Journal of Immunological Methods 243 (2000) 77–97
`
`www.elsevier.nl/locate/jim
`
`A practical approach to multicolor flow cytometry for
`immunophenotyping
`
`b ,*
`a ,1
`Nicole Baumgarth , Mario Roederer
`aDepartment of Genetics, Stanford University Medical School, Beckman Center B-007, Stanford, CA 94305-5318,USA
`bDepartment of Stomatology,UCSF, San Francisco, CA,USA
`
`Abstract
`
`Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to
`simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced
`multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They
`provide a large amount of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell
`populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving
`crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such
`identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry
`comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful
`results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and
`artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the
`11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one
`fluorescent dye is used.
`2000 Elsevier Science B.V. All rights reserved.
`
`Keywords: Flow cytometry; Immunofluorescence; Immunophenotyping; Multiparameter flow cytometry
`
`1. Introduction
`
`Abbreviations: APC, allophycocyanin; A595, Alexa 595; CasB:
`Cascade Blue; CasY, Cascade Yellow; Cy5.5APC, Cy7APC,
`Cy5.5, Cy7 conjugates of allophycocyanin; Cy5PE, Cy5.5PE,
`Cy7PE, Cy5, Cy5.5, and Cy7 conjugates of phycoerythrin;
`Cy5.5PerCP: Cy5.5 tandem conjugate of PerCP; FITC, fluores-
`cein; PBP: phycobiliprotein (i.e., PE or APC); PE, phycoerythrin;
`PerCP: peridinin chlorophyll protein; PFC, polychromatic flow
`cytometry; PMT, photomultiplier tube; TR, Texas Red; TRPE,
`Texas Red-conjugated phycoerythrin; SA, streptavidin
`*Corresponding author. Tel.: 11-415-514-0395; fax: 11-415-
`476-4204.
`E-mail address: roederer@drmr.com (M. Roederer).
`1
`Present address: Center for Comparative Medicine, University
`of California, Davis, CA 95616, USA.
`
`Since the earliest application of flow cytometry to
`the study of cells, there has been a drive to increase
`the number of distinct measurements for each cell.
`This developmental effort blossomed in the late
`1990s, when the number of independently measur-
`able ‘colors’ (each color corresponds to a distinct
`fluorescence-based measurement of a cellular protein
`or function) increased from five to 11 (Roederer et
`al., 1997; Bigos et al., 1999).
`The success of this developmental effort was due
`to the coordinated development of new hardware,
`new fluorochromes, and new software analysis tools
`
`0022-1759/00/$ – see front matter
`PII: S0022-1759( 00 )00229-5
`
`2000 Elsevier Science B.V. All rights reserved.
`
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`
`that significantly increase the quality and quantity of
`measurements. This increase comes with a price,
`however, as this new technology has its own set of
`technical problems and difficulties that users must be
`aware of and must overcome in order to derive
`meaningful results. Nonetheless, once these hurdles
`are overcome, this new technology is well worth the
`effort, as the information obtained from the measure-
`ments is not only novel but could not be obtained
`otherwise using standard flow cytometric techniques.
`As we outline below, setting up multicolor flow
`cytometry is not simply achieved by adding new
`reagents to existing reagent combinations, but re-
`quires a more involved process of quality control,
`optimization, and ‘debugging’. Therefore,
`to dis-
`tinguish multicolor flow cytometry with its unique
`benefits and technical problems from current stan-
`dard flow cytometric technologies (i.e., using four or
`fewer fluorescent dyes), we refer to it as ‘poly-
`chromatic flow cytometry’ (PFC) and will use the
`term throughout this manuscript.
`Flow cytometers capable of collecting data for
`more than three or four colors are now becoming
`more prevalent, as manufacturers have recognized
`the significant demand for the types of analysis
`afforded by this technology. Given the bewildering
`array of fluorochromes, lasers, hardware, and soft-
`ware that might be used in PFC, we outline here the
`fundamental requirements,
`interactions, and prob-
`lems associated with setting up this technology, so
`that users can make educated decisions about instru-
`ment requirements and the design of their experi-
`ments. Finally, we provide some examples in which
`this technology has been of particular benefit. We
`hope to provide with this brief review some practical
`tips and encouragement for those thinking of expand-
`ing their current flow cytometric measurements. The
`benefits of true multicolor flow cytometry make this
`technique a particularly useful and probably soon an
`irreplaceable tool for the study of cell biology and
`immunology.
