Flow cytometry
John F. Dunne and Holden T. Maecker
Introduction
The technologies associated with watching cells
flow through an image plane have developed in sev-
eral important directions over the last 30 years. This
chapter will focus on flow cytometers,tools that have
optimized fluidics,electronics and optics to generate
extraordinary measurement precision and dimen-
sionality on samples of cells in suspension. Outside
the scope of this discussion remains the continuum
of devices ranging from real optical microscopes
that create pictures of cells moving in situ like blood
cells flowing in a capillary, to simple counting
devices that monitor the passage of particles in a
sensing region.
While the fundamental fluidics constraints of
flow cytometers have not changed, the equipment
available to analyze the particles as they pass
through a flow cell has been enhanced dramatical-
ly with developments in lasers, digital electronics,
fluorescence chemistries and computer power.
Analysis rates have migrated from hundreds to tens
of thousands of cells per second, but more impor-
tantly, the number of colours commonly simultane-
ously measured has gone from one or two in the
1970s, to four becoming common in the 1990s, to
the eight to twelve colour experiments being pub-
lished recently [1]. These polychromatic experi-
ments create a new kind of information about flow
samples, as well as a new set of technical chal-
lenges. The optimization of instrument and experi-
ment protocols and the efficient analysis of this
kind of data requires mind-stretching exercises in
multidimensional thinking. Useful highly dimen-
sional commercial tools are beginning to enter
research laboratories, and scientists are using them
with some satisfaction.As a result, the technology is
B3
now bounded more by the complexity of thought
than by the availability of sophisticated experimen-
tal devices.
Mechanistic principles
Fundamentally, flow cytometers measure fluorescent
or scattered light emitted by a cell during its illumi-
nation in bright light, typically a highly focused laser
beam [2].In most cases,the cell stream is positioned
using hydrodynamic focusing,in which a thin core of
cell suspension is limited to the centre of a larger
flowing sheath fluid.The cells thus arrive sequential-
ly into the laser beam at thousands of cells per sec-
ond.
Light is scattered by the cells, and this scattered
light is usually measured at narrow angles just above
and below the laser beam (commonly called “FOR-
WARD SCATTER”), and by a separate detector at wider
angles orthogonal to the beam (commonly called
“SIDE SCATTER”). FORWARD SCATTER is descriptive of the
size of the cell, while SIDE SCATTER is proportional to
size and granularity. Thus in commonly analyzed
populations of blood cells, platelets, lymphocytes,
monocytes and granulocytes can be distinguished
reasonably well simply on the basis of their intrinsic
light-scattering properties (Fig. 1).
More valuably, cells can be stained with fluores-
cent reporter molecules, and the binding of these
reporters can describe extremely subtle phenotypes.
The most common class of reporters is fluorescently
conjugated monoclonal antibodies (mAb). Thou-
sands of these products are commercially available.
The antibody protein binds very specifically to par-
ticular EPITOPES in proteins present on the cell sur-
face, or if the cell is permeabilized the antibody can
enter the cell and bind to EPITOPES within. Since the
184 Flow cytometry

FIGURE 1. DISCRIMINATION OF LEUKOCYTE SUBSETS BY LIGHT SCATTER

The labeled subsets of leukocytes were fluorescently stained with specific mAb (not shown), and the specific mAb-
stained populations were coloured as indicated. These populations were then displayed in a dot plot of forward ver-
sus side scatter. A threshold was set on forward scatter, excluding much of the platelet population, in order to prevent
the collection of small debris that would otherwise interfere with the analysis. Note that the coloured populations are
reasonably well resolved from each other. However, the relative positions of these populations will depend upon the
treatment of the sample (in this example, formaldehyde-fixed and detergent-permeabilized cells were used).

antibody is covalently linked to a fluorescent mole-
cule, the antibody binding is directly correlated to
the fluorescent signal emanating from the cell dur-
ing laser excitation. In a well-developed assay with
binding properties and chemistries well understood,
the brightness associated with antibody binding can
be a direct measure of the abundance of the relevant
antigenic protein (see Box 1). By using several mAb,
each conjugated to fluorescent dyes with character-
istic colours, combinations of antibody binding can
characterize populations of cells that may coexist in
suspension.These and other commonly used fluores-
cence strategies will be discussed in more detail
below.
 Classification/types of assay 185

BOX 1. WHY USE FLOW CYTOMETRY AS AN ANALYTIC METHOD?

