by Stewart and Stewart (2001a).
Materials
Lymphoid cell suspension of interest (see Support Protocol 1)
Fc receptor (FcR)-blocking immunoglobulin (Ig) solution, 200 µg/ml
Fluorochrome-conjugated antibody solutions to be tested
Immune Cell Centrifuge fitted with a microtiter plate adapter
Phenotyping
Using Flow Vortex mixer fitted with a microtiter plate adapter
Cytometry Flow cytometer
18.8.10
Supplement 23 Current Protocols in Toxicology
 1. Add 2 × 106 lymphoid cells to each of five wells in a 96-well microtiter plate.
2. Centrifuge cells for 3 min at 200 to 1500 × g, 4◦ C, in a centrifuge fitted with a
microtiter plate adapter.
3. Block FcR-mediated binding by adding 50 µl FcR-blocking Ig to each well, vortexing
gently using a vortex mixer fitted with a microtiter plate adapter, and then incubating
samples for 10 min on ice.
4. Without removing the FcR-blocking Ig added in step 3, add 0.125, 0.25, 0.5, 1.0,
and 2.0 µg of MAb, respectively, to the five wells containing lymphoid cells. Vortex
cells gently and incubate for 10 min on ice.
5. Acquire immunofluorescence data using a flow cytometer
6. Create a two-parameter histogram of forward scatter versus side scatter and establish
an appropriate gating region that encompasses the majority of viable cells.
7. Display fluorescence data from viable cells in single-parameter histograms. Establish
one region that encompasses the negative population and a second region that brackets
the positive population. Determine the median channel of fluorescence (MCF) for
each region.
8. Compute the signal-to-noise (S/N) ratio, treating the positive population as the signal
(S) and the negative population as the noise (N). Plot the calculated S/N ratios as a
function of MAb concentration.
9. Identify the optimal MAb titer (i.e., the titer that produces the highest S/N ratio).
Once the optimal titer is determined, it is not necessary to increase the amount of antibody
used for staining unless the number of cells per sample exceeds 20 million. Even then,
the amount of antibody needed for staining will only increase by a factor of two or
three. However, it cannot be stressed enough that time and effort should be taken to
optimize staining conditions (staining temperature, antibody concentration, staining time,
and cell number) prior to any study. Doing so can also save money, as the manufacturer’s
recommended staining concentration is often higher than necessary.

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent for all recipes and protocol steps. For common stock
solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

ACK lysing buffer

0.15 M NH4 Cl
10 mM KHCO3
0.1 mM disodium EDTA
Adjust pH to between 7.2 and 7.4 using 1 M HCl
Filter sterilize using a 0.2-µm filter
Store at room temperature
If the buffer is stored for an extended period, check its pH before use.

HBSS, pH 7.2, supplemented with 5% (v/v) FBS

1× Hanks’ balanced salt solution (HBSS; Sigma) containing:
1 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES)
5% (v/v) fetal bovine serum (FBS), low-endotoxin, characterized (HyClone)
1 mM sodium pyruvate
Adjust pH to 7.2 using 1 N NaOH
Immunotoxicology
Store up to 1 month at 4◦ C
18.8.11
Current Protocols in Toxicology Supplement 23
 PBS, pH 7.2, supplemented with sodium azide and BSA
Dulbecco’s PBS, calcium- and magnesium-free (e.g., Life Technologies),
containing:
0.1% (w/v) sodium azide
1% (w/v) BSA
Adjust pH to 7.2 using 1 N NaOH or 1 N HCl
Filter solution through 0.22-µm filter paper
Store up to 1 month at 4◦ C
EDTA (1 mM) can be added to reduce cellular aggregation.
CAUTION: Be extremely careful when handling sodium azide.

COMMENTARY
Background Information
Secondary anti-Ig antibodies
Although indirect staining with secondary
fluorochrome-conjugated anti-Ig antibodies
is the least desirable staining method, due
to problems with nonspecific staining, there
are certain precautions that can be exercised
to minimize these problems. The secondary
anti-Ig antibody is usually a polyclonal anti-Ig
antibody that is specific for a large number of
epitopes on the primary antibody. Since poly-
clonal secondary antibodies will be directed
against all the Ig isotypes (e.g., IgG, IgA,
IgM) found in the serum of the animal from
which they were derived, these antibodies
should be purified by affinity chromatography
so that their binding is restricted to the heavy
chain of the appropriate Ig isotype. The
binding of the secondary anti-Ig antibody can
be further restricted by selecting a reagent
with no light-chain activity (i.e., one that is Fc
fragment–specific). This will further ensure
that the anti-Ig antibody is highly specific for a
particular class of Ig (e.g., IgG) and thus min-
imize cross-reactions with other Ig isotypes.
In addition, it is essential to use the F(ab) or
F(ab )2 fragment of the affinity-purified sec-
ondary antibody to minimize FcR-mediated
binding and, hence, reduce background stain-
ing. For example, a goat F(ab )2 anti–mouse
IgG antibody (heavy and light chain–specific,
purified by affinity chromatography) is
specific for the heavy chain of mouse IgG but
will also bind to other Igs that have the same
light chain as IgG (e.g., IgA, IgM). Therefore,
this reagent is not specific for mouse IgG at
all. The authors have had great success using
polyclonal secondary antibodies purchased
from Jackson ImmunoResearch Laboratories.
The binding of a secondary antibody can be
Immune Cell
Phenotyping further restricted to a specific Ig isotype and
Using Flow subclass. For example, if a rat IgG1 MAb spe-
Cytometry cific for CD4 were being used as the primary
18.8.12
Supplement 23
MAb, one could select a secondary MAb spe-
cific for rat IgG1 , if available.
In all cases, it is necessary to determine the
optimum antibody titer as well as assess the
binding specificity of the secondary anti-Ig an-
tibody prior to a study. First, the secondary
antibody should not react with any cellular
epitope. This can be assessed by simply incu-
bating the cells of interest with the secondary
antibody alone. The signal produced by this
staining should be nearly identical to the signal
yielded by unstained cells. If not, the reagent
is inappropriate and should be replaced.