`
`2. Fluorochromes
`
`The ability to measure multiple parameters of each
`cell is limited by the number of fluorochromes that
`
`can be simultaneously measured. The 11-color PFC
`that is currently in routine use at Stanford uses dyes
`excited by three different laser lines. The excitation
`and emission spectra of these dyes and the filters that
`were chosen to collect the emitted light from these
`dyes are shown in Fig. 1.
`
`2.1. Characteristics of useful fluorochromes
`
`When designing experiments for the flow cytome-
`ter that include the use of new dyes, careful consid-
`eration must be given to the choice of fluorochromes.
`Desirable fluorochromes for cytometric technologies
`have several properties:
`they (i) are biologically
`inert;
`(ii) have high cell-associated fluorescence
`intensities (‘bright’); (iii) exhibit little spectral over-
`lap amongst each other; and (iv)
`for
`immuno-
`phenotyping, are easily conjugated to monoclonal
`antibodies.
`
`2.1.1. Biological inertness
`Most of the fluorochromes that are currently in use
`are biologically inert:
`i.e.,
`they do not affect
`the
`cells, nor do they bind directly to cellular elements.
`There are, however, exceptions to this:
`the most
`common example is that of the ‘background’ binding
`of Cy5PE (and other Cyanine–PBP tandem dyes) to
`monocytes and B cells. This background binding is
`variable between species and can be extremely high
`in some instances. For example, Cy5PE binds strong-
`ly to B cells in mice with autoimmune disorders
`(e.g., non-obese diabetic mice). While there are
`methods available to reduce this background, it is of
`concern when the particular cell types being studied
`are those that
`interact
`‘nonspecifically’ with the
`fluorochrome.
`
`2.1.2. Cell-associated fluorescence intensity
`With regard to the high fluorescence intensities or
`the ‘brightness’ of a fluorochrome, it should be noted
`that the characterization of a fluorescence signal as
`‘bright’, i.e., the difference between the unstained
`and the stained cells,
`is still empirical. ‘Bright’
`signals result from fluorochromes with the following
`characteristics: (1) a high extinction coefficient; (2) a
`high quantum yield; (3) an emission spectrum over-
`lapping as little as possible with cellular auto-
`
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`79
`
`Fig. 1. Shown are excitation and emission spectra of the 11 dyes that we currently use for polychromatic flow cytometry (PFC). Indicated
`are the wavelengths of the different laser lines used for their excitation (407, 488 and 595 nm; other laser lines that can be used for some of
`the dyes are shown for reference), and the filters chosen for optimal light collection and minimal spillover. The fluorochromes, laser lines
`and filters are currently in routine use on a modified three-laser hybrid Becton-Dickinson/Cytomation flow cytometer, described previously
`(Roederer et al., 1997; Bigos et al., 1999).
`
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`fluorescence; (4) measurable with high sensitivity
`detectors; and (5) the ability to conjugate multiple
`fluorochromes to each detecting unit (e.g., a high
`ratio of fluorochrome to antibody).
`The differences in brightness can be quantitated
`experimentally by conjugating the same monoclonal
`antibody to the various fluorochromes at optimized
`ratios. Fig. 2 shows histograms of human peripheral
`blood lymphocytes that were stained with 11 differ-
`ent conjugates of an anti-CD8 monoclonal antibody,
`one conjugate for each of the 11 fluorochromes that
`we currently use in PFC. As shown in this figure, the
`‘brightness’ is affected not only by the intensity with
`which the positive cells are stained, but also by the
`background of the negative cells. Although some of
`the conjugates appear to stain much ‘brighter’ than
`
`others, all of these fluorochromes are useful for
`clearly distinguishing the positively stained cells
`from the unstained cells.
