1.'
From its e a r k t days US the COulter COUTtter, the flow CytOPneter has evolved into a pOWe?$Xl clinical tool for studying the intrinsic and extrinsic properties of individual cells
phyll) . Extrinsic propertiecare measured using reagents and include surface antigen content; lectin binding; total protein content; DNA, RNA, and chromosomal content; calcium influx; intracellular PH; and nuclear or intracellular antigen content. Applications for flow cytometry range from clinical testing of
Alice Gilman-Sachs Finch University of Health Sciences and The Chicago Medical School 700 A Analytical Chemistry, Vol. 66, No. 13, July 1, 1994
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0 1994 American Chemical Society
hematopoietic cells to sophisticated analysis of chromosome content for basic research. The earliest flow cytometer, the Coulter counter, was used for accurate determination of the number of white or red blood cells, based on electronic impedance measurements as each cell passed the measurement point (I). Today, more sophisticated flow cytometers combine state-of-the-art advances in computers and laser technology to obtain an objective and precise measurement of multiple, characteristic parameters of an individual cell at one time. These parameters include size, volume, granularity (refractive index), and fluorescence caused by interactions of specific probes with cell surface antigens or cytoplasmic molecules. Flow cytometers now range from instruments with as many as three or four different lasers designed for research applications (3),which cost as much as $250,000, to clinical instruments with one argon laser, which are less expensive and versatile but can provide rapid and quantitative clinical information (1-7). The use of monoclonal antibodies with flow cytometry for the analysis of hematopoietic cells has greatly advanced the field of diagnostic pathology; new concepts for diagnosis and classification based on quantitative measurements of cellular parameters and the expression of specific differentiation antigens on the surface of cells have been established (1, 3,5,8).In this Report, a description of the principles of flow cytometry and a brief overview of the major applications of flow cytometry will be presented. Principles of flow cytometry
A flow cytometer has several components, including a light source, usually a laser; a sample chamber, flow cell, and sheath
fluid stream; a photodetector or photomultiplier tubes (PMTs) that collect light and convert it to electronic signals; a signal processing system that converts analog signals to digital signals; and a computer to direct operations, store the collected signals, and display data (Figure 1) (I).When a cell passes in front of the laser beam, the light scattered or the fluorescence emitted from the cell is converted to an electronic signal that is proportional to a specific parameter for that cell. The information from a population of cells is displayed on a computer screen as a frequency histogram. To analyze cells, a preparation of single cells is suspended in a stream of fluid (usually saline). The cells are then hydrodynamically focused into the center of a surrounding sheath fluid by passage through a narrow orifice of a flow cell specially designed to produce a laminar flow. The cells pass in front of an extremely narrow coherent monochromatic beam of laser light; the intersection of the cells in the center of the stream and the laser beam must be precisely aligned to obtain the correct information from the analysis. The most commonly used laser is an argon laser, which can emit a very narrow elliptical beam of light ranging from 16 x 40 pm to 40 x 700 pm, depending on the beam-shaping optics used in the flow cytometer (6).This laser can be tuned, using a set of prisms at each end, to emit wavelengths of light ranging from 365 to 514 nm. The wavelength most often used is 488 nm, the excitation wavelength for two common fluorochromes. Although the earliest flow cytometers used powerful (5 W) water-cooled Ar lasers, in recent years less powerful (15 mw) air-cooled lasers have proved satisfactory for many applications. Other types of lasers (HeNe, Kr, dye, and He-Cd), however, can be
used in combination with certain dyes for special applications, as shown in Table 1 (7).
When a cell passes in front of the elliptically shaped narrow laser beam, light is scattered in all directions. Depending on the direction in which the light is scattered and detected by strategically placed photodetectors (either photocells or PMTs), information about a designated cellular parameter is obtained (Figure 1). For instance, it has been shown empirically that light scattered by cells at small angles (0.5-2.0") in the forward direction (FALS, or forward angle light scatter) to the intersection of the sheath stream and the laser is proportional to cell size. Small cells do not scatter as much light as larger cells do. Similarly, light scattered and collected at right angles to the intersection of the sheath fluid stream and the laser is proportional to the granularity or internal complexity of the cell. Therefore, cells with fewer granules or less internal structure do not scatter as much light at 90" (90" LS, or light scatter) as do cells with more granules. Likewise, cells with dim fluorescence do not emit as much light as cells with bright fluorescence. FALS and 90" LS are used to measure intrinsic properties of cells (i.e., size or granularity). Fluorescence emitted by cells is usually attributable to the interaction of cells with dye or fluorescent antibodies and is used to measure extrinsic properties such as surface antigen or DNA content (2,5,8). The amount of light falling on each PMT or photocell is converted by each photodetector into a proportional electronic signal that can be observed as an electronic pulse on an oscilloscope. The pulse height, which is proportional to the light, is measured and converted by analog-to-digital converters into a number
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Figure I.Main components of a flow cytometer. The PMTs shown at 90" to the intersection of the sheath fluid stream and the laser detect either light proportional to granularity (PMT-90" light scatter) or fluorescence (PMT- 1 = green, PMT-2 = orange, PMT-3 = red), depending on the optical filter assembly in front of each of them. (Adapted with permission of Coulter Corp.)
