Flow Cytometry

Mario Roederer, David R Parks, Leonore A Herzenberg and Leonard A Herzenberg,

Department of Genetics, Stanford University, Stanford, California, USA

History

By the time the prototype of the fluorescence-acti-vated cell sorter (FACS) went into operation (at Stanford in the early 1970s), immunofluorescence studies with the light microscope had already demonstrated that lymphocytes could be broadly subdivided according to whether they expressed surface immunoglobulin (Ig) or surface Thy-1. These visually-defined subpopulations, which we now know as B cells and T cells, were presumed to be responsible for different lymphocyte functions; however, the lack of methods for cleanly separating one subpopulation from the other confounded attempts to establish this point.

During the next few years, while the FACS was undergoing its initial metamorphosis into a commercially-available single-laser instrument, a variety of physical and immunocytotoxic methods were developed that could selectively deplete B cells, T cells or T cell subpopulations from a cell suspension; for example, passage through nylon wool, passage through an affinity column, or complement-dependent killing with antibodies to distinctive cell surface molecules. These depletion methods have proven extremely useful; however, they are mainly restricted to depleting unwanted cell types from a population and thus do not provide for the specific isolation of subpopulations, which is necessary for definitively characterizing their functions.

FACS-based cell isolation methods, in contrast, are well suited to this task, as they provide the potential for sorting and testing viable subpopulations. A significant advantage over mechanical separation methods is that the populations isolated by FACS are usually highly pure. Whereas mechanical separations usually result in purities of 60-90% (with the remaining cells being unwanted subsets), single FACS-separation runs can achieve over 99% purity -with re-sorting resulting in essentially 100% purity. In addition, the electronic control of the sorting in FACS gives the researcher the ability to sort only a single, viable cell - the quickest and most efficient cell cloning technology extant.

The major advantage of FACS-based separations, however, resides in the multiparametric nature of the measurement. A wide array of characteristics can be used, individually or in combination, to define sub-populations. For example, cell size, surface pheno-type, mitotic stage, and intracellular levels of enzymes and metabolites. Thus, as the capabilities of this newly-developed instrument became known, it found increasing use in studies of the functional and developmental relationships among subpopulations of lymphocytes, as well as other cells.

In the last decade, the power of the FACS has been utilized for a much greater range of samples than leukocytes. All kinds of unicellular organisms, from bacteria to yeast to paramecia, have been separated by FACS. This technology has also been applied to the separation of subcellular particles: for instance, sorting endosomes, lysosomes or mitochondria. In addition, the FACS has been used to separate individual eukaryotic chromosomes - a technique that has been pivotal in the establishment of chromosome-specific DNA libraries, an important tool in the human genome project.

In recent years, the application of FACS to biology has taken another significant turn - that of single-cell functional characterization. A variety of assays have been developed to quantitate the functional capacity of individual cells by flow cytometry. These include assays for proliferation or cell cycle progression (e.g. total DNA, RNA and protein contents, as well as number of divisions measured by dyes permanently retained by DNA or cellular membranes), activation (e.g. expression of activation markers on the surface of cells), cytokine expression, apoptosis, calcium mobilization, acidification of internal compartments, changes in reduction-oxidation potential, metabolic activity and various enzymatic activities.

The power of these assays by FACS comes through two unique capabilities. First, since the assay is single-cell based, the resulting measurements provide a view of the distribution of activities within a population, rather than an average across the entire population. Since few biological processes are homogeneous across all cells, this view can be critical in assessing the true functional nature of cells. Second, the functional assays can be combined with standard immunofluorescence assays to phenotype the cells;

thus, the functional quantitation can be restricted to any desired subpopulation of cells.

While such functional assays are still reserved primarily for the basic researchcr, more and more of them are finding clinical utility. As the instrumentation and data analysis becomes routinely automated, these assays progress into clinical settings, such as prognosis in human immunodeficiency virus (HIV) disease. Over the next 10 years, many of these single-cell FACS-based functional assays will become routine in the clinic because they provide information that cannot be obtained by standard bulk assays.

Elucidation of lymphocyte subpopulations by FACS

Much of the early work centered around the characterization of B cell phenotypes, functions and precur-sor-progeny relationships. The first significant FACS studies in these areas, published in 1972, demonstrated that antigen-binding cells are precursors of cells that secrete antibodies reactive with the bound antigen. Other studies, begun shortly thereafter, demonstrated that the surface Ig molecules expressed on B cells are restricted to individual allotypes and isotypes and that these surface Ig molecules reflect the allotype and isotype commitment of the cell and its antibody-producing (plasma cell) progeny.