`It is important to point out that the brightness of a
`fluorochrome will differ depending on the instrument
`used. For example, conjugates of antibodies to PE
`generally result in much brighter staining when cells
`are analyzed with typical benchtop cytometers using
`flow cells as compared to cytometers using jet-in-air
`detection. This is due to a number of differences in
`the optical paths that affect the efficiency with which
`emitted light is collected by the detectors; however,
`other differences (notably the laser power) also
`influence sensitivity. Therefore, reagent ‘brightness’
`is a relative measurement and must be assessed by
`users on any given instrument.
`
`Fig. 2. An anti-human CD8 monoclonal antibody was conjugated at optimal fluorochrome–antibody ratios to each of the indicated
`fluorochromes (Roederer, 1997a,b). Human peripheral blood lymphocytes, purified by gradient centrifugation over Ficoll, were stained with
`each of the indicated anti-CD8 conjugates and with an antibody to the pan-T cell marker CD3, conjugated either to FITC (for CasB and
`CasY graphs) or to CasB (all others). Shown are histograms of cells that stained positively for CD3. The dashed line indicates levels of
`staining for cells stained with anti-CD3 only, and the solid line indicates the anti-CD8 fluorescence for cells stained with both antibodies.
`Empirically, the relative ‘brightness’ of any of these fluorophores can be estimated based on the separation of CD8 positive and negative
`cells. The apparent difference in the number of cells between the positive and negative peaks for these dyes is a visual artefact of the scaling
`(note that the ‘CD8-dim’ cells appear to be more prevalent in those plots where the CD8 peak is higher). There was no difference in the
`percent dim or bright-positive cells for any of distributions.
`
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`81
`
`2.1.3. Spectral overlaps
`The spectral overlaps that exist between dyes
`currently represent the biggest hurdle in PFC analy-
`sis. Due to the similarities and overlaps in the
`emission spectra of different dyes (see Fig. 1), it is
`not possible to choose emission filters that uniquely
`measure only one of
`the dyes in a multicolor
`experiment. The appropriate choice of filter, how-
`ever, can greatly reduce collection of light from
`other fluorochromes (i.e., reduce ‘spillover’). Note
`from Fig. 1 that a set of up to four dyes excited by
`three different
`lasers can be chosen that exhibit
`essentially no spectral overlap; thus, four- or fewer-
`color experiments can be carefully designed to avoid
`the problems caused by spillover. However,
`the
`necessity of having three lasers for a four-color
`experiment renders this an impractical solution.
`Because of
`the spectral overlap, each fluoro-
`chrome will contribute a signal to several detectors,
`therefore the contribution in detectors not assigned to
`that fluorochrome must be subtracted from the total
`signal in those detectors. This process, termed ‘com-
`pensation’, is discussed in detail below.
`
`2.1.4. Conjugation to antibodies
`For many applications of flow cytometry, a further
`requirement for a fluorochrome is that
`it can be
`readily conjugated to antibodies. The 11 dyes we
`commonly use (Figs. 1 and 2) meet this criterion.
`There are, however, varying degrees of difficulty
`involved in carrying out the conjugation reactions. It
`is likely that any laboratory carrying out flow
`cytometric analyses with five or more dyes will need
`to conjugate antibodies, as commercial conjugates
`are not yet available for a number of these dyes.
`Even if all of the dyes become available as antibody
`conjugates, it is unlikely that every combination of
`antibody and fluorochrome necessary for each user
`will be available.
`Detailed procedures for conjugating the dyes can
`be found on the web (Roederer, 1997a) or from
`commercial vendors who supply conjugation kits.
`Briefly, the procedures for conjugating these dyes
`fall into three classes. (1) Small organic molecules
`(FITC, TR, A595, A430, CasB, and CasY) are
`conjugated to antibodies in relatively simple, short
`reactions that
`require only one or
`two column
`separations (or other desalting procedures). (2) Sin-
`
`gle protein molecules (PE, APC) require a slightly
`more complex procedure, but reactive fluorochromes
`can be easily prepared, are stable for long periods of
`time, can be used in a simple conjugation procedure.