for display on a frequency histogram. The greater the amount of light scattered or fluorescence emitted by a cell, the higher the electronic pulse signal and consequently the larger the digitized signal (number). The more sophisticated the flow cytometer, the more photodetectors and ultimately the more parameters that can be measured for each cell. Photocells (used to convert light scatter) or PMTs (used to convert fluorescence or 90" LS)can be used to measure light scatter proportional to the size, granularity, or fluorescence of each cell; some advanced instruments can be used to measure as many as six parameters at once on a single cell (1,6, 7). To quantitate the information obtained for a population of cells, these digitized signals or numbers are displayed on a o n e parameter frequency histogram. A finite number of channels (usually 256) for each parameter is displayed on the x-axis and indicates increasing fluorescence, size, or granularity, depending on the parame-
ter being determined (Figure 2). The number of cells in each of the 256 channels is displayed on the y-axis. The electronic signal (either the pulse height or an integrated pulse area) is measured for each cell and then converted to a number ranging from 0 to 255, the range of channels on the frequency histogram, by an analogto-digital converter connected to each photodetector. After the digitized number is obtained for each cell, individual cells are tallied in one of the 256 channels of the frequency histogram. Over a very short time (some flow cytometers can analyze up to 10,000 cells per second), a one-parameter histogram is displayed in real time (i.e., while the specimen is running through the flow cytometer) on the computer screen. By simple integration and the use of various computer programs, it is possible to quantitate the number of cells in a population that are positive for that parameter. A flow cytometer can detect and measure low levels of fluorescence on the sur-
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face of or within cells. The x-axis of a oneparameter histogram used to measure fluorescence intensity can be either a log or a linear scale. When measuring bright fluorescence, a linear scale is often used; however, a log scale is used for dim fluorescence. The decision to use either a log or a linear scale depends on the application (6). The example described above is based on the analysis of a single parameter. Multiparameter analysis for a population of cells is a more powerful tool because heterogeneous populations of cells can be resolved into subpopulations based on two or more parameters such as size and granularity (1).In these more complex analyses, two parameters can be combined for analysis and displayed at the same time. Each cell plotted on the histogram is designated by a dot (Figure 3). The denser or darker the population on a two-parameter histogram, the more cells present. With multiparameter analysis and sophisticated computer programs, a particular subset of cells can be selected from a heterogeneous population on the basis of one or two parameters, such as size and cytoplasmic granularity, and the expression of other parameters in this population can be determined (1).This process, called gating or mapping, allows for the rapid and objective analysis of subsets of cells without having to physically isolate that population of cells. The numerous probes used in flow cytometry range from fluorochromes conjugated directly to monoclonal or polyclonal antibodies, such as fluorescein (FITC) or phycoerythrin (PE) ,to stains that bind specifically to a particular cellular component, such as propidium iodide (PI), which binds specifically to DNA in the presence of RNase within the nucleus of cells [Table 1, (9)].After each probe is excited by the light at its excitation wavelength, it returns to the ground state by emitting light of a longer wavelength; this emission is then detected as fluorescence (6). As mentioned above, an air-cooled Ar laser tuned to emit light at 488 nm is commonly used in most clinical flow cytometers; it is relatively inexpensive compared with the more powerful water-cooled laser and can be used with the FITC, PE, and PI fluorescent probes. Each probe is
excited at 488 nm and emits photons of longer but distinct and separable wavelengths-FITC emits at 530 nm, PE at 570 nm, and PI at 630 nm. Each emitted wavelength can be collected by using a series of optical filters placed in front of the path of emitted light to separate and direct light of the appropriate wavelength to the designated PMT for that fluorescent probe (Figure 1) (6, 7). Practically speaking, some factors must be recognized for optimal analysis of cells by flow cytometry. First, the intersection of the laser beam and the cells within the center of the sheath fluid must be perfectly aligned so that light scatter is not lost and an optimal electronic signal is determined by the PMT. Usually this alignment is accomplished by using a preparation of latex beads coupled to fluorochromes (FITC, PE) as a control sample. These beads, which simulate what is commonly seen when cells are analyzed by flow cytometry, must be used daily to maximize excitation, ensure that emitted light is collected properly, and reduce the day-to-day variation (10). Second, the cells to be analyzed must be prepared properly. If a monoclonal antibody is used, saturating conditions are necessary when cells are mixed with the antibody. Although this is usually predetermined with a commercial monoclonal