Second, if this secondary antibody is used in
multicolor analyses, it should not cross-react
with any other MAb used for staining. To
test for potential reagent interactions, incubate
two samples of cells with the fluorochrome-
conjugated MAb, followed by washing. Next,
add the secondary anti-Ig antibody to one sam-
ple and PAB to the other sample. The fluo-
rescence pattern of the sample stained with
the secondary anti-Ig reagent should be nearly
identical to the pattern produced by the sample
stained with PAB. If the fluorescence pattern
is different, the secondary antibody in question
should not be used in multicolor analyses.
Fluorochrome choice
Fluorochrome choice is highly contingent
on the availability of the laser lines avail-
able on a particular flow cytometer. For in-
stance, the Beckman Coulter XL flow cytome-
ter is equipped with a single argon ion laser
tuned to a wavelength of 488 nm, the most
common wavelength used in flow cytometry.
This instrument can handle up to four fluo-
rescent emissions, all from dyes that can be
excited by the 488-nm wavelength, but it can-
not process the signal generated by a MAb
conjugated to allophycocyanin (APC), which
must be excited by a 633-nm laser line. How-
ever, APC signals can be processed on any
benchtop analyzer, such as the FACScalibur
Current Protocols in Toxicology
(BD Biosciences) or the FC500 (Beckman
Coulter), that is equipped with a HeNe laser.
For multicolor analyses, there are a number
of factors to be considered when selecting a
MAb conjugated to a particular fluorochrome.
This selection is particularly important when
using antigens that are expressed at low
density, as some fluorochromes have higher
absorption coefficients and/or higher quantum
yields than others, making them more suitable
for detecting weakly expressed antigens. In
order to achieve the highest possible staining
intensity, select antibodies conjugated to the
brightest possible fluorochromes, e.g., in
order, from brightest to dullest—PE, PE-Cy5,
PE–Texas Red, APC, and FITC. In general,
the fluorescence emitted by MAbs conjugated
to PE, PE-Cy5, and APC is 5 to 10 times
brighter than the fluorescence emitted by
FITC conjugates. PE is often the fluorochrome
of choice when examining the expression of
an antigen that is expressed at low levels (e.g.,
CD25, CD69) or detecting the presence of
intracellular cytokines.
New fluorochromes are continually being
developed, and they can be used in addition
to or instead of the dyes mentioned above.
Molecular Probes (2004b) has produced a new
series of dyes, called the Alexa Fluor dyes,
that exhibit more intense fluorescence than
do other spectrally similar conjugates. For
example, Alexa488, whose fluorescence spec-
trum is nearly identical to that of fluorescein
isothiocyanate (FITC), is considered by many
to be the best FITC-like reagent. A number
of MAbs conjugated to Alexa488 can be
obtained from commercial sources. If the ex-
periment necessitates the use of a second-step
reagent, the authors use fluorochrome-labeled
SA, which is readily available from commer-
cial sources, to stain biotin-labeled MAbs.
For simple one- or two-color analyses, the
second-step reagent of choice for the authors
is PE-labeled SA, which will stain at the
highest possible level. For multicolor studies,
the authors routinely use a tandem conjugate
PE–Texas Red–labeled SA. It is possible that
PE-Alexa610-labeled SA, by virtue of its low
background staining, may be a better option
than the PE–Texas Red fluorochrome, which
can exhibit the high background staining that
is typical of Texas Red dyes. The authors
rarely use FITC-labeled SA but have had great
success with Alexa488-labeled SA, which is
far superior to its FITC-labeled counterpart.
All MAbs should be stored in the dark,
as most fluorochromes are photosensitive.
PE-Cy5 and PE-Cy7 tandem conjugates are
Current Protocols in Toxicology
particularly sensitive to photodegradation.
This phenomenon can easily be recognized by
an increase in the PE signal and a concomitant
decrease in the Cy5 or Cy7 signal. Use of
cyanine tandem dyes can result in increased
background staining, as these dyes can bind to
monocytes and B cells. However, this binding
is highly species-dependent; for instance,
background binding can be high in mice
with autoimmune disorders. Thus, again, it is
prudent to assess the background staining of
the reagents used prior to a large study.
Multicolor staining combinations for
routine immunophenotyping of murine cells
Table 18.8.1 presents examples of possible
staining strategies that can be used to pheno-
type various cell populations in the mouse. It
is becoming increasingly clear that no single
marker can be used to specifically identify a
unique subset of cells. For instance, expression
of CD4 and CD8a is not restricted to T cells, as
these molecules are also expressed on subsets
of dendritic cells (CD11c+ ). In addition, CD4
is expressed on a subset of natural killer (NK)
cells. Thus, in order to ensure that only T
cells are analyzed, a MAb specific for CD3e,
a marker that is expressed on thymocytes
and mature T cells, can be included in the
MAb cocktail. The authors have also found
that the correlated expression of two markers,
CD11b and Gr-1, is needed to phenotype either
macrophages or granulocytes in mouse spleen.
Both of these cell types express CD11b and
Gr-1; however, the level of Gr-1 is lower on
macrophages than on granulocytes. The two
populations are more clearly resolved by in-
cluding additional markers that are expressed
on the macrophage, such as F4/80, CD86, or
CD54 (Choi et al., 2003). It is essential to
recognize that many markers can be expressed
at varying levels on different cell types.
Critical Parameters
Autofluorescence
Cellular autofluorescence is most often
associated with myeloid cells, due to the
presence of intracellular flavins in such cells.
These flavins are easily excited by the 488-nm
laser line, with peak emission occurring
around 525 nm. Therefore, any signal gener-
ated by autofluorescent cells will be processed
by the same photomultiplier tube (PMT) that
processes FITC fluorescence. It is obvious
that the presence of highly autofluorescent
cells can contribute an unwanted addition
Immunotoxicology
to the FITC signal. Autofluorescence can
18.8.13
Supplement 23
 Table 18.8.1 Examples of Possible Antibody Combinations for Staining Mouse Leukocytes
Using Five Fluorochrome-Labeled Monoclonal Antibodies in a Staining Cocktail, with Analysis
Performed on an FC500 Flow Cytometer (Beckman Coulter) with Dual Laser Excitation at 488
nm and 633 nma

Cell type(s) Possible combination of stains

T cells, NK cells FITC-anti-CD4

PE-anti-CD3e
Biotin-anti-NK1.1b
PE-Cy5–anti-CD8c
PE-Cy7–anti-CD45d,e
Viable T cells FITC-anti-CD4
PE-anti-CD3e
Biotin-anti-NK1.1b
7-AADf
PE-Cy7–anti-CD8
Granulocytes, dendritic cells, B cells, macrophages FITC-anti-Gr-1
PE-anti-CD11c
ECD-anti-B220g
PE-Cy5–anti-CD11b
PE-Cy7–anti-CD45
Activated T cells FITC-anti-CD8
PE-anti-CD25
ECD-anti-CD4
PE-Cy5–anti-CD62Lh
PE-Cy7–anti-CD8
a Abbreviations: 7-AAD, 7-aminoactinomycin D; APC, allophycocyanin; FITC, fluorescein isothiocyanate; MAb, mon-
oclonal antibody; NK, natural killer; PE, phycoerythrin.