`2
`(3) Tandem dyes
`(Cy5PE, Cy5.5PE, Cy7PE,
`Cy5.5APC, Cy7APC, Cy5.5PerCP), require the use
`of more complex procedures which involve first the
`generation of the reactive tandems that can then, in a
`second step, be used for conjugation to antibodies.
`Generating the reactive tandems requires careful
`testing of different conditions for each batch of
`tandem dye to optimize the ratio of the two dyes
`used. However, once the chemistry has been opti-
`mized, a large batch of reactive dye can be prepared
`for use in many conjugations.
`
`2.2. Other fluorochromes.
`
`New fluorochromes for immunophenotyping appli-
`cations are constantly being developed. At the time
`of writing, several dyes exist
`that are useful for
`immunophenotyping applications and which can be
`used in addition to or instead of some dyes listed in
`Fig. 1.
`
`2.2.1. PerCP
`This is a chlorophyll-like protein that can be
`directly conjugated to antibodies. It has an emission
`spectrum similar to Cy5PE, but with no excitation in
`the red, and hence very little spillover into the APC
`detector compared to Cy5PE. On the other hand, it
`has several disadvantages: it is easily ‘bleached’ by
`lasers, limiting its utility to low-power instruments
`such as benchtop instruments; it is less bright than
`Cy5PE; and it is only available in conjugates from a
`single vendor (Becton-Dickinson, San Jose, CA).
`
`2.2.2. Cy5.5PerCP
`A relatively new tandem conjugate of PerCP that
`has excitation and emission spectra very similar to
`Cy5.5PE. Unlike PerCP, it can be used in high power
`laser instruments as it does not ‘bleach’ easily, but is
`also only available in conjugated form through
`Becton-Dickinson.
`
`2
`
`Cy5PE is the same as ‘Tricolor’, Cy7APC is the same as
`‘Allo-7’ and ‘PharRed’, and Cy5.5PerCP is the same as ‘TruRed’.
`
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`2.2.3. Alexa 430,595
`A series of new dyes termed ‘Alexa dyes’ from
`Molecular Probes (Eugene, OR) includes two that we
`have tested for their usefulness in PFC. A430 is an
`excellent replacement for CasY, in that it has less
`non-specific interaction with CasB and is otherwise
`roughly similar in ‘brightness’ (Baumgarth, unpub-
`lished). A595 is spectrally similar to Texas Red (TR)
`but does not have its non-specific binding charac-
`teristics, leading to a somewhat better separation of
`positive and negative signals than that seen with
`many TR conjugates.
`
`2.2.4. TRPE
`This dye, also called ‘Red 613’ or ‘ECD’ is
`excited by a 488 nm laser line and can replace
`Cy5PE in a multicolor staining combination, as it has
`considerably less overlap into the APC channel than
`Cy5PE. It is however less bright than Cy5PE and it
`has somewhat more overlap into the PE channel; it
`also displays considerably higher background bind-
`ing typical of TR conjugates.
`There are a number of other dyes that are com-
`monly used to directly stain cellular components
`such as DNA (e.g., Hoechst, propidium iodide),
`cellular membranes (e.g., CFSE, nile red), lysosomes
`(e.g., CFDA), and dyes that can measure metabolic
`activity of the cell (e.g., rhodamine 123). It is beyond
`the scope of this manuscript to cover fluorochromes
`that are not used for immunophenotyping. For a
`detailed description of these therefore refer to (Sha-
`piro, 1994). Importantly, however, the use of any
`combination of dyes, irrespective of the measure-
`ment for which it is used, will need to address the
`same issues such as reagent ‘brightness’, spillover,
`and compensation as described here for antibody
`conjugates.