It is possible to use many different methods for quantifying the proteins expressed by a population of cells. In addition
to flow cytometry, Western blotting, immunoprecipitation, ELISA, RIA, enzyme-linked immunospot (ELISPOT), fluores-
cence microscopy, and immunohistochemical staining are all methods used to quantify cellular proteins. Of these, only
flow cytometry, ELISPOT, fluorescence microscopy, and immunohistochemical staining provide information on a single-
cell basis.The others are bulk assays that do not quantify the number or phenotype of cells that are expressing the pro-
tein of interest (though they may provide other information,like the size of the targeted protein).Of the single-cell assays,
flow cytometry is unique in the number of fluorescence parameters that have been combined in a single assay.Thus, it
provides the richest information, on a per-cell basis, of the current generation of assays for cellular protein analysis.

Classification/types of assay rescently conjugated CD4 mAb are added to whole

Immunophenotype
In the 1980s, with the early propagation of FLOW
CYTOMETRY and mAb,there was some expectation that
the protein surfaces of hematopoietic cells could be
mapped with an ever-increasing complexity to reveal
changes in the types of cells present or their relative
abundance that might correlate with clinical disease
onset or progression. In the intervening 20 years, with
thousands of fluorescent antibodies commercially
available, the management of only two patient class-
es is substantially driven by these kinds of flow
assays, HIV disease and leukemia/lymphoma.
Flow immunophenotyping in HIV disease
It’s now clear that one of the most proximate and dra-
matic effects of HIV infection is an eventually lethal
loss of CD4+ T cells [3].This was one of the first easi-
ly recognized manifestations of infection, indeed the
first clinical test that helped identify AIDS patients
[4], and has been one of the most useful analytical
tools available to characterize mechanisms of HIV
pathologies as well as therapeutic benefits of anti-
HIV pharmaceuticals and other treatment modali-
ties. More recently augmented by assays that quanti-
fy viral load, CD4 counting remains the standard
method of monitoring disease progression [4].
The CD4 counting assay is relatively simple
among FLOW CYTOMETRY assays. Most commonly, fluo-
blood, generally as part of a cocktail of 2 to 4 anti-
bodies which together uniquely identify those lym-
phocytes that bear the CD4 antigen.With specialized
software tools and sample preparation procedures
that are fully validated and approved as in vitro diag-
nostics, the flow cytometer can report the number of
CD4 cells remaining per microlitre of blood. While
normal ranges can vary around 1000 cells,AIDS pro-
gression commonly results in levels that are half of
that, and profound disease is usually correlated with
CD4 counts at or below 200 CD4 cells per µl.
While the assay is commonly available to AIDS
practitioners in the developed world, technical and
financial barriers have thus far prevented the propa-
gation of the current CD4 assays in environments
with less well-funded medical infrastructures.
Technology providers are now working with govern-
ments and other funding organizations to develop
viable alternatives and/or equivalents to address this
critical world health issue.
Flow immunophenotyping in leukemia/
lymphoma
The general observation that the immunopheno-
types commonly found in peripheral blood samples
are relatively stable may have been disappointing in
some regards, but it was helpful in that it allows the
recognition of unusual phenotypes characteristic of
transformed cells. Especially in the case of trans-
formed cells of hematopoietic lineages, these unusu-
186 Flow cytometry
al phenotypes have now been mapped into more or
less broadly recognized categories of malignant dis-
ease,and flow cytometric assays are commonly used
to help diagnose and monitor leukemias and lym-
phomas.
Several clinical research consortia have pub-
lished consensus documents describing the gener-
al utility, practical aspects and interpretation of
combinations of markers and their distribution on
cancer cells [5–7]. Still, no commercially available
or otherwise standard kit has been approved for
diagnostic use, and common practice is for clinical
centres to use their own discretion to implement
and validate such assays. Nevertheless, the standard
of care of patients suffering from these malignan-
cies has been improved, sometimes dramatically,
largely based on practitioners’ ability to detect and
characterize leukemias and lymphomas by FLOW
CYTOMETRY.
Immunotoxicology immunophenotyping
While most common disease states do not change
the frequency of normal blood cell components, the
toxicity associated with novel pharmacophores or
industrial pollutants can have profound effects.
Recent work establishing consensus protocols for
the evaluation of immunotoxity of such chemical
entities documents a growing utility for this class of
assays [8]. The foci of these assays are typically
rodent,dog or non-human primate models,and mAb
reagents are less commonly available or less well
characterized. The normal ranges of various lym-
phoid cell types, other environmental factors that
can influence these ranges, and the magnitude of
changes in these cell types that should be used as
sentinels for concern or to prohibit the use of such
chemicals in various exposure scenarios, all repre-
sent ongoing work.
Functional assays
An important additional class of flow assays consists
of those assays that experimentally induce a func-
tional response in cells of interest, then use FLOW
CYTOMETRY to measure the response. One of the most
common assays of this type is the measurement of
CYTOKINE production in lymphocytes in response to
antigenic stimulation. The production of a CYTOKINE
can be easily measured using well-established
reagents and protocols, and is a hallmark of lympho-
cyte function. With multicolour FLOW CYTOMETRY, the
frequency of responsive cells, the amount and kinet-
ics of CYTOKINE production, and the combination of
various CYTOKINES can very richly describe an
immunological response qualitatively and quantita-
tively.These assays will be discussed in detail below,
and by way of comparison, other functional assays
will be addressed briefly (see Box 2).
Proliferation
Another hallmark of lymphocyte function is cellular
proliferation. Several flow assays have been well
established in the literature that have been correlat-
ed more or less well to traditional 3H-thymidine
incorporation associated with DNA synthesis. Per-
haps the most directly correlated of these is the use
of bromodeoxyuridine (BrdU), an orthologue of
thymidine used by DNA synthase and incorporated
in the nuclei of proliferating cells, and detected by
FLUOROCHROME-conjugated specific mAb [9]. Anoth-
er proliferation assay utilizes the dilution of a cytoso-
lic dye, commonly carboxyfluorescein diacetate,
which results from CYTOKINESis [10]. One of the earli-
est flow assays for proliferation, which is still in com-
mon use, is the direct measurement of DNA content
using a quantitative DNA stain such as propidium
iodide or the vital dye Hoescht 33258 [11, 12].This is
especially useful for peripheral blood lymphocytes,
which are natively arrested with a 2c DNA content in
the G0 phase of the cell cycle, upon stimulation can
enter S phase, and progress to the 4c DNA content
typical of G2/M.This doubling of DNA is easily recog-
nized flow-cytometrically, and the percentage of
cells with more than 2c DNA can be roughly related
to traditional metrics like the mitotic index.Another
nucleic acid stain, acridine orange, has the unusual
property of shifting its emission spectrum depend-
ing on whether it is bound to single- or double-
stranded nucleic acid (roughly speaking, RNA or
 Components/construction of assays 187