b This biotinylated MAb necessitates the use of streptavidin labeled with PE–Texas Red or equivalent.
c PE-Cy5-labeled MAb could be replaced by a MAb conjugated to APC or equivalent.
d PE-Cy7-labeled MAb could be replaced by a MAb conjugated to APC-Cy7 or equivalent.
e CD45 is a pan-leukocyte marker that is very useful in discriminating leukocytes from contaminating red blood cells,
especially in the phenotyping of peripheral blood cells. A gating region can be established on the CD45+ leukocyte
population as defined in a two-parameter histogram of side scatter versus CD45 expression.
f 7-AAD is a DNA dye used as a viability indicator in unfixed cell samples. Its peak emission wavelength is 660 nm.
g ECD is the trade name for the PE–Texas Red tandem conjugate produced by Beckman Coulter. Very few mouse
marker–specific MAbs that are directly conjugated to this fluorochrome are available.
h Because CD62L can easily be shed from the surface of cells, it is imperative to keep cells on ice throughout staining
and data acquisition. The authors recommend that samples stained with anti-CD62L be kept on ice until loaded manually
onto the flow cytometer for data collection. The use of a multisample carousel is not recommended when analyzing cells
stained for CD62L, since cells cannot be kept cold while on such a device.

be particularly bothersome when measuring
weak FITC fluorescence signals. If there are
excessive numbers of myeloid cells in the
experimental samples, the resulting high level
of autofluorescence may make it difficult to
interpret data. A sample of unstained cells
can be used to determine the contribution of
cellular autofluorescence to the background
fluorescence signal as well as the minimum
level of fluorescence exhibited by cells.
There are several approaches that can be
Immune Cell
Phenotyping used to circumvent the problems associated
Using Flow with cellular autofluorescence. When study-
Cytometry ing myeloid cells, it may be prudent to use
18.8.14
Supplement 23
MAbs that are conjugated to APC or APC-
Cy7, as these dyes can be excited by a HeNe
laser and emit at wavelengths greater than
600 nm, beyond the emission spectrum for cel-
lular autofluorescence. When immunopheno-
typing lymphocytes in a sample containing el-
evated numbers of myeloid cells, one could
include a MAb that specifically identifies the
myeloid cells (such as one specific for Gr-1, a
marker of myeloid/granulocyte lineage). The
use of such an antibody allows one to gate
out granulocytes and macrophages, thereby
eliminating cellular autofluorescence from
analysis.
Current Protocols in Toxicology
Blocking FcR-mediated binding
There are two ways that an antibody can
specifically bind to cells. Antibodies can bind
to a specific antigen (epitope-specific binding)
through their antigen-binding sites, the F(ab )
fragment, or they can bind to myeloid cells
that express receptors for the Fc portion of the
Ig. Even though FcR-mediated binding is not
antigen-specific, it is highly specific and needs
to be eliminated in order to assess antigen-
specific staining.
FcR should be blocked with normal Ig prior
to staining with epitope-specific MAbs. The
authors typically incubate cells for 10 min
at 4◦ C with purified normal Ig (Jackson
ImmunoResearch Laboratories) taken from
the same species that produced the antibodies
used for staining. For instance, if a rat IgG2a
anti-CD4 MAb is used, FcR-mediated binding
should be blocked with an excess of purified
rat IgG. The use of 10 µg of normal IgG in a
staining volume of 50 µl (final concentration,
200 µg/ml) should be sufficient to block FcR.
After 10 min of blocking, the MAbs are added
directly to the samples without removing the
FcR block, so that epitope-specific staining can
proceed in the presence of FcR-block. Another
approach to blocking FcR is to incubate cells
with an unconjugated MAb that is specific for
the FcR (CD16/CD32). It should be noted that
this approach is more expensive than the for-
mer approach. In any case, do not block FcR if
there is interest in assessing FcR expression.
Isotype controls
Isotype controls are also used to determine
the minimum level of cellular fluorescence
following staining and to assess the effective-
ness of FcR blocking. An isotype control must
match the primary MAb in terms of isotype
and subclass, as well as fluorochrome label and
concentration. For example, the appropriate
isotype control for cells incubated with 0.5 µg
FITC-labeled rat IgG2a MAb (specific for
mouse CD4) is 0.5 µg FITC-labeled normal
rat IgG2a . The appropriate isotype control
for cells stained with biotin-labeled rat IgG1
MAb (specific for CD8) is treatment with
biotin-labeled normal rat IgG1 , followed by
incubation with fluorochrome-conjugated SA.
For multicolor analyses, one isotype
control sample can be set up to assess all
the MAb isotypes in a single tube. It is not
necessary to analyze each isotype control
separately. For example, a sample of cells can
be incubated, in the presence of an FcR block,
with a cocktail of isotype-matched Igs for each
specific MAb used in the staining procedure.
Current Protocols in Toxicology
The fluorescence pattern of the isotype control
should be nearly identical to that of the
autofluorescence control. When this is not the
case, it is indicative of an improperly titrated
MAb, a poor-quality MAb, or both. This
could have a profound effect on one’s ability
to interpret positive fluorescence, especially
when studying antigens that are expressed
either transiently or at very low levels.