`
`2.3. Fluorochrome combinations
`
`Out of the dozen or more fluorochromes that can
`be used for immunophenotyping (by virtue of their
`spectral and chemical properties discussed above), it
`is important
`to select
`the best combination for a
`particular experiment under consideration. This is
`true whether the experiment requires four or 11
`colors. To a large extent, the constraints on which
`fluorochromes can be used will depend on the
`
`availability of laser lines on the machine in use (i.e.,
`is a 595-nm dye laser line available, or a 407-nm
`violet-enhanced krypton laser line?). The useable set
`of dyes also depends on the number of detectors that
`are available on the instrument to collect the excited
`light (i.e., how many colors can be collected that are
`excited by the first, second, and third laser line?).
`Once those constraints have been determined, there
`can still be a wide range of possible combinations.
`Table 1 lists a number of such combinations, given
`some constraints of laser and detector availability
`often seen in currently available machines.
`
`3. Hardware
`
`3.1. Lasers
`
`One of the single largest component costs of
`cytometry instrumentation is the cost of the lasers.
`Newer diode lasers are becoming prevalent and these
`can be significantly cheaper than the older gas ion
`lasers. When considering a solid-state laser,
`it
`is
`important to choose one that has a long life span and
`provides a consistent power output, which therefore
`excludes the very cheap solid-state lasers that are
`currently available. In addition, for current stream-in-
`air instrumentation it is desirable to have at least 50
`mW of power for each laser line in use, since the
`fluorescence signal (and thus sensitivity) increases
`with laser power. For most purposes,
`it
`is not
`necessary to have much more power
`than this,
`because most fluorochromes will saturate (are maxi-
`mally excited) at 100–150 mW. Indeed, additional
`power may actually reduce relative signal to back-
`ground staining, because the fluorophore will satu-
`rate at much lower powers than background fluores-
`cence. Table 2 lists some of the currently available
`lasers and the lines that are often used in flow
`cytometry, their approximate costs and the dyes that
`they can excite.
`
`3.2. Optical setup
`
`requires
`Sensitive detection of fluorochromes
`selection of appropriate filters that are placed before
`each detector, or photomultiplier tube (PMT). Filters
`must be selected so as to collect as much emitted
`
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`83
`
`Table 1
`Sample fluorochrome combinations
`
`Dyes
`
`a
`
`4
`
`5
`
`6
`
`7
`
`Lasers
`
`b
`
`2 (4881647)
`
`1 (488)
`
`2 (4881595)
`
`2 (4881632)
`
`1 (488)
`
`2 (4881633)
`
`2 (4881595)
`
`1 (488)
`
`2 (4881632)
`
`2 (4881595)
`
`Combinations
`
`c
`
`FITC, PE, Cy5.5PE or Cy7PE, APC
`FITC, PE, Cy5PE, Cy5.5APC or Cy7APC
`FITC, PE, Cy5PE, APC
`FITC, PE, TRPE, APC
`FITC, PE, Cy5PE, Cy5.5PE or Cy7PE
`
`FITC, PE, Cy5.5PE or Cy7PE, TR, APC
`FITC, PE, Cy5PE, TR, Cy5.5APC or Cy7APC
`FITC, PE, Cy5PE, TR, APC
`FITC, PE, Cy7PE, APC, Cy5.5APC
`FITC, PE, Cy5.5PE, APC, Cy7APC
`FITC, PE, Cy5PE, Cy5.5APC, Cy7APC
`FITC, PE, Cy5PE, APC, Cy5.5APC or Cy7APC
`FITC, PE, Cy5PE or Cy7PE, APC, Cy7APC
`FITC, PE, Cy5PE, Cy5.5PE, Cy7PE
`
`FITC, PE, Cy5.5PE, Cy7PE, APC, Cy5.5APC or Cy7APC
`FITC, PE, Cy5PE, Cy5.5PE or Cy7PE, Cy5.5APC, Cy7APC
`FITC, PE, Cy5PE, Cy5.5PE or Cy7PE, APC, Cy5.5APC or Cy7APC
`FITC, PE, Cy5.5PE, Cy7PE, TR, APC
`FITC, PE, Cy5PE, Cy7PE, TR, Cy5.5APC
`FITC, PE, Cy5PE, Cy5.5PE, TR, Cy7APC
`FITC, PE, Cy5PE, Cy5.5PE or Cy7PE, TR, APC
`FITC, PE, Cy5PE, TRPE, Cy5.5PE, Cy7PE
`
`FITC, PE, Cy5.5PE, Cy7PE, APC, Cy5.5APC, Cy7APC
`FITC, PE, Cy5PE, Cy5.5PE or Cy7PE, APC, Cy5.5APC, Cy7APC
`FITC, PE, Cy5.5PE, Cy7PE, TR, APC, Cy5.5APC or Cy7APC
`FITC, PE, Cy5.5PE or Cy7PE, TR, APC, Cy5.5APC, Cy7APC
`
`a Any of the listed combinations can be augmented with a 407 nm Krypton laser and the fluorochromes CasB and CasY (or A430) to
`increase the number of colors by two without significantly affecting compensation requirements.