BOX 2. DIFFERENT ASSAYS FOR MEASURING T CELL FUNCTION

In this chapter, measurement of intracellular cytokines by flow cytometry is described in some detail as a method for
determining T cell functional responses after short-term stimulation. However, alternative methods are also in common
use. Some are flow cytometry assays, such as MHC-multimer staining [35], or cytokine capture assays [36]; others use
different analytical platforms, such as ELISPOT [37].The main advantage of flow cytometry as an analytical platform is
its multiparameter capability (see Box 1).Of the flow cytometry methods available for measuring T cells in a short-term
assay, MHC-multimer staining is unique in that it measures T cell specificity rather than function, and thus requires no
activation at all. Multimeric forms of MHC molecules with bound antigenic peptides are produced and labeled with a
fluorochrome such as PE.These can then be used to stain T cells (via their T cell receptor) in much the same way as a
fluorochrome-conjugated antibody. However, their use requires a knowledge of the particular peptide and MHC restric-
tion pattern of a T cell immune response; as such, they are not useful for quantifying the overall T cell response to a
pathogen,especially in a MHC-diverse population.Cytokine capture assays,like intracellular cytokine staining,measure
responses without regard to MHC restriction.The cytokine capture assay is especially useful if one wishes to maintain
the cells in a viable state for sorting and further analysis (since it does not require fixation and permeabilization). Still
newer assays, such as measures of granule exocytosis [38], can also be used on viable cells, and add to the armamen-
tarium of tools available for dissecting T cell responses.