In general, the authors have found that some
isotype Igs (e.g., IgM, IgG2b ) exhibit more
background staining than others, which can
complicate the analysis of antigens expressed
at very low levels. This is particularly rele-
vant in analysis of intracellular antigens. In
these cases, a cold block control is performed
to verify antibody specificity. This can eas-
ily be accomplished by comparing the fluo-
rescence of two cell samples, with one be-
ing pretreated with a cold blocking MAb (i.e.,
the same clone as the experimental MAb, but
with no conjugate) for 15 min prior to the ad-
dition of the fluorochrome-conjugated MAb.
The binding of the fluorochrome-conjugated
MAb should be completely inhibited by the
cold block. For example, to assess the expres-
sion of CD25, which can be present at low
levels on activated T cells, cells are pretreated
with unlabeled purified anti-CD25 MAb (at
a 3-fold higher concentration compared with
the conjugated MAb) for 15 min prior to the
addition of the fluorochrome-conjugated anti-
CD25 MAb. This sample should represent a
true negative control for CD25 staining. Back-
ground staining for cell surface expression of
CD69, another T cell marker that is transiently
up-regulated upon activation, can be assessed
similarly. Due to the complexities involved in
multicolor analyses, it is prudent to invest a lit-
tle time to test these conditions prior to a large
study.
Background staining exhibited by cells
stained with multiple reagents is very different
from that seen in unstained cells or isotype con-
trols, due to the broadening artifacts produced
by compensation (see below) and/or reagent-
reagent interactions. Therefore, the best neg-
ative control for any given marker in a mul-
ticolor stain is a sample of cells stained with
all but one reagent (commonly referred to as
“fluorescence minus one,” or FMO). For ex-
ample, when assessing CD25 expression on
antigen-specific D011.10 T cells, the best neg-
ative control for CD25 expression is a cocktail
containing MAbs to CD3, CD4, and KJ-126
(to identify the antigen-specific T cells in the
D011.10 model) plus an isotype control for the
Immunotoxicology
CD25 MAb. In this way, the level of negative
18.8.15
Supplement 23
 staining for CD25 expression can be assessed
by gating on the antigen-specific T cells.
Dead cells
Cell viability must be assessed in each
experimental study. Since most MAbs freely
pass through the cell membranes of dead and
damaged cells, dead cells will stain positively
even if there is no specificity for a particular
MAb. This nonspecific fluorescence can inter-
fere with data analysis, and if ignored, it can
result in erroneous interpretation of the data.
For exclusion of dead cells, vital DNA dyes
such as PI, 7-AAD, and TO-PRO-3 can be
added to unfixed cell samples prior to analysis.
These dyes cannot be used to assess the viabil-
ity of cells that are permeabilized (as for intra-
cellular staining) or fixed, since they will leak
out of fixed cells. On the other hand, the vital
dye ethidium monoazide (EMA; final concen-
tration, 1 to 5 µg/ml) can be used to label dead
cells prior to fixation (Reidy et al., 1991). Once
inside a dead cell, EMA can be photochemi-
cally cross-linked to the DNA by exposure to
visible light. This cross-linking allows EMA
to be retained by fixed cells.
Compensation
One of the most critical issues in perform-
ing multicolor phenotyping is dealing with
overlaps in the spectral emissions of different
fluorochromes. Once excited by the laser, a
fluorochrome will emit a broad band of light.
Even though a band-pass filter is often placed
in front of a PMT to restrict the wavelengths
of light that are transmitted to the tube, it is
nearly impossible to exclude all of the light
emitted by the other fluorochromes used in
multicolor staining. Because of these spectral
overlaps, each fluorochrome will contribute
an unwanted light signal to several PMTs that
are not assigned to detect that fluorochrome.
For example, there is significant overlap in
the spectral emissions of FITC and PE, so
much so that some of the FITC emission
spills over into the PE PMT and it must be
eliminated from this PMT before analyzing
PE fluorescence. By the same accord, PE
contributes an unwanted signal to the FITC
PMT, albeit at much lower levels, and this
signal must be eliminated from the FITC PMT.
If not eliminated, these spillovers will make a
“false” contribution to the data. The process by
which these false contributions are eliminated
electronically is called compensation.
Immune Cell Compensation is one of the least understood
Phenotyping processes in multicolor analyses. It is impor-
Using Flow tant to consider compensation not only from
Cytometry
the standpoint of applying it correctly but also
18.8.16
Supplement 23
from the perspective of designing and analyz-
ing multicolor experiments. Investigators need
to be able to recognize data that has resulted
from improper compensation, as failure to do
so can easily result in data misinterpretation.
Any spillover signal not completely compen-
sated (i.e., undercompensated) out of a PMT
can result in an overestimation of the pos-
itive population. Proper compensation is al-
ways essential when measuring antigen den-
sity by MCF.
Compensation is often considered a process
by which unwanted fluorescent signals are
“subtracted” from the true fluorescent signal.
However, compensation is not a subtraction
process, but rather a straightforward applica-
tion of linear algebra by which a proportion of
the unwanted signal is eliminated from the true
fluorescent signal. For some fluorochrome
combinations, such as FITC and PE-Cy5 or
APC, there is little, if any, overlap in spectral
emissions; therefore, compensation is negligi-
ble. However, there is considerable overlap in
the emission spectra of the PE, PE–Texas Red,
and PE-Cy5 dyes, enough to make compen-
sation quite challenging even for investigators
who routinely perform multicolor analyses.
In order to set up compensation properly, a
sample of cells must be stained with a repre-
sentative reagent for each fluorochrome used
in the experiment. The number of compensa-
tion controls will be equivalent to the number
of fluorochromes used in staining. For exam-
ple, five-color staining requires five compen-
sation controls, one for each fluorochrome. It
is not necessary to use the same MAbs in the
experiment as in the compensation controls.
This is especially true when a particular exper-
imental reagent is expected to stain just a small
subset of cells. For example, the frequency of
antigen-specific T cells (KJ1-26+ CD4+ ) adop-
tively transferred in the D011.10 model can be
as low as 0.3% one day after injection with
ovalbumin. Thus, it would be very difficult, if
not impossible, to properly set compensation
levels using the conjugated MAb that stains
KJ1-26+ cells. What is important is using the
brightest possible reagents for compensation
controls. Therefore, the authors routinely set
up compensation controls using CD45, CD8a,
CD19, or CD45R MAbs conjugated to the ap-
propriate fluorochromes. These markers are
expressed at high levels on most immune cells,
making it much easier to properly set com-
pensation levels. In order to set up compensa-
tion properly, the compensation control sam-
ple must contain at least two populations—
specifically, one bright population and one
Current Protocols in Toxicology
negative or not-so-bright population. When ex-
amining spleen cells, there will be two distinct
populations that stain for MAbs to CD8a and
CD45R; one is positive, and the other is nega-
tive. Because CD45 stains all leukocytes, it is
necessary to add an aliquot of unstained cells
to the CD45 compensation control after the
staining process but just prior to flow analysis
to artificially create a negative population.