`b Number of lasers, followed by excitation lines (see footnote a).
`c Combinations are listed in rough order of increasing quality of measurements. i.e., for any given set of combinations, the first ones will
`tend to have lower compensation requirements and higher brightness. There are many more possible combinations than can be listed in this
`table. The order does not take into account commercial availability or ease of conjugate preparation. Substitutions: PerCP can be substituted
`for Cy5PE on lower laser-power instruments; Cy5.5PerCp can be substituted for Cy5.5PE on any system; A595 can be substituted for TR on
`any system. TRPE could be added to most combinations not employing 595 lasers or used as a substitute for Cy5PE. Where two dyes are
`listed as ‘A or B’, either can be used in the combination listed, but ‘A’ is preferred for reasons of brightness or compensation.
`
`light from the primary fluorochrome for high sen-
`sitivity, but as little as possible from other fluoro-
`chromes to reduce the compensation required. In
`general, these two criteria work against each other;
`therefore, for any given set of fluorochromes used in
`an experiment, the optimal filter set may be different.
`However, we have found that the filter set shown in
`Fig. 1 and Table 3 is close to optimal for many
`experimental setups that are currently in use.
`A further
`important selection criterion is the
`ability to block scattered light from the laser lines.
`Modern filters are considerably better than the old
`
`the FITC
`filters, allowing for example to collect
`signals with a much wider bandpass filter than was
`previously possible (old, 530/30; new, 525/ 50)
`while still blocking scattered light from the 488-nm
`laser line.
`For experiments using a small subset of these
`fluorochromes, we found that using the widest
`possible bandpass that still excludes as many other
`fluorescences as possible yields the best
`results.
`Nonetheless, switching to wide-open filters will
`generally only increase the measured signal by 20–
`100% compared to the bandpass filters shown in
`
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`Table 2
`Lasers for flow cytometry
`
`a
`
`Laser type
`
`Argon Ion
`
`Cooling
`
`Air
`
`Krypton
`
`Water
`
`Violet-enhanced
`b
`Krypton ion
`
`c
`
`Dye
`
`Doubled Nd-YAG
`
`HeNe
`
`Diode
`
`Water
`
`Water
`
`Air
`
`Air
`
`Air
`
`Line (nm)
`
`Approx. cost
`
`Fluorochromes
`
`488
`
`568
`
`647
`
`4071413
`
`595
`
`532
`
`632
`
`635
`
`$10K
`
`$30K
`
`$50K
`
`$15K
`
`d
`
`$12K
`
`$6K (50 mW)
`
`$1K (10 mW)
`
`FITC, PE, TRPE, PerCP,
`Cy5PE, Cy5.5PE, Cy5.5PerCP,
`Cy7PE, A488
`
`PE, TRPE, PerCP, Cy5PE,
`Cy5.5PE, Cy5.5PerCP, Cy7PE,
`A568, TR, A595
`APC, Cy5APC, Cy5.5APC,
`Cy7APC (Cy5PE, Cy5.5PE,
`e
`Cy7PE)
`
`CasB, CasY, A430
`
`TR, A595, APC, Cy5,
`Cy5.5APC, Cy7APC (TRPE,
`e
`Cy5PE, Cy5.5PE)
`
`PE, TRPE, PerCP, Cy5PE,
`Cy5.5PE, Cy5.5PerCP, Cy7PE
`
`APC, Cy5, Cy5.5APC,
`
`Cy7APC (Cy5PE, Cy5.5PE)
`
`e
`
`a Except as noted, lasers are rated at 50 mW output or greater at the given line.