DNA) and has been used to simultaneously report
DNA doubling and the increase in mRNA character-
istic of lymphoid stimulation [13].
Bead matrix immunoassays
Recently bead-based immunoassays have been
developed for flow cytometric assessment of various
soluble ANALYTES. Each member of a set of beads is
commonly identified by specific fluorescence level
and/or size, and acts as an ANALYTE capture platform
using covalently bound specific mAb.The amount of
captured ANALYTE is measured using a second anti-
body specific for an alternate site of the same ANA-
LYTE, this second antibody being conjugated to a flu-
orescent reporter. Since the various beads can be
recognized independently, and since each captured
ANALYTE can be quantified independently, this assay
format is well suited to multiplexed analysis. Thus
small volumes of biological fluids (as little as 10 µl of
tears, for example [14]) can be inspected for quan-
tification of soluble proteins or other LIGANDS for
which specific antibody pairs are available.Common
implementations of this strategy include the simulta-
neous quantification of 5–20 different immunomod-
ulatory proteins including CYTOKINES, chemokines,
growth factors and hormones. Commercially avail-
able kits range from completely integrated systems
to basic platforms that can be developed for custom
ANALYTE sets, and are used broadly in basic research
and drug discovery proteomics projects. No patient
management tools in this class have yet been
approved for in vitro diagnostic use as of this writing.
Components/construction of assays
Traditionally, FLOW CYTOMETRY sample preparation
and processing has been constrained by the fact that
flow cytometers were designed to accept tubes, and
those tubes were loaded onto the instrument manu-
ally, one at a time. A second constraint came from
instrument set-up and data analysis, which were typ-
ically time-consuming and required a certain knowl-
edge base. Both of these constraints are now chang-
ing with new instrumentation and software. Current
cytometers can often handle racks of tubes that are
automatically run in a walk-away mode. Plate load-
ers are also available that can feed samples from 96-
well plates directly to the cytometer, again in a walk-
away mode.These developments have been comple-
188 Flow cytometry
mented by software that can either: (1) perform data
analysis simultaneously with acquisition; or (2) per-
form a batch analysis routine that analyzes all of the
data from an experiment at once. The usefulness of
such analysis routines is further augmented by flexi-
ble analysis templates that can accommodate
changing data without repetitive adjustment of set-
tings by the operator [15].
The impact of these changes in FLOW CYTOMETRY
hardware and software have opened up the use of
FLOW CYTOMETRY to larger pre-clinical and clinical
studies that might involve hundreds of specimens.
However, the steps involved in sample preparation
can still be complex, as detailed below. Automation
of these steps will further allow the use of FLOW
CYTOMETRY in high-volume settings where it was pre-
viously considered too cumbersome.
Antibodies for cell staining
Since the late 1970s, polyclonal antisera have been
increasingly replaced by mAb for most immunologi-
cal applications,including FLOW CYTOMETRY.MAb can
be produced in unlimited quantities, have better lot-
to-lot reproducibility, and tend to have lower back-
grounds than polyclonal antisera [16].
Another trend in FLOW CYTOMETRY has been the
increased use of direct FLUOROCHROME conjugates,
rather than indirect fluorescence analysis using sec-
ond-step antibodies or other reagents that carry the
FLUOROCHROME tag.While indirect staining can some-
times amplify the fluorescence signal compared to
direct staining,it may be at the expense of increasing
background fluorescence [17]. Also, multiparameter
FLOW CYTOMETRY is much more difficult using indirect
staining methods,due to the potential for interaction
between multiple second-step conjugates and the
primary antibody TARGETS.
Some of the more common FLUOROCHROMES used
in FLOW CYTOMETRY today are listed in Table 1. Not all
of these are compatible with all cytometers, or even
with all other FLUOROCHROMEs,as can be seen by their
excitation and emission spectra (see for example
www.bdbiosciences.com/spectra). Other considera-
tions of relative brightness and compatibility with
certain experimental parameters (intracellular ver-
sus surface staining, for example) will also steer the
choice of certain FLUOROCHROME conjugates over oth-
ers. For a more complete discussion of the issues of
designing multiparameter experiments, see refer-
ences [18, 19].
Cell types
FLOW CYTOMETRY can be performed on any cell type
that can be rendered into a single-cell suspension.
Since blood cells already exist in this state, they have
been most widely studied by the technique. Howev-
er, adherent cells or tissues can also be analyzed if
they are dissociated from each other and/or their
SUBSTRATE using either enzymatic (e.g., trypsin) or
non-enzymatic (e.g., EDTA) treatments. Where possi-
ble, non-enzymatic dissociation protocols are prefer-
able, because they do not cleave cell-surface pro-
teins that might be TARGETS of the flow cytometric
analysis.
Erythrocyte lysis
Whole blood consists of relatively HOMOGENEOUS ery-
throcytes, and a much more complex collection of
leukocytes. The latter can be stained with FLUO-
ROCHROME-conjugated antibodies in the context of
whole blood, but analysis of whole blood is difficult
because of the light-scattering properties of the ery-
throcytes, which are so numerous as to obscure the
illumination of the leukocytes. Fortunately, erythro-
cytes are differentially sensitive to hypotonic lysis,
and can be removed by incubation of blood with
ammonium chloride in water. Leukocytes are more
resistant to osmotic damage than erythrocytes,
which cannot exclude this salt, and subsequently
take up water and are lysed.Typically, whole blood is
treated for 10 minutes with a large volume of ammo-
nium chloride solution either before or immediately
after staining with FLUOROCHROME-conjugated anti-
bodies. An alternative to ammonium chloride lysis
involves hypotonic lysis in the presence of a fixative
(e. g., formaldehyde), whereby the erythrocytes are
preferentially lysed while LEUKOCYTES are fixed in a
single incubation of 10 minutes or so. Solutions for
 Components/construction of assays 189