There are two ways to perform compen-
sation. Hardware compensation is performed
during data acquisition, whereas software
compensation is performed during data analy-
sis. Because of the complexity of five-color
analyses, the authors routinely collect flow
cytometric data without performing hardware
compensation in such analyses. Instead, prior
to analyzing the experimental data using of-
fline software, the data collected from compen-
sation controls are used to perform software
compensation using WinList software (Ver-
ity). The authors have found no disparity in the
results obtained using software compensation
as compared with hardware compensation.
Compensation controls must be evaluated
in every possible pairwise combination us-
ing two-parameter histograms. For example,
in studies using FITC, PE, and PE-Cy5 fluo-
rochromes in a single sample, the FITC com-
pensation control must be evaluated using
the following two-parameter histograms: FITC
versus PE and FITC versus PE-Cy5. Two-
color samples require the assessment of just
one pairwise combination, three-color samples
require three combinations, four-color samples
require six combinations; and five-color sam-
ples require ten combinations. Thus, it should
be readily apparent that the complexity of com-
pensation increases substantially with the ad-
dition of each fluorochrome. To properly set
compensation levels, the center of the positive
cell population (i.e., as determined by the me-
dian fluorescence intensity) is lined up with the
center of the negative cell population. This is
accomplished by adjusting compensation until
the median channel for the positive population
is equal to the median channel for the negative
population. It is important to ensure that the
fluorescence distribution for the negative pop-
ulation is not piled up on the baseline axis, as
this will make it nearly impossible to determine
the median channel and, thus, very difficult to
set up compensation properly. An in-depth dis-
cussion on compensation can be found online
(Roederer, 2000).
Not all flow cytometers can handle all
the possible pairwise combinations that may
be necessary for hardware compensation. For
Current Protocols in Toxicology
example, the Beckman Coulter FC500 and XL
flow cytometers are capable of handling the
pairwise compensation involving FITC and
PE-Cy5, but the BD FACSCalibur is not. It can
be argued that, in general, there is minimal, if
any, FITC emission that spills over into the PE-
Cy5 PMT. However, when using carboxyflu-
orescein diacetate succinimidyl ester (CFSE),
a low but measurable signal from this dye can
spill over into the PE-Cy5 PMT. Thus, when
using the BD FACSCalibur, in lieu of hardware
compensation, one must rely on software com-
pensation to eliminate unwanted CFSE signal
from the PE-Cy5 detector.
It is possible to minimize the need for com-
pensation by carefully selecting reagents that
are conjugated to fluorochromes whose emis-
sions have little or no spectral overlap. For
two-color analyses, the authors often select
MAbs that are conjugated to FITC and ei-
ther APC or PE-Cy5, since there is little, if
any, overlap between the emission spectrum
of FITC and the emission spectra of the lat-
ter two dyes. Beyond two-color analyses, flu-
orochrome choice gets more complicated with
each additional fluorochrome. In general, PE–
Texas Red–conjugated MAbs are used by the
authors only when performing five-color im-
munophenotyping, as this fluorochrome has
considerable spectral overlap with both PE
and PE-Cy5 emissions, making compensation
quite challenging. Of lesser importance, there
are very few MAbs that are directly conju-
gated to PE–Texas Red, and two-step staining
procedures are therefore often required. If a
HeNe laser is available, the authors often se-
lect APC- or APC-Cy7-labeled reagents over
PE-Cy5 conjugates.
Control samples
In order to ensure that data are collected and
interpreted correctly, several control samples
must be faithfully evaluated prior to running
the experimental samples. These include con-
trols to assess cellular autofluorescence, non-
specific binding, FcR-mediated binding, and
compensation.
Sometimes, the number of essential control
samples exceeds the number of experimental
samples. However, it is only through the proper
use of all appropriate control samples that data
can be collected and interpreted correctly. Con-
trols can be performed for each animal, but
this can easily lead to excessive numbers of
control samples. For large studies, a pool of
cells is created for each treatment group, and
this pool is used to test for autofluorescence
Immunotoxicology
and isotype staining. In this way, a pool will
18.8.17
Supplement 23
 contain representative cells from each animal
in a given treatment group. For example, in
an experimental study in which the effects of
three different dose levels of a chemical are ex-
amined, there will be four pools of cells, each
representing a particular treatment group (one
for the vehicle control group and one for each
of the three dose levels). Each treatment pool
will have its own autofluorescence control and
its own isotype control, resulting in a total of
four autofluorescence controls and four iso-
type controls.
To assess autofluorescence, a sample of un-
stained cells (with no MAbs added) is pro-
cessed in a similar fashion as for all exper-
imental samples. This sample should always
be analyzed first in an experiment. To begin,
the voltages for the photodetectors collecting
the light-scatter signals (both forward scatter
and side scatter) are adjusted to define the cells
of interest. Second, the voltage of each PMT is
adjusted to place the negative cell population
in the first log decade of the histogram. It is im-
portant to apply enough voltage to ensure that
the majority of cells are off of the baseline; this
assures that any positive staining will fall be-
yond the first log decade. In essence, this con-
trol sample provides the baseline representing
the minimum (or negative) fluorescence exhib-
ited by cells.
The next control to be analyzed is the iso-
type control. The fluorescence pattern of the
isotype control should be similar to that of
the unstained control if FcR-mediated binding
has been blocked effectively. If not, one would
need to evaluate the isotype controls used in the
experiment. Like the autofluorescence control,
the isotype control also determines the mini-
mum level of fluorescence that is exhibited by
cells.
For multicolor experiments, compensation
controls must be analyzed in order to account
for any spectral overlap not removed by filters.