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antibody, in research applications it is important to determine an appropriate concentration with each antibody. In addition, positive and negative controls should be used on all daily runs. In clinical assays for the determination of lymphocytes in a population of white blood cells, a monoclonal antibody that reacts with all lymphocytes is used as a positive control to ensure that the bit map or gate is proper and that all, and only lymphocytes, are in the bit map (11).An isotype matched control, usually IgG conjugated with the appropriate fluorochrome, is used as the negative control. The sample must also be prepared so that fluorescence will not be lost (Le., by staining rapidly and keeping the stained cells in the dark, by fixing the cells to prevent them from losing the fluorescent monoclonal antibody or dye, and by treating the cells to prevent the formation of debris by accidentally lysing some cells). These factors are important for the analysis of clinical specimens (8,11,12)and for research applications (3). When leukocytes from blood are analyzed by flow cytometry, if 90" LS and FALS are displayed on a two-parameter histogram, three populations of white blood cells can be detected (Figure 3). The lymphocyte population consists of the smallest and least granular of the leuko-
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Figure 2. Examples of one-parameter histograms. The x-axis of each histogram consists of 256 channels, each relating to increasing electronic pulse. (a) Negative control of lymphocytes stained with FITC-lgG. (b) Positive assay of lymphocytes stained with FITC-anti-CD4 Ab. The positive cells consist of 53.05% of the lymphocytes.
cytes, whereas the granulocyte population is the largest and most granular. The monocyte population is intermediate in size and granular content. For analysis by flow cytometry, maps can be drawn around each population by using electronic circuitry and special computer software programs. Each population can then be analyzed independently for other parameters such as surface marker expression, using fluorochrome-conjugated monoclonal antibodies, or DNA content, using special stains specific for DNA (1,5, 11).The percentage of cells within each bit map or gate is then determined by integration and comparison with a negative control. In more sophisticated analyses, two color combinations of monoclonal antibodies can be reacted with cells to obtain information such as whether an individual cell in a heterogeneous population expresses two different surface antigens. Many flow cytometers can store the information obtained from one test run and the user can play this information over again simply by using a computer program. This method of storing data on a computer, called list mode, allows a researcher to reanalyze one assay multiple times by either re-gating or using different parameters. By moving a bit map around to improve the resolution, one can obtain
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better data analysis. Most users, however, collect information in real time. The isolation of pure populations of cells, “cell sorting,’’ can also be accomplished with a flow cytometer (13). Any cell that can be identified and measured in the usual manner can be sorted and separated from a mixed population. Cells can either be sorted into test tubes or cloned individually into microtiter plates. In sorting flow cytometers, as the sheath fluid leaves the nozzle, it is vibrated at a high frequency and thus breaks into droplets (Figure 1).Ideally, each droplet contains one cell. When it is determined that a droplet contains the desired cell, a charge is put on the droplet. As the charged droplet passes between two charged plates, it is deflected and sorted to the right or the left into a collection tube. Extremely pure populations of cells may be isolated from heterogeneous populations at the rate of 5000 per second, but only a limited number of cells can be purified: it could take all day to sort millions of cells. Applications
Cellular subsets. The most common application of flow cytometry is determining the percentage of specific subsets of cells in a population using fluorescent monoclonal antibodies. Polyclonal antibodies that recognize human white cells and subsets have been available for a number of years, but the ability to produce mono-
Figure 3. Two-parameter histogram or dot plot of peripheral blood leukocytes. Three populations can be resolved: Map 1, lymphocytes; Map 2, monocytes; and Map 3, granulocytes.