TABLE 1. COMMONLY USED FLUOROCHROMES FOR ANTIBODY-COUPLED FLOW CYTOMETRY

Fluorochrome Type of molecule Typical excitation laser Approximate emission peak

Fluorescein isotyocyanate (FITC) Small organic 488 nm 518 nm

AlexaFluor 488 Small organic 488 nm 518 nm
Phycoerythrin (PE) Protein 488 or 532 nm 574 nm
PE-Texas Red Protein tandem 488 or 532 nm 615 nm
PE-Cy5 Protein tandem 488 or 532 nm 665 nm
Peridinin chlorophyll protein (PerCP) Protein 488 or 532 nm 676 nm
PerCP-Cy5.5 Protein tandem 488 or 532 nm 695 nm
PE-Cy7 Protein tandem 488 or 532 nm 776 nm
Allophycocyanin (APC) Protein 633 nm 659 nm
AlexaFluor 647 Small organic 633 nm 667 nm
AlexaFluor 700 Small organic 633 nm 718 nm
APC-Cy7 Protein tandem 633 nm 784 nm
Pacific Blue Small organic 405 nm 454 nm
AmCyan Protein 405 nm 487 nm

this procedure are commercially available. After
lysis/fixation, the cells may be directly analyzed by
FLOW CYTOMETRY (“no-wash” assays); or they may be
subjected to washing to remove the residual red cell
debris and unbound fluorochrome-conjugated anti-
bodies before analysis (“washed” assays). Washed
assays typically allow better signal-to-noise discrimi-
nation of fluorescently stained populations, and bet-
ter visualization of lymphocyte scatter properties.
However, no-WASH-ASSAYS have become popular in
the clinical marketplace because of their simplicity;
and by detecting cells based on threshold staining
for a common leukocyte antigen,such as CD45,these
assays can adequately resolve subpopulations of
lymphocytes.
Density gradient separation
As an alternative to erythrocyte lysis of whole blood,
mononuclear LEUKOCYTES (lymphocytes and mono-
cytes) can be directly isolated from whole blood prior
to staining. Solutions of high molecular weight carbo-
hydrates, such as FICOLL, are used for this separation,
which is accomplished on the basis of density [20].
By underlaying whole blood (usually diluted 1:1 with
buffer or tissue culture media) with a similar volume
of a FICOLL solution, a density gradient is formed.This
biphasic solution is then subjected to centrifugation
(typically at about 400 x G for 15-20 minutes). Erythro-
cytes and granulocytes, which have the greatest buoy-
ant density, are pelleted to the bottom of the FICOLL
layer. Lymphocytes and monocytes, which are less
dense, collect at the interface of the plasma and
FICOLL,where they are collected by pipetting.Platelets,
the least dense leukocytes, will remain in the plasma
layer. Successive centrifugation of the mononuclear
cells collected from the interface is then carried out
at 250 × G to remove residual FICOLL and further
deplete the mononuclear cells of platelets.
Density gradient separation techniques are a stan-
dard, albeit time-consuming method for the isolation
of mononuclear cells from small to large volumes of
blood. However, simpler alternatives are also available
that consist of a gel matrix pre-dispensed into a blood
collection tube or centrifuge tube. By adding whole
blood and centrifuging at a prescribed speed, the
mononuclear cell layer can be collected from the top
of the gel interface, while erythrocytes and granulo-
cytes are forced through the gel plug. Such systems
190 Flow cytometry
are available from commercial vendors, and provide
generally equivalent results with greater convenience
than the FICOLL separation technique [21].
Activation
As described briefly above, functional assays are
those in which the cell types of interest are stimulat-
ed in vitro and allowed to manifest some kind of
response, the quantity and quality of which is meas-
ured in the flow cytometer. Lymphocyte activation is
a particularly valuable class of functional assays,and
typically requires specific antigen or polyclonal
MITOGEN as a stimulus. A commonly measured
response to this stimulus is CYTOKINE production,
which can be detected in a time period of as little as
4 hours (for IFN-γ,IL-2,TNF-α,and IL-4) [22].Visualiza-
tion of CYTOKINE production is accomplished with the
aid of a secretion inhibitor, such as brefeldin A [23]
or monensin [24], which allows for intracellular
accumulation of the CYTOKINES where they can be
specifically stained after cell permeabilization. Typi-
cal antigen-activation protocols involve 6 hours of
stimulation in the presence of brefeldin A (for pep-
tides or MITOGENS),or 6 hours of stimulation with pro-
tein antigens, the last 4 hours in the presence of
brefeldin A [25]. The latter protocol allows for pro-
teins to be processed and presented by MHC mole-
cules prior to inhibition of the secretory pathway by
brefeldin A [26].Activation is carried out at 37 °C,but
can be terminated by cooling the cells prior to fur-
ther processing [22]. Thus, programmeable water
baths or similar devices can be used to automatical-
ly time the activation process, even if it is initiated at
the end of a workday.
Because activation of lymphocytes can induce
adhesion molecule expression, a brief treatment
with EDTA at the end of the activation period is rec-
ommended to dislodge adherent cells from the cul-
ture vessel [26].
Fixation/permeabilization
For staining of intracellular EPITOPES such as CYTO-
KINES, cells are first fixed (usually with a formalde-
hyde-based buffer) and then permeabilized (usually
with a detergent-based buffer). Commercial reagents
containing fixatives and detergents are readily avail-
able and allow for reproducible fixation and perme-
abilization protocols.
Cell staining
For activation assays designed to measure CYTOKINE
production,intracellular staining is required; but cell-
surface staining is usually also performed in the
same sample to allow phenotyping of the respond-
ing cell population. Depending upon the antibodies
and EPITOPES targeted, this surface staining can some-
times be done together with the intracellular
CYTOKINE staining, if the targeted EPITOPES are resistant
to the conditions of fixation and permeabilization
[22, 26]. If they are not, a surface staining step is car-
ried out after activation but before fixation and per-
meabilization.
For most EPITOPES, surface and/or intracellular
staining can be done by incubation with a cocktail
of FLUOROCHROME-conjugated antibodies for 30–60
minutes at room temperature.Titration of antibodies
is required for optimal staining (although many man-
ufacturers offer pre-titred antibodies and cocktails of
antibodies). The optimal titre for surface staining
(unfixed cells) is often different from that for intra-
cellular staining (after fixation and permeabiliza-
tion).
As noted above, simple surface staining of whole
blood can be done using a no-WASH-ASSAY format.But
more complex assays, such as intracellular CYTOKINE
staining, require washing, both after fixation and per-
meabilization steps, and after antibody staining.
Increasing automation and throughput
The relative complexity of sample preparation, espe-
cially for the more complex functional assays, has
inhibited their application to studies of large num-
bers of animals or large clinical trials. However,
robotic sample preparation devices are available
and more are in development that would significant-
ly aid in this process. For example, robotic worksta-