The number of compensation controls will be
equivalent to the number of fluorochromes
used in staining. Compensation is greatly sim-
plified in four- or five-color analyses when the
PMTs are balanced, with their voltages set at
nearly identical levels. However, it is crucial to
remember that once compensation is set, any
change in the PMT voltages will necessitate
repetition of the entire compensation process.
Standardization of flow cytometric data
Immune Cell There are three sources of error that can be
Phenotyping introduced during immunophenotypic analy-
Using Flow sis: specifically, instrumental, technical, and
Cytometry
biological. However, the amount of error
18.8.18
Supplement 23
introduced by the instrument can easily be
minimized. The instrument’s overall perfor-
mance should be routinely evaluated prior
to data acquisition using standardized beads,
such as Beckman Coulter Flow-Check beads.
These beads will assess the instrument’s flu-
idics and optics to ensure proper alignment
of the laser with the sample stream. Once
the fluidics and optics have been checked,
the authors routinely run a sample of Flow-
Set beads (Beckman Coulter) to further stan-
dardize the instrument’s settings. These beads
mimic the immunofluorescence exhibited by
human and mouse cells and can be used at the
voltages required for immunophenotypic anal-
yses. The voltages and gain settings for each
photodetector are adjusted to position the flu-
orescence emitted by the beads within a pre-
scribed region, a process commonly referred to
as channel positioning. In this way, any appar-
ent change in marker expression over time can
be expected to be biologically relevant, and not
a result of changes in the instrument.
Once it has been established that the instru-
ment is performing under the expected exper-
imental conditions, negative controls (autoflu-
orescence and isotype) can be analyzed to en-
sure that the negative population falls within
the first log decade for each channel being mea-
sured. Next, compensation controls should be
performed to remove any unwanted fluores-
cence from the various PMTs due to overlaps
in the spectral emissions of the fluorochromes
used. It should be noted that improper com-
pensation can result in false-positive or false-
negative results, so great care should be exer-
cised when performing these controls.
Once the instrument’s settings have been
established, the authors routinely run a con-
trol sample, referred to as the verification con-
trol, in order to determine whether the sys-
tem will provide accurate information. This
control sample is stained with a set of MAbs
that are conjugated to the same fluorochromes
used in the experimental study but are specific
for well-recognized markers. For example, in
a five-color staining experiment (using MAbs
conjugated to FITC, PE, PE–Texas Red, APC,
and APC-Cy7), the verification tube would
contain MAbs specific for CD4, CD8, CD3,
CD45R, and Gr-1, with each of these MAbs
being conjugated to one of the fluorochromes
used in the study. In general, spleen cells from
a naive mouse are used in these controls. Not
only are the authors familiar with the stain-
ing profiles of these markers, but they have
established a historical record that they can
use for comparison with the observed relative
Current Protocols in Toxicology
frequencies. In short, this verification control is
expected to yield easily recognizable data that
enable the authors to make any necessary fi-
nal changes to compensation values. Once the
verification control has produced the expected
results, one can be confident that the flow cy-
tometer will provide accurate results for the
experimental samples.
Data analysis
The authors routinely acquire listmode data
on 10,000 cells of interest when performing
multiparametric analysis. For example, when
immunophenotyping spleen cells for lineage-
specific markers (such as markers of B cell,
T cell, macrophage, or granulocyte lineage),
they typically collect flow cytometric data on
10,000 viable spleen cells. If the authors are
interested in analyzing cell surface markers
gated on a discrete subset, such as antigen-
specific CD4+ cells in the D011.1 model
(which can represent as little as 0.5% to 4%
of the total population), they collect data on
10,000 antigen-specific CD4+ cells. Following
data acquisition, flow cytometric data can be
displayed in either single-parameter or two-
parameter histograms to monitor the cells of
interest. In this way, gating and analysis strate-
gies can be fine-tuned.
Figure 18.8.1 illustrates one approach to the
analysis of five-color flow cytometric data. The
data represented in this figure are derived from
studies monitoring the events of T cell activa-
tion in the graft-versus-host (GvH) response.
T cells (both CD4+ and CD8+ ) were purified
from the spleens of C57Bl/6 mice (H-2b ; re-
ferred to as donors) and injected into C57Bl/6
(H-2b ) × DBA/2 F1 mice (H-2d ; referred to
as hosts). The donor T cells recognize the
H-2d antigens contributed by the DBA/2 strain
in the F1 host and become activated. Two days
after the injection of these donor T cells, the
F1 mice were sacrificed, and their spleens were
processed into single-cell suspensions. Spleen
cells were then stained simultaneously with a
cocktail of five MAbs specific for CD4, H-2Dd ,
CD28, CD25, and CD62L.
As shown in Figure 18.8.1A, a cluster of
spleen cells was first identified by its light scat-
ter profile (FS vs. SS), as is customary in flow
cytometry. A rectilinear region was then estab-
lished to exclude dead cells, debris, and cell
aggregates from further analysis. This process
is referred to as gating. The FS-versus-SS pro-
file of dead cells, as determined by PI uptake,
indicated that these cells were located below
the gating region (data not shown). In gen-
eral, the authors find that the viability of their
Current Protocols in Toxicology
splenic suspensions often exceeds 97%. When
immunophenotyping blood cells, the authors
routinely include a MAb to the pan-leukocyte
marker CD45, which allows them to set a
gate around all the leukocytes present, thereby
eliminating, or gating out, residual RBCs and
platelets; this is most often performed by set-
ting a gate around the appropriate cell clusters
as defined by their CD45-versus-SS profiles.
Two days after adoptive transfer into F1
mice, one can easily identify a small but dis-
tinct cluster of cells that represent the donor
CD4+ cells (Fig. 18.8.1B). These cells are
identified by their expression of CD4 and their
lack of expression for H2Dd . As shown in this
two-parameter histogram, the majority of cells,
which are the host cells, express H2Dd . Only
a small population of cells lacks H2Dd expres-
sion, representing the donor T cells. An ellip-
tical region has been set to establish a gate for
these CD4+ donor cells so that their expres-
sion of the T cell activation markers CD28,
CD62L, and CD25 can be monitored through-
out the course of the GvH response.