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clonal antibodies to human leukocytes has revolutionized immunohematology and biomedicine. Monoclonal antibodies are the products of the somatic cell hybridization between antibody-forming cells with a finite lifespan and myeloma cells capable of replicating indefinitely in vitro. The successfully fused hybridoma cell line represents an unlimited source of an antibody that is immunologically homogeneous and secretes antibody molecules of the same specificity indefinitely (14). (In contrast, polyclonal antibodies are a heterogeneous mixture of antibodies and differ in specificity from animal to animal and sometimes from species to species.) Monoclonal antibodies to human leukocyte differentiation antigens (CD, or cluster of differentiation,antigens) have been produced in numerous laboratories. They are constantly being exchanged and compared to identify those monoclonal antibodies that react with the same surface molecules on different populations of human leukocytes (14, 15).Monoclonal antibodies that react with the same differentiation antigen on the surface of human leukocytes are assigned to the same CD group (14, 15). At the 5th International Conference on Human Leukocyte Differentiation Antigens in 1993, groups of monoclonal antibodies were assigned to 125 CD designations (15). The CD antigens identified in these 78 clusters are present on lymphocytes, monocytes, granulocytes, and even platelets. In combination with flow cytometry, these monoclonal antibodies allow one to identify and quantitate populations and subpopulations of cells in whole blood or tissues. For instance, monoclonal antibodies to CD4 are used to quantitate CD4 helper T-lymphocytes in patients with AIDS. The decrease in this cell population occurs during the course of infection and is used to determine the health status of patients (11, 12). In 15-30 s, 5000 lymphocytes stained with fluorescent antiCD4 Ab can be analyzed and the percentage with CD4 quantitated. This assay, an immunophenotype, is used to determine whether lymphocytes have normal numbers of cells within a given subset. Just as an immunophenotype is needed for a patient with AIDS, the immunophenotype of blood from an individual with leu-
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kemia is important for classifying the type of leukemia. The immunophenotype for leukocytes in specimens from normal individuals is well characterized (16-19). Patients with acute leukemia have immunophenotypes representing a more primitive cell usually found in bone marrow (16). Chronic leukemia exhibits a more restricted expression of CD antigens than that commonly seen in normal blood (17). The expression of immunophenotypes relates to the type of cell or subset that has become malignant. These findings are used to provide a more definitive diagnosis. Also, lymphoid leukemias are different from myeloid leukemias with regard to the expression of CD antigens; these differences also help in determining treatment and prognosis (18, 19). The detection of surface molecules on cells using a monoclonal antibody is relatively simple and rapid (10). For example, to measure the percentage of CD4 and CD8 lymphocytes in whole blood, a sample (usually 50-100 pL) of whole anticoagulated blood is incubated with saturating amounts of FITC-anti-CD4 antibody and PE-anti-CD8antibody for 10-15 min. The specimen is then washed to remove unreacted antibodies, the red blood cells are lysed, and the remaining white blood cells are examined by flow cytometry. First, a two-parameter histogram based on FALS and 90” LS is obtained. By using multiparameter analysis, a bit map or gate is placed around the smallest and least granular population, the lymphocytes (Figure 3, Map 1).Each lymphocyte in this population is then assayed for green fluorescence (FITC) equal to CD4 expression and for red fluorescence (PE) equal to CD8 expression. The results can be displayed as a one-parameter histogram of either green (FITC) or red fluorescence (PE) alone or as a two-parameter histogram of green versus red fluorescence. A computer program based on the integration of the number of cells in the positive channels is used to determine the percentage of green CD4 and red CD8 cells (Figure 2). Normal values for these lymphocytes are 50%CD4 and 25%CD8. In patients with AIDS the percentage of CD4 lymphocytes may be as low as 1%at the end stage of the illness (20). Monoclonal antibodies give typical patterns of reaction with cells when assayed N
Figure 4. Three-dimensional plot of calcium influx. The influx is measured in lymphocytes pulsed with Indo-1 and incubated with ionomycin, which induces calcium influx. Note the increasing ratio of violet to blue fluorescence on the x-axis with increasing time.