tions are commercially available that can aliquot
whole blood into staining tubes, add antibodies,
incubate, and add erythrocyte lysis buffer. There are
also workstations that can perform cell washing, and
thus can automate most of the steps of a washed
assay. Integration of these robotic workstations can
not only increase the number of samples handled in
these applications, but can also lend considerable
standardization to processes that otherwise are high-
ly operator-dependent.
Truly high-throughput sample processing, howev-
er, is best accomplished with multiwell plates. Both
phenotypic and functional assays can be performed
in 96-well plates; in fact, protocols for activation, pro-
cessing, and analysis of samples for intracellular
CYTOKINE staining in a single 96-well plate have been
published [15]. With the availability of plate-based
loaders for flow cytometers, much larger numbers of
samples can be processed in a single run, with mini-
mal incremental technician time. Further automa-
tion of plate-based sample processing can also be
accomplished using robotic workstations. For exam-
ple, a robotic workstation has been described that
integrates a plate-based liquid handling robot (for
dispensing cells and reagents) with an incubation
station, plate-based indexing centrifuge, and a flow
cytometer equipped with a plate loader [27].
Transfer of plates to and from the various stations is
done with a robotic arm, and software controlling
the robotic arm also integrates the various compo-
nents. Further development of such systems will
broaden the ability to use FLOW CYTOMETRY for highly
parallel studies potentially involving thousands of
samples.
Data acquisition and analysis
Although sample preparation can be made parallel
using multiwell plates and automation, sample
acquisition is still a serial process. Fortunately, tube-
and plate-loaders are available that can automate
acquisition, and software can be used to annotate
data files before they are run.
Data analysis involves the setting of “gates”or regions
in one- or two-dimensional data space, followed by
further analysis of the fluorescence properties of
Components/construction of assays 191
cells within those gates.Typically, viable cells of inter-
est are first identified by their light scatter properties,
using forward and SIDE SCATTER parameters. This may
be followed by gating on subsets of lymphocytes, for
example, CD3+ (T cells), CD19+ (B CELLS), etc. Subsets
of T cells (CD4+,CD8+) may be identified through fur-
ther gating. In the case of functional assays, a respon-
sive population, e.g., CYTOKINE-positive, is quantified
from the subset of interest, e.g., CD8+ T cells.
The PRECISE placement of fluorescence and light
scatter gates is historically done by eye, based upon
recognition of typical patterns of staining (negative,
dim, bright, etc.). However, cluster-finding algorithms
are available in some current FLOW CYTOMETRY soft-
ware programmes that allow these gates to be drawn
in a semi-automated way, and to be responsive to
changes from one data file to another [15]. Such
changes might include slight differences in staining
intensities in different donors, between different
CYTOKINE antibodies used, etc. By using such cluster-
finding algorithms, a gating template can be con-
structed that can then process all the data of a given
experiment without requiring visual checking of
each data file. The ungated “raw” data is stored, and
the gating template can be applied to the raw data
files either contemporaneously with data acquisi-
tion, or in a batch analysis mode at the end of data
acquisition. If data analysis strategies change as a
study unfolds, the raw data can be subsequently
reprocessed through revised gating templates. This
requires considerably less operator time than tradi-
tional methods of data analysis in which the user
places gates manually and checks/adjusts them for
each data file, then has to re-do all the work manual-
ly if strategies change.
A final component of data analysis that is often
overlooked is the incorporation of flow cytometric
data into a database that may also link it with other
types of measurements (patient clinical data, etc.).
Fundamentally, this requires the ability to extract
measurements of interest from the FLOW CYTOMETRY
data files into a spreadsheet along with any annota-
tion associated with the data files.This can be done
with modern FLOW CYTOMETRY software packages,
such that manual entry of data into a spreadsheet is
not necessary.This is a fundamental requirement for
192 Flow cytometry