Gating on the donor CD4+ cells (10,000)
as shown in Figure 18.8.1B, one can gener-
ate single-parameter histograms that illustrate
the expression of the T cell activation markers
(Fig. 18.8.1C). The immunofluorescent profile
for each marker is illustrated in the filled his-
tograms, while the open histograms illustrate
negative staining as determined by their appro-
priate isotype controls. Two populations can
easily be distinguished on the basis of the dif-
ferential expression of CD62L, CD62Lpos , and
CD62Lneg . However, the staining profiles for
CD25 or CD28, as seen in single-parameter
histograms (Fig. 18.8.1C), are more homoge-
neous.
Figure 18.8.1D illustrates the power of mul-
tiparametric analysis. Multiple subpopulations
can be resolved by displaying the correlated
expression of two markers in two-parameter
histograms. While there is a hint of two pop-
ulations in each of the single-parameter his-
tograms for CD25 and CD28 expression, one
could easily identify two cell clusters by cor-
relating the expression of these two markers
in a two-parameter histogram. It is interest-
ing to note that three distinct cell clusters can
be distinguished on the basis of the correlated
expression of CD62L and CD25 expression.
One strategy is to “color-event” the events of
interest. As shown in Figure 18.8.1D, two re-
gions have been set around the cells that have
down-regulated their expression of CD62L,
one population expresses CD25 (color-evented
Immunotoxicology
red), and another does not express CD25
18.8.19
Supplement 23
 Figure 18.8.1 Legend at right.

Immune Cell
Phenotyping
Using Flow
Cytometry

18.8.20
Supplement 23 Current Protocols in Toxicology
(color-evented blue). The cells that have re-
tained their CD62L expression have been
color-evented gray. In this way, the expression
of all their markers can be easily visualized for
each population in all the histograms. For in-
stance, the red population also expresses high
levels of CD28 and elevated levels of CD4. In
contrast, the blue population expresses inter-
mediate levels of CD28 and CD4. By defini-
tion, both the red and blue cells are consid-
ered activated T cells since they have down-
regulated their expression of CD62L; however,
the red cells are further along in the activa-
tion pathway since they have also up-regulated
their expression of CD25, which occurs only
after several cell divisions. The gray popula-
tion represents CD4+ cells that have not be-
come activated by day two of the GvH re-
sponse.
There is a new approach in visualizing flow
cytometric data, i.e., changing the traditional
four-decade logarithmic scale for immunoflu-
orescent data displayed in two-parameter his-
tograms to a new Logicle axis (Tung et al.,
2004). The plot axes have been redefined to al-
low the log-transformed scale to have a zero as
well as a negative region. There are several ad-
vantages to this approach. Once data have been
properly compensated, a large number of neg-
ative cells pile up in the first channel, against
the axis, as these cells exhibit little to no flu-
orescence. Using the new Logicle scale, these
negative cells can be visualized as distinct cell
clusters. In addition, this new approach also
makes it easier to assess compensation. Both
FlowJo (Tree Star) and WinList (Verity Soft-
ware House) offer the option to display flow
cytometric data using the new Logicle axis.
Statistics
The data shown in Figure 18.8.1 represent
the response of one individual animal. In most
immunotoxicological studies, the authors use
four to six animals per treatment group. In or-
der to determine a treatment effect, data de-
rived from the treatment group must be com-
pared with data from the vehicle control group.
The authors will often include a group of naive
animals (N = 2 to 3) in order to monitor
the antigen-induced response of the control
animals.
In general, the authors prefer to present flow
cytometric data in the form of histograms and
use statistics to state the level of confidence in
the accuracy of each result. Flow cytometric
data are usually presented as the frequency of
any given population that expresses a particu-
lar marker and/or as a measure of the intensity
of the fluorescent or light scatter signals. There
are three measures commonly used to describe
the central tendency when assessing fluores-
cence intensity: mean, median, and mode. The
mean fluorescence can be significantly skewed
higher or lower by the presence of a few out-
liers. Therefore, when possible, one should use
the median fluorescence of the positives, since

Figure 18.8.1 (at left) Five-color analysis of T cell activation in a graft versus host (GvH) model.
T cells purified from the spleens of C57Bl/6 mice (H-2b ; referred to as donors) were injected
into C57Bl/6 (H-2b ) x DBA/2 (H-2d ) F1 mice. These donor T cells recognize the H-2d antigens
contributed by the DBA/2 strain in the F1 host and become activated. Two days after the injec-
tion of donor T cells, the F1 mice were killed, and their spleens were processed into single-cell
suspensions. The spleen cells were stained simultaneously with MAbs to CD4, H-2Dd , CD28,
CD25, and CD62L. Cells were analyzed using an FC500 flow cytometer (Beckman Coulter). (A)
Viable cells were identified on the basis of their forward scatter (FS)–versus–side scatter (SS)
profiles. (B) CD4+ donor cells (elliptical region) were identified by their expression of CD4 and
their lack of expression of H2Dd . In this sample, the donor CD4+ cells represent 1.9% of the to-
tal spleen cell population. (C) Expression of the T cell activation molecules CD28, CD25, and
CD62L was assessed in single-parameter histograms by gating on 10,000 donor CD4+ cells. Cells
with positive staining for the specified MAbs are represented by the gray-filled histograms. Open
histograms represent negative staining associated with the isotype control antibodies. (D) Cor-
related expression of activation molecules on donor CD4+ cells as illustrated in two-parameter
histograms. Donor CD4+ cells that have up-regulated expression of CD25 are color-evented as
red events. Using color-eventing, a distinct cluster of donor CD4+ CD25+ cells that also express
low levels of CD62L and high levels of CD28 is readily identifiable. When the data are displayed
in a two-parameter histogram, the presence of at least three CD4+ subpopulations distinguished
by their differential expression of CD25 and CD62L is evident (far left histogram). It is interesting
to note that not all cells with low CD62L expression exhibit high CD25 expression. Also of interest
is that CD4+ CD25+ cells have up-regulated expression of CD4, as shown in (B). This black and
white facsimile of the figure is intended only as a placeholder; for full-color version of figure go to
http://www.interscience.wiley.com/c p/colorfigures.htm Immunotoxicology

18.8.21
Current Protocols in Toxicology Supplement 23
 the median is generally a more robust estimate
of central tendency than the mean.