By 1976, sorting and transfer studies with Ig allotype congenic mice placed this basic description of 'allelic exclusion' in its current context by showing that all Ig heavy chains expressed by an individual B cell or its progeny are encoded on the same Ig heavy chain chromosome (haplotype). These findings, which provided the operational and theoretical framework for a large series of subsequent B cell development studies, also laid the groundwork for current molecular and FACS studies defining the genetic organization of the Ig heavy chain (IgH) chromosome region and the mechanisms involved in IgH gene rearrangements and class (isotype) switching.

FACS contributions to T cell studies during this period were less dramatic, largely because of difficulties encountered in obtaining antibodies (and hence FACS reagents) that specifically detected individual cell surface antigens. At this time, antibodies for use in FACS (and other) studies were prepared from conventional antisera, which typically contain a mixture of antibodies reactive with the target antigens and an abundance of irrelevant Ig that can stick nonspecifically to cells. Anti-Ig reagents, such as those used in the B cell studies cited above, were relatively easy to prepare because lg-specific antibodies can be bound to (and eluted from) secreted Ig coupled to an insoluble matrix. Specific reagents for detecting surface determinants other than Ig, however, were substantially more difficult to produce, as they could not readily be isolated by binding and elu-tion methods. By and large, these reagents had to be produced from antisera (or fractionated Ig) from which contaminant antibodies were removed by absorption with appropriate cells. Thus, FACS T cell work proceeded slowly until the late 1970s, when monoclonal antibodies were introduced as FACS staining reagents.

The development of monoclonal reagents for detecting the major T cell surface determinants in mouse and humans went hand in hand with the development of FACS methods for identifying and sorting the major T cell subpopulations. In essence, the availability of highly specific and easily purified antibodies that could be readily coupled with fluorochromes made it possible to use the quantitative expression of cell surface antigens to recognize lymphocyte (and other) subpopulations and to sort these subpopulations to chart their functions.

Initial studies with this monoclonal-based methodology defined the murine Ly-1 (CD5) and Lyt-2 (CD8) lymphocyte surface antigens and characterized the expression of these antigens on T cell sub-populations in mouse spleen, lymph node and thymus. These FACS characterizations were used to discover monoclonal antibodies that detected the human Leu-1 (CDS) and Leu-2a (CD8) homologs of the murine genes (Figure 1). In addition, these early studies used these (and other) monoclonal antibodies to identify and characterize the so-called 'helper/inducer' and 'suppressor/cytotoxic' subsets of human T cells.

At present, nearly all FACS studies characterizing the expression of cell surface antigens are conducted with monoclonal antibody reagents. The shift to this virtually exclusive use of these reagents occurred extremely rapidly and was accompanied by an equally rapid expansion in FACS utilization, particularly in studies of human and murine lymphocytes. Surface phenotypes for helper and suppressor T cell subsets were further defined and new subpopulations within these subsets were identified. B cell FACS studies also prospered and marked advances were made in clinical FACS application. In one striking example, leukemia phenotyping opened the way to a rationalization of treatment protocols that substantially improved patient survival. In another dramatic instance, measurement of helper and suppressor sub-population frequencies in normal human adults laid the groundwork for the current widespread use of such measurements as an index of disease pro-

Figure 1 Two-parameter FACS: Leu-2a and Lyt-2 expression on (A) human and (B) mouse lymphoid cells. Early single-color FACS analysis, in conjunction with biochemical studies, demonstrated that human Leu-2a, which is found only on suppressor/cytotoxic cells, is expressed in a very similar pattern as the murine Lyt-2 surface antigen. The Leu-2a antigen was detected by indirect staining with SK1 monoclonal antibody supernatant followed by fluorescein-conjugated goat antimouse lgG1. The Lyt-2 antigen on mouse cells was detected by direct staining with fluorescein-conjugated 53-6.7 monoclonal antibody. Both of the determinants are found on 80-90% of the thymus cells (light lines) and 20-40% of the spleen T cells (dark lines). These antigens are now known as CD8.

Figure 1 Two-parameter FACS: Leu-2a and Lyt-2 expression on (A) human and (B) mouse lymphoid cells. Early single-color FACS analysis, in conjunction with biochemical studies, demonstrated that human Leu-2a, which is found only on suppressor/cytotoxic cells, is expressed in a very similar pattern as the murine Lyt-2 surface antigen. The Leu-2a antigen was detected by indirect staining with SK1 monoclonal antibody supernatant followed by fluorescein-conjugated goat antimouse lgG1. The Lyt-2 antigen on mouse cells was detected by direct staining with fluorescein-conjugated 53-6.7 monoclonal antibody. Both of the determinants are found on 80-90% of the thymus cells (light lines) and 20-40% of the spleen T cells (dark lines). These antigens are now known as CD8.

gression in acquired immune deficiency syndrome (AIDS).