by flow cytometry. Some of these give a peak of bright fluorescence, some give a peak of dim fluorescence, and some give both a bright and a dim pattern (bimodal) with cells. The concept of brightness relating to density of expression can be used to quantitate surface antigens and compare expressions of the same molecule on populations of the same cells from different individuals (21).Usually the comparison is made by measuring the mean channel fluorescence for two different specimens stained under the same conditions. A specimen with a higher density of an antigen will give a higher mean channel fluorescence than a specimen with a lower density of antigen (21). In addition to human leukocytes, monoclonal antibodies are available that recognize cells from many species ranging from the mouse to the pig. These antibodies have been used in combination with flow cytometry to identify and quantitate surface antigens, cytoplasmic molecules, increased expression of receptors following activation of cells, and a myriad of other molecules that can be identified with specific dyes or antibodies. Calcium in$ux. The measurement of intracellular ionized calcium concentrations in living cells using Indo-1 is another valuable application of flow cytometry (22).When cells are activated through ligand binding to surface receptors, calcium influx (an increase in intracellular
calcium concentration) occurs; the influx is thought to represent one signal transduction pathway. Indo-1 dye chelates calcium and exhibits changes in the wavelength of fluorescence emission upon binding to calcium. It is loaded into living cells as an acetoxymethyl ester, which diffuses through the membrane within 1h. The dye can be excited between 330 and 350 nm; its use requires a flow cytometer equipped with either an Ar ion laser or a Kr ion laser that can be tuned to UV lines. In the absence of calcium, the dye emits blue fluorescence (480 nm). Chelated to calcium, the dye emits a violet fluoresence (405 nm) . Light at each wavelength can be detected in individual cells using two different PMTs with the appropriate bandpass filters to collect the signals. The intensity of blue or violet fluorescence is measured as the mean channel fluorescence intensity on a linearly scaled one-parameter histogram. Data for cells that have been stimulated is displayed as a ratio of violet to blue fluorescence for each cell over time. A two-parameter histogram with the ratio of violet to blue fluorescence on one axis and time on the other is used to show whether calcium influx occurs in the population under investigation (Figure 4). The results are quantitated by measuring the percentage of cells exhibiting calcium influx (i.e., those in which the ratio increased over time). Flow cytometers equipped with a second laser make it possible to map one population of cells using monoclonal antibodies that fluoresce green or red (FITC or PE) when excited at a wavelength of 488 nm by an Ar laser and to measure the calcium influx in these same cells with Indo-1 using UV wavelengths from a second Kr laser (23).This technique has been used to study the effects of stimulation of receptors on subsets of cells and the accompanying calcium influx. DNA content. The DNA content of individual cells in solid tissues or cell lines can easily be determined (5,24-28). Single cells must be used; monodisperse preparations of tissues are usually produced by enzymatic digestion. The single cells are then made permeable and stained with a fluorescent dye that binds stoichiometrically to DNA in the nucleus. Cells that are resting (in the GO/G1 phase) bind less dye than cells that have replicated their
DNA and are ready to divide (the G2M phase). Cells that are in the process of r e p licating DNA take up intermediate amounts of stain (S-phase). After the amount of fluorescence in each cell is determined by flow cytometry, a linearly scaled one-parameter histogram is obtained, and the percentage of cells in each phase of the cell cycle, based on dye u p take, is determined (Figure 5). The dye most commonly used to stain DNA is PI, which intercalates into cells in amounts proportional to the amount of DNA present. This dye excites at a wavelength of 488 nm and emits at about 630 nm. A two-color application using PI to measure DNA and an FITC antibody to detect cellular proteins can be performed to examine cell cycle kinetics and associated molecules. PI also binds to RNA, however; therefore, RNase treatment of permeable cells is necessary to digest RNA and eliminate the concomitant measurement of these molecules with the DNA Other DNA binding dyes usually require excitation by UV wavelengths and are not easy to use. Some are specific for certain base pairs; others bind to both RNA and DNA (Table 1). Many tumors are either DNA aneuploid (they have extra chromosomes) or are rapidly proliferating. The measure-
Figure 5.One.parameter histogram to measure DNA content. (a) Tumor cells. (b) Diploid control cells. The cells are stained with propidium iodide to measure DNA content. An aneuploid population is present in the tumor cell histogram but is absent from the control cells.