The precision in cell counting, or the ex-
tent to which replicate samples agree with each
other, is highly dependent on the number of
events sampled. The statistic which is most of-
ten used to measure precision is the coefficient
of variation (CV), which can be determined
using the following simple equation:
√
CV = [( N)/N] × 100%
where N is the number of events sampled and
the square root of N represents the expected
standard deviation. As the difference between
two populations becomes smaller, better and
better precision (i.e., a lower CV) is needed.
For example, the CV for 25 cells is 20%; for
100 cells, 10%; and for 10,000 cells, 1%. In
the authors’ laboratory, in order to attain high
precision, flow cytometric data are routinely
collected on a minimum of 10,000 cells of in-
terest, whether one is acquiring 10,000 viable
cells or 10,000 antigen-specific T cells (such as
the CD4+ donor cells shown in Fig. 18.8.1B).
Anticipated Results
The objective in flow cytometric analyses is
to resolve epitope-positive cells from epitope-
negative cells with a high degree of precision
and accuracy. Sometimes, it is necessary to se-
lect a MAb that is labeled with a fluorochrome
such as PE in order to maximize the resolu-
tion of positive cells from negative cells, es-
pecially when assessing dim immunofluores-
cence. However, there are several procedures
that can be employed to ensure the collection
of accurate results.
Proper setup will help to eliminate instru-
mental error as a source of variation within an
experiment. The systematic process by which
an instrument’s performance is validated is
known as quality control, and this process
should be conducted for every experiment.
There are several sequential quality control
steps that should be followed (as discussed in
Critical Parameters): (1) the use of standard-
ized beads, such as Flow-Check fluorospheres
(Beckman Coulter), to check laser alignment
as well as the fluidics and optics of the sys-
tem; (2) the use of standardized beads, such
as Flow-Set fluorospheres (Beckman Coulter),
for channel positioning (to ensure that changes
observed in time-course studies are the result
Immune Cell of biological effects and not of changes in the
Phenotyping instrument); (3) the use of compensation con-
Using Flow trols to remove unwanted fluorescence from
Cytometry
fluorochromes with overlapping spectral emis-
18.8.22
Supplement 23
sions; and (4) the use of a verification con-
trol to establish the accuracy of the results
yielded by the system. Once the instrument
has been validated, there are several technical
issues that must be addressed, such as cellular
autofluorescence, FcR-mediated binding, and
the presence of dead cells, which can result
in the collection of inaccurate data and possi-
bly lead to data misinterpretation or erroneous
conclusions.
It is easy to obtain high precision in flow
cytometric data; this can be done by simply
collecting data on a minimum of 10,000 cells
of interest. As an example, when assessing ac-
tivation markers on CD4+ T cells, data should
be collected by gating on 10,000 of these cells.
The precision of a given measurement is usu-
ally characterized by the statistic CV, and for
10,000 events, the expected CV would be 1%.
Thus, the assessment of 10,000 cells of inter-
est will help to ensure that small differences
between two populations may still be biologi-
cally significant.
It is only through these steps that one can
be confident that the results obtained are accu-
rate, and this can ensure the validity of data
interpretation. This is of utmost importance
when assessing treatment effects in toxicologi-
cal studies. Treatment effects can be discerned
by changes in the relative frequency of a par-
ticular lineage-specific population (e.g., CD4+
T cells, B cells, macrophages) or by changes
in the relative frequency of a specific sub-
set within these major leukocyte populations
(e.g., activated CD4+ T cells, cytolytic T effec-
tor cells). Sometimes it is necessary to report
changes in the MCF for a particular marker,
especially when all the cells of interest homo-
geneously express that marker (e.g., CD11a,
CD54). However, the bottom line in flow cyto-
metric analysis is that each result is expected to
accurately reflect the cell population(s) being
sampled. In this way, one can be assured that
the data can be correctly interpreted and valid
conclusions derived. In-depth discussions on
the issues presented can be found in Stewart
and Stewart (2001b) and Shapiro (2003).
Time Considerations
The time required to process, stain, and
analyze samples is contingent on many fac-
tors, including the number of animals in
the experiment and the number of stain-
ing combinations. The time needed to pre-
pare single-cell suspensions from lymphoid
tissues depends on the organ to be pro-
cessed (spleen, thymus, and/or lymph nodes).
Spleen cell suspensions will require an extra
Current Protocols in Toxicology
step in sample processing to remove RBCs,
whereas thymus and lymph node cell suspen-
sions will not. The use of microtiter plates
will greatly expedite the staining of large
numbers of samples derived from lymphoid
organs. Even though blood samples require no
further processing once the blood is collected,
the staining process requires the use of test
tubes or microcentrifuge tubes, making sam-
ple manipulation more time-consuming. The
time required for cell staining is dependent on
the number of cell samples to be stained and
the staining method employed (direct or in-
direct). In general, the direct staining method
will take 10 min for FcR blocking and 10 min
for incubation with fluorochrome-conjugated
MAbs, followed by 10 to 20 min for wash
steps. Indirect staining will require an addi-
tional 20 min to incubate cells with the second-
step reagent, followed by 10 to 20 min for wash
steps. The time necessary to acquire flow cy-
tometric data will vary depending on cell con-
centration and on the total number of events
to be collected. For example, it should take
less than 1 min per sample to collect 10,000
to 20,000 events when determining the vari-
ous lineage-specific populations in a sample
of spleen cells. However, it will definitely take
longer to collect 10,000 events when gating
on a smaller population. Instrumental setup
will take much longer for multicolor analy-
ses as compared with one-color analyses, as
proper compensation will need to be estab-
lished in multicolor studies. The time involved
in data analysis is contingent on the number of
fluorochromes used in each MAb cocktail as
well as the number of samples processed. Data
analysis can take as little as just a few min-
utes per sample for samples stained with one
fluorochrome-conjugated MAb and as many
as several hours for samples stained with five
different colors.
Acknowledgement
The authors would like to acknowledge
Castle Funatake for her valuable editorial as-
sistance. This unit was supported by the Cell
and Tissue Analysis Facilities and Services
Core of the Environmental Health Sciences
Center, Oregon State University, grant num-
ber P30 ES00210, National Institute of Envi-
ronmental Health Sciences, National Institutes
of Health.
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Contributed by Julie A. Oughton and
Nancy I. Kerkvliet
Oregon State University
Corvallis, Oregon
Current Protocols in Toxicology