Multiparameter FACS studies

Methods for labeling antibodies with different fluorochromes (e.g. fluorescein and rhodamine) were developed while the FACS was still in its infancy. Fluorescence microscope studies demonstrated rhat these 'differently-colored' antibodies could be independently detected on the same cell and thus could be used to distinguish subpopulations on the basis of qualitative or semiqualitative correlations in the expression of two or more surface antigens. In essence, these studies showed that lymphocyte sub-populations distinguished initially by the expression (or nonexpression) of one surface marker could be further subdivided according to the expression (or nonexpression) of a second marker, etc.

Developing an array of fluorescent dyes for reagent labeling and efficient FACS systems that could capitalize on the availability of differently-colored antibody reagents produced with these dyes took some time. Early FACS instruments were equipped with a basic two-color capability that allowed quantitative evaluation of the expression of pairs of cell surface markers on individual cells; however, these initial multiparameter studies were severely restricted by the limited dye combinations, the single-laser FACS instruments and the simplistic computer support available at the time. In fact, studies with these early instruments basically provided a tantalizing glimpse of how valuable multiparameter FACS methods could be; relatively little was actually accomplished with these methods until the introduction of the dual-laser FACS, the phycobiliproteins (phycoerythrin (PE) and allophycocyanin (APC)) and the sophisticated FACS software that together characterize the multiparameter analysis and sorting methods in use today.

Strictly speaking, multiparameter FACS studies

(defined as the measurement of more than one parameter per cell) began with the prototype FACS, which was built with two sensors: one for fluorescence, and the other for low-angle light scatter ('forward scatter'). The forward scatter measurement on this initial instrument was intended solely to signal the arrival of a cell-sized object in the light path; however, studies with cell populations from lymphoid organs soon showed that the forward scatter signal could also be used as a meaningful measure of

Flow Scatter Immune Cells

Figure 2 Fluorescence spectra of molecules conjugated to antibody reagents. The excitation and emission spectra of eight fluorescent molecules that can be conjugated to immunoglobulins and used in immunofluorescence studies are shown. All eight can be simultaneously and independently measured. Dashed lines, excitation spectra; solid lines, emission spectra. Spectra are uncorrected for detector sensitivity and are scaled for presentation purposes. Also shown are the excitation lines from the 406 nm krypton line, the 488 nm argon line, and the 595 nm dye laser line. Finally, the stippled boxes represent the transmission areas for each of the filters used to detect that fluorescent molecule by FACS.

Figure 2 Fluorescence spectra of molecules conjugated to antibody reagents. The excitation and emission spectra of eight fluorescent molecules that can be conjugated to immunoglobulins and used in immunofluorescence studies are shown. All eight can be simultaneously and independently measured. Dashed lines, excitation spectra; solid lines, emission spectra. Spectra are uncorrected for detector sensitivity and are scaled for presentation purposes. Also shown are the excitation lines from the 406 nm krypton line, the 488 nm argon line, and the 595 nm dye laser line. Finally, the stippled boxes represent the transmission areas for each of the filters used to detect that fluorescent molecule by FACS.

cell size and could distinguish, for instance, lymphocytes from monocytes. In addition, the forward scatter signals were shown to be useful for discriminating live cells from dead cells, and for excluding ('gating out') dead cells during analysis and sorting. Thus, as the FACS came into use initially for biological studies, two parameters - forward scatter and fluorescence - were routinely measured and recorded for individual cells.

The introduction of methods for measuring two additional parameters - large-angle light scatter ('side scatter') and a second fluorescence color -moved FACS measurements closer to the current definition of multiparameter studies, i.e. those in which at least two fluorescence measurements and one light scatter measurement are taken per cell. Most flow cytometers are now capable of measuring forward and side scatter as well as at least three fluorescence colors. State of the art instrumentation at Stanford is capable of detecting the two scatter signals as well as eight different, independently-quantifiable fluorescence measurements. The advance to such ten-parameter, three-laser instrumentation has required development efforts at a multitude of levels: 1) chemistry - the continuing development of fluorescent dyes with spectra suitable for flow cytometry (e.g. excitation by commonly avail-

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