FIGURE 2. CD4 T CELL ENUMERATION ASSAY

Cells and counting beads are successively gated as described in the text, and the percentages and absolute counts of
various lymphocyte subsets are automatically reported by the analysis software.

any large studies using FLOW CYTOMETRY as one of
their components.
Examples and their application
Immunophenotyping assays
An example of an IMMUNOPHENOTYPING assay to quan-
tify T cell subsets is shown in Figure 2. Note that this
was done as a “no-wash” assay in four colours, along
with fluorescent beads to allow absolute counting.
Fifty microlitres of whole blood were stained with
antibodes to CD45, CD3, CD4, and CD8. The blood
was subjected to fixation/erythrocyte lysis, then run
on a four-colour flow cytometer. An ACQUISITION
THRESHOLD was set on CD45 fluorescence, in order to
identify leukocytes. All cells above this threshold
were collected, with acquisition halted at 20,000
CD45+ events.
Analysis of this assay was done as follows.An ini-
tial gate was set on CD3+ cells, in order to identify all
T cells. CD4+ and CD8+ T cells were then quantified
from a plot gated on CD3+ cells.Note that this sample
also contained counting beads which are identified
by very high fluorescence in all colours. Because a
known number of these beads was added to a
known volume of the blood sample, a simple calcu-
lation can convert the percentages of each cell sub-
population to an absolute count of cells per micro-