`b 405-nm diode lasers with sufficient power for cytometry will be available soon; currently, 4-mW lasers cost about $4K.
`c Currently, 5-mW HeNe lasers with a 594-nm line can be obtained for about $3K; this power level is probably too low except for
`bench-top (flow cell) cytometers.
`d A dye laser also requires a 5-W, 488-nm pump laser, adding another $20K to the cost of this laser. However, the 488-nm line of the
`pump laser (running in ‘all lines’ mode) can be split off and used as the primary laser beam in the instrument, obviating the need to purchase
`the primary Argon laser.
`e Dyes in parentheses will also be excited by this laser, although their primary excitation is by other laser lines.
`
`Table 2. For flow cytometric experiments, 2-fold
`increases in signal-to-background levels are marginal
`compared to the optimizations that can be achieved
`in other parts of the experiment.
`
`4. Compensation
`
`4.1. What is compensation?
`
`Compensation is the process by which the spectral
`overlap between different fluorochromes is mathe-
`matically eliminated. The algorithm of compensation
`is a straightforward application of linear algebra, and
`should not be thought of as a subtraction process.
`Compensation between detectors can be performed
`either by hardware, after signal detection but before
`
`logarithmic conversion and /or digitization, or post-
`collection by software.
`While compensation is one of the most important
`processes required for proper data analysis in flow
`cytometry, it is also perhaps the least well under-
`stood. In order to properly design, implement, and
`analyze multicolor experiments, users must be aware
`of the effects of compensation, understand how to
`apply it correctly, and recognize when data are not
`properly compensated. While we will discuss some
`of these topics here in brief, the reader is strongly
`encouraged to read the texts (Roederer, 1999) and
`web pages (Roederer, 1997b) devoted to this topic
`for more in-depth information.
`
`4.2. Compensation complications
`
`Currently, most instruments provide the capability
`
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`

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`
`85
`
`Table 3
`Optimal interference filters for fluorochromes
`
`a
`
`Fluorochrome
`
`CasB
`CasY, A430
`FITC
`PE
`Cy5PE
`PerCP
`Cy5.5PE, Cy5.5PerCP
`Cy7PE
`TRPE, TR, A595
`APC
`Cy5.5APC
`Cy7APC
`
`Filter
`
`b
`
`440/40
`545/90
`525/50
`575/25
`665/30
`680/30
`720/45
`785/45
`625/40
`660/40
`705/50
`d
`750LP
`
`c
`
`d
`
`c
`
`a Optimization is a function of maximizing the amount of signal
`collected while minimizing the amount of spillover from other
`fluorochromes. Different filters may be optimal for particular
`applications; see text.
`b Filters are given as ‘x/y’, where x is the wavelength in
`nanometers of the center of the bandpass and y is the width in
`nanometers of the band. LP, long pass filter. Filters have an extra
`coating to block scattered light.
`c
`Use of a 720/45 filter for Cy5.5PE is optimal when Cy5PE is
`also to be measured. For measuring Cy5.5PE (or Cy5.5PerCP) in
`the absence of Cy5PE, then a 705/ 50 is better. Because APC has
`a lower emission wavelength than Cy5PE, the 705/50 works well
`for Cy5.5APC in combination with APC. However, if Cy5.5APC
`is to be measured with Cy5, then the 720/45 filter should be
`selected.
`d
`The 750LP and the 785/50 are roughly equivalent
`collecting Cy7 emission; either can be used.
`
`for
`
`for compensating between several spectrally adjacent
`pairs of detectors: for example, between FITC and
`PE, and between PE and Cy5PE. For a two-color
`analysis, such pairwise compensation is sufficient
`and complete. However, the introduction of a third
`fluorochrome introduces the potential for complex
`interactions that cannot by fully corrected by such
`pair-wise compensations. For example, FITC has low
`but measurable emission into the Cy5PE detector.