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ment of the proliferation index (percentage of cells in the Sphase) and the detection of aneuploid populations in tumors provide valuable information that can be used to identify proper treatment for cancer patients and, in some cases, evaluate their prognosis (25-27). In general, tumors that are more aggressive and rapidly cycling have higher percentages of cells in the S-phase and indicate a higher grade malignancy. Tumors that have aneuploid populations (because of the presence of an abnormal number of chromosomes) may also be considered more malignant (25). These abnormalities may be identified using flow cytometric techniques. Tumors are heterogeneous populations of cells. Some cells belong to the malignant population; others are normal. The degree of aneuploidy in a tumor cell population may be expressed as the DNA index (DI). The DI is the ratio of the mean or mode channel of fluorescence in a sample GO/G1 population (tumor cell population) divided by the mean or mode channel of a diploid reference cell population (26). It is important to calculate the DI to avoid confusing abnormal populations with normal cellular populations in the tumor. Although it is relatively easy to determine the DI, calculating the proliferation index or percentage of cells in the Sphase is more complex (25,26). Many algorithms and computer programs can be used to calculate an accurate percentage of the cycling cells in a tumor cell population. However, because both malignant and normal diploid cells are present in most tumors, it is necessary to make the appropriate subtractions and to determine where the GO/G1 peak ends and the Sphase peak begins. It is difficult to make such a determination with complete certainty, especially in tumors with aneuploid populations. One approach to this problem is to pulse growing cells or tumor explants with bromodeoxyuridine (BrdU) (28). Cells will incorporate this molecule into their replicating DNA during the S-phase. These cells are then detected with a FITC antibody to BrdU. By staining cells with PI to label DNA and BrdU incorporation (to detect cycling cells), it is possible to use two-parameter analysis for more accurate determination of which cells are in
the S-phase. It is necessary, however, to pulse cells and sample them periodically for accurate measurement of the cycling cells. Also, this analysis cannot be performed on cells that are dead or incapable of being cultured. Many studies have been performed to correlate the DNA content of different types of tumors with the outcome or treatment (5, 25, 26). These studies have been facilitated by the ability to perform analysis of DNA content on paraffin-embedded tumors. Some of these specimens have been stored for years in pathology laboratories. A retrospective investigation of the relationship between the DNA content in these specimens and outcome and treatment has resulted in new approaches to treatment.
Chromosomes have also been karyotyped with flow cytometry and even sorted to produce chromosome-specific gene libraries (31).After chromosomes are isolated, they.are stained with either PI or DNA stains specific for bases using, for example, chromomycin, which is G-C specific, and Hoechst 33342, which is A-T specific. Although a good karyotype of human chromosomes can be resolved by staining only with PI (one-parameter histogram), better resolution can be obtained by two-parameter analysis using a combination of stains such as chromomycin and Hoechst 33342. This application requires a laser with UV capability. Future directions
New applications for flow cytometry are developed every year. For example, an accurate and objective reticulocyte count can now be obtained by reacting whole blood with an RNA-specific stain, thiazole orange, and quantitating the percentage of reticulocytes containing RNA by flow cytometry. Immunoglobulin bound to the surface of platelets or other cells (red blood cells in blood transfusions or preformed antibodies to leukocytes in organ transplants, such as flow cross-matches) may also be detected. In patients with low platelet counts, platelet-bound immunoglobulin can be determined. The platelets are separated from the red blood cells Recently flow cytometric techniques and then reacted with fluorescein-conjuhave been used to detect and measure the gated antibody to human immunoglobulin. The percentage of platelets with green expression of nuclear proteins during the fluorescence, caused by the reaction of cell cycle. Some, but not all, antibodies to nuclear antigens are suitable for flow cyto- surface-bound immunoglobulin and the FITC indirect reagent, is then determined. metric techniques (29,3U). Thus antibodies to c-myc, TAG, c-mos, p53, c-myb, Many other applications exist for flow PCNA, Ki-67, P105, and others have been cytometry: rare event analysis to measure cells present in amounts < 1%such as feused to identify nuclear proteins in permeable cells. Two-parameter analysis of tal cells in maternal blood, measurement cells that are proliferating, permeable, of the percentage of cells undergoing a p and then stained with FITC-anti-c-mycanti- optosis (programmed cell death, to rebody and PI allows the identification of move unwanted cells during developthe portion of the cell cycle in which ment), determination of drug resistance, c-myc is highly expressed; also, the effect detection of DNA or RNA with fluorescent of activators or other cellular proteins on in situ hybridization, determination of the expression of the nuclear oncogene cell cycle regulation, and monitoring of can be measured with this method. One drugs used for therapy. Advances in flow must be careful, however, to determine cytometry are made each year as new the best method for detecting the oncoprobes and lasers, as well as more powergene product because the conditions used ful computers, become available (32). to fix cells and make them permeable Three- and even four-color applications may result in protein destruction (29). will certainly lead to new applications in
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