litre of blood. Since percentages are relative to other
populations, absolute counts have become a stan-
dard readout for reporting these types of results.
Commercial software packages can perform these
calculations in an automated fashion for this partic-
ular application.
Functional assays
An example of a functional assay (identifying intra-
cellular CYTOKINE production) is shown in Figure 3.
This assay was done by stimulation of whole blood
with peptides derived from human cytomegalovirus
(HCMV),a common herpesvirus that causes chronic,
latent infection of various tissues. HCMV causes non-
pathological infection in immunocompetent hosts,
but is an opportunistic pathogen in immunocompro-
mised hosts [28].
The peptides used for stimulation in this assay
were a mixture of 138 15-mers, overlapping by 11
amino acid residues each, and spanning the
sequence of the pp65 glycoprotein of HCMV. Pp65 is
an immunodominant protein which contains EPI-
TOPES that stimulate CD4 and CD8 T cell responses in
a variety of HLA haplotypes [29]. By using such over-
lapping peptides,both CD4 and CD8 T cell responses
can be stimulated with relative efficiency [25]. The
blood was stimulated with this peptide mixture for
six hours in the presence of brefeldin A, followed by
EDTA treatment, fixation/erythrocyte lysis, permeabi-
lization, and surface/intracellular staining. The anti-
bodies used included anti-IFN-γ,CD69,CD4,and CD3.
Because this was a washed assay, it was not neces-
sary to use a reagent like CD45 for fluorescence trig-
gering. Instead, a threshold was set on FORWARD SCAT-
TER to eliminate small debris,and an acquisition gate
was set around the cluster of small lymphocytes in
forward versus SIDE SCATTER. Acquisition was stopped
when 40,000 CD4+ T cells were collected.
Analysis of this assay was done as follows.An ini-
tial gate was set on CD3+ lymphocytes in a CD3 ver-
sus SIDE SCATTER plot. Secondary gates were set on
CD3+CD4+ and CD3+CD4– cells, respectively. From
each of the latter gates, plots of CD69 versus IFN-γ
expression were displayed,and cells positive for both
of these activation markers were quantified. All of
Examples and their application 193
these gates were set using a cluster-finding algorithm
that allows the gate to automatically shift with the
population from sample to sample [15]. An excep-
tion is the final region quantifying IFN-γ+CD69+ cells,
which was a user-defined rectangle, but which was
“tethered” to the negative population, so that it too
would shift in response to changes in the back-
ground level of fluorescence from sample to sample.
In this way,even very rare populations of IFN-γ+CD69+
cells could be quantified,whether they formed a rec-
ognizable cluster or not.
If desired, one could combine the information
from the above two assays, in order to express the
HCMV pp65-responsive CD3+CD4+ or CD3+CD4– cells
as an absolute number of cells per microlitre.Again,
this might provide more standardized results in pop-
ulations that have varying numbers of CD4 and CD8
T cells (such as HIV patients).
What is the usefulness of identifying and quantify-
ing intracellular CYTOKINE responses? One major
application is the development of new vaccines (see
Chapter C.1) that are designed to elicit cellular
immunity [30, 31]. These include both prophylactic
and therapeutic vaccines for HIV, cancer, and certain
other viral and intracellular bacterial pathogens.
Establishing biomarkers of immunogenicity of vac-
cines (and eventually surrogate markers of protec-
tion) would greatly facilitate the comparison of differ-
ent vaccine constructs and strategies,and allow more
rapid improvement of vaccines for these diseases.
In the area of immunotoxicology (see Chapter
D.1), quantification of functional T cell responses,
whether to specific antigens or to MITOGENS, could
also provide valuable information [30]. While
IMMUNOPHENOTYPING can potentially uncover gross
changes in cellular subsets in response to a drug,
functional assays can uncover much more subtle
changes. Suppression (or augmentation) of antigen
or MITOGEN responses could give important clues to
immunological side-effects of drugs. This could be
done as part of the early screening of drug candi-
dates, before expensive animal studies are undertak-
en. Functional FLOW CYTOMETRY assays could also be
used to monitor the dosing of immunomodulatory
drugs in settings such as transplantation and autoim-
munity [32, 33]. By standardizing, automating, and
increasing the ease and throughput of these assays,
194 Flow cytometry

FIGURE 3. CYTOKINE FLOW CYTOMETRY ASSAY

(A) Cells are initially gated to identify CD3+CD4+ and CD3+CD4– lymphocytes (the latter population being substan-
tially equivalent to CD8+ T cells). (B) Functional responses from each of these populations are then quantified as the
percentage of the gated population that is positive for both CD69 (a surface marker of activation) and IFN-γ (a T cell
cytokine). The response from an antigen-stimulated sample (top plots) is then compared to that from a control, unstim-
ulated sample (bottom plots). Note that even a very low percentage of positive cells, e.g., 0.1%, can be easily differ-
entiated from the control, when the percentage of positive cells in the control sample is low enough, as in these exam-
ples. The statistical significance of the difference between control and test samples can also be calculated [34].

they can become ever more potent tools for
immunological research, immunotoxicology, and
clinical medicine.
Summary
FLOW CYTOMETRY is a powerful technique for analyz-
ing cells in suspension, and has been extensively
applied to the analysis of lymphocytes and their sub-
sets. Flow cytometric assays can be divided into
IMMUNOPHENOTYPING assays and functional assays,the
latter requiring in vitro stimulation of cells in order to
read out a response, such as CYTOKINE production.
Such assays are being applied to the monitoring of
clinical disease states and responses to vaccination
or immunomodulation,and they have great potential
to be used in measuring immunotoxicology as well.
Selected readings
Givan AL (2001) Flow cytometry: First Principles. J. Wiley,
New York
Robinson JP (ed) (1999) Current Protocols in Cytometry
(Current Protocols Series). J.Wiley, New York
Purdue cytometry bulletin board: http://www.cyto.purdue.
edu/hmarchiv/cytomail.htm (Accessed November
2004)
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