`This overlap may not be completely corrected by the
`the FITCfi PE and PEfi Cy5PE
`combination of
`compensation settings, causing what appears to be
`artefactual Cy5PE fluorescence when using a very
`bright FITC stain. Obviously, as more than three
`colors are used, these problems increase.
`Indeed, for a typical 11-color stain, one would
`potentially need to use up to 100 pair-wise com-
`pensation controls to fully compensate all colors (in
`
`reality only about 40 of these are non-zero). Further-
`more, because of the multiple interactions between
`these controls, some settings may actually be less
`than zero; something that current hardware controls
`do not allow. Finally, since setting interacting pair-
`wise compensation controls is an iterative process, it
`would take virtually forever to manually set them
`properly.
`An additional complication to compensation is
`brought about by the use of tandem dyes (e.g.,
`Cy5PE, Cy7APC, etc.). Tandem dyes are covalently
`linked combinations of ‘donor’ (e.g., PE) and ‘accep-
`tor’ (e.g., Cy5) dyes. The ratio of acceptor to donor
`is usually in the range of 3:1 to 10:1, depending on
`the optimization strategy used by the manufacturer.
`In fact, different tandem-conjugated antibodies from
`the same manufacturer often have slightly different
`ratios of acceptor to donor. The problem is that the
`compensation required to correct tandem emissions
`in other fluorescence channels strongly depends on
`this ratio. Therefore, a different compensation setting
`may be needed for every different tandem conjugate
`used in a particular experiment. In essence,
`this
`means that
`the compensation setting needs to be
`adjusted for every different staining combination in
`use.
`that
`is apparent
`it
`Given these complications,
`complete compensation on multicolor systems re-
`quires computer intervention. Currently no instru-
`ment is commercially available that provides users
`with the ability to automatically adjust compensation
`settings on a per-stain basis, nor even to provide
`complete compensation among all pairs of fluores-
`cence detectors. Therefore, the only solution as yet is
`the use of software analysis programs that are
`applied after data collection is completed for com-
`pensation of the data. Examples of such software
`include WinList (for PC) and FlowJo (for Macin-
`tosh).
`The use of software to set compensation simplifies
`the process considerably. Computers can set
`the
`compensation correctly (there is no subjective criter-
`ion applied) and can be instructed to appropriately
`vary the compensation matrix according to the
`specific fluorochromes used in any given sample.
`However, there are significant limitations to software
`compensation (see below). In the interim before
`complete automated hardware compensation is avail-
`
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`
`able, the optimal solution is to combine both hard-
`ware and software compensation to minimize the
`limitations of each.
`
`4.3. Limitations of compensation
`
`Both available hardware and software compensa-
`tion have significant limitations that place restrictions
`on the utility of PFC. Hopefully, some of these
`limitations will soon be obviated by new technology
`developments, which would enable the easier set-up
`of new reagent combinations for multicolor flow
`cytometric measurements. Until then, it is important
`to understand these limitations and how they may be
`best addressed.
`One limitation that is shared by both hardware and
`software compensation is due to the low precision of
`measurements in the far-red channels, Cy7APC,
`Cy5.5APC, and to a certain extent APC. In these
`channels, for dimly stained cells, the actual number
`of photoelectrons detected by the PMT can be very
`low, due to the inefficiency of the currently available
`PMT to detect light at this wavelength. Even the
`‘deep red-sensitive’ PMTs now available do not
`overcome this problem. The unavoidable counting
`errors inherent in the measurement can lead to the
`introduction of significant artefacts. For example, if
`only five photoelectrons are collected, the counting
`]˛
`error is
`5, or 45%. This error propagates to other
`channels that require compensation from this detec-
`tor. The result is that the ‘broadening’ of populations
`that occurs due to compensation (Roederer, 1997b,
`1999) is significantly worse for the far-red emitting
`fluorochromes.
`Hardware compensation operates on the analog
`signals collected from the PMT, before further signal
`processing is done. For detectors with sufficient
`si