Cell Cycle Analysis by Flow and Laser-Scanning Cytometry
The cytometric methods for cell cycle analysis can be grouped into three categories. The first comprises methods that rely on a single time point ("snapshot") measurement of the cell population. This analysis may be either univariate, based on the measurement of cellular DNA content alone (Crissman and Hirons, 1994), or multivariate (multiparameter), when in addition to DNA content another cell attribute is measured (Darzynkiewicz et al., 1996; Endl et al., 2001; Larsen et al., 2001). The measured attribute is expected to provide information about a particular metabolic or molecular feature(s) of the cell that correlates with a rate of cell progression through the cycle or is a marker the cell proliferative potential or quiescence. While the single time measurement reveals the proportions of cells in G1 vs S vs G2/M, it provides no direct information on cell cycle kinetics. However, if duration of the cell cycle (or time of doubling of cells in the culture) is known, the length of G1, S, or G2/M phase can be estimated from the percentage of cells in the respective phase.
In the second category are methods that combine time-lapse measurements of populations of cells synchronized in the cycle or whose progression through the cycle was perturbed, e.g., halted by the agent arresting them at a specific point of the cycle. These methods reveal kinetics of cell progression through the cycle. A classical example of this group is the stathmokinetic approach where cells are arrested in mitosis, e.g., by vinblastine or colcemide, and the rate of cell entrance to mitosis ("cell birth" rate) is estimated from the slope representing a cumulative increase in the percentage of mitotic cells as a function of time of the arrest (Darzynkiewicz et al., 1987).
Methods of the third category rely on analysis of DNA replication concurrent with measurement of DNA content. They may be either single time point measurements or use the time-lapse strategy to measure cell cycle kinetics. Incorporation of the thymidine analogue and the S phase marker 5'- bromo-2'-deoxyuridine (BrdUrd) is detected either cytochemically, based on the use of DNA dyes such as Hoechst 33258, whose fluorescence is quenched by BrdUrd (Poot et al., 2002), or immunocytochemically using fluoresceinated BrdUrd antibodies (Dolbeare and Selden, 1994). Still another method of this category detects incorporated BrdUrd by the increased sensitivity of DNA to photolysis: utilizing terminal deoxynucleotidyl transferase, the photolytically generated strand breaks are then labeled with fluorochrome- tagged deoxynucleotides (Li et al., 1996). Because the latter method escapes the harsh conditions used to induce DNA denaturation (heat or acid), it is applicable in conjunction with immunocytochemical detection of intracellular proteins. The time-lapse measurements of the cohort of BrdUrd-labeled cells allows one to estimate their rate of progression through different points of the cell cycle (Terry and White, 2001).
The methods described in this article, representative of each of the three categories, can be applied both to cells measured by flow cytometry and to cells mounted on slides. The latter can be analyzed by laserscanning cytometer (LSC), the microscope-based cytofluorimeter that measures the fluorescence of individual cells deposited on slides rapidly, with sensitivity and accuracy comparable to that of flow cytometry (Kamentsky, 2001). One advantage of LSC is that the cells are stained and measured while attached to microscope slides. This eliminates cell loss that inevitably occurs due to repeated centrifugations during sample preparation for flow cytometry. Another advantage stems from the possibility of relocation of particular cells on slides for their visual inspection or morphometry, following the initial measurement of a large cell population and electronic selection (gating) of cells of interest. The instrument thus combines advantages of both flow and image cytometry.
Only a few selected methods are presented in this article. More detailed descriptions of these and other methods, their applicability to different cell systems, and advantages and limitations are provided elsewhere in books devoted specifically to the cell cycle (Gray and Darzynkiewicz, 1987; Fantes and Brooks, 1993; Studzinski, 1995, 1999) or flow cytometry (Darzynkiewicz et al., 1994, 2001; Gray et al., 1990).
II. MATERIALS AND INSTRUMENTATION
The materials listed for each of the different procedures can be purchased from the following sources: Triton X-100 (Cat. No. T 9284), Pipes (Cat. No. P 3768), RNase A (Cat. No. R 5000), and 5'-bromo-2'- deoxyuridine (Cat. No. B 5002) are from Sigma Chemical Co.; DAPI (4',6'-diamidino-2-phenylindole; Cat. No. D 1306), propidium iodide (PI, Cat. No. P-1304), and high-purity acridine orange (AO, Cat. No. A-1301) are from Molecular Probes; and methanol-free formaldehyde (Cat. No. 4018) is from Polysciences Inc.
The greatest selection of monoclonal and polyclonal antibodies applicable to cell cycle analysis is offered by DACO Corporation, Sigma Chemical Co., Upstate Biotechnology Incorporated, B.D. Biosciences PharMingen, and Santa Cruz Biotechnology, Inc.
A variety of models of flow cytometers of different makers can be used to measure cell fluorescence following staining according to the procedures listed in this article. The manufacturers of the most common flow cytometers are Becton Dickinson Immunocytometry Systems, Beckman/Coulter Inc., DACO/ Cytomation, and PARTEC GmbH. The multiparameter laser-scanning cytometer is manufactured by CompuCyte Corp.
The software used to deconvolute DNA content frequency histograms, to estimate the proportions of cells in the respective phases of the cycle, is available from Phoenix Flow Systems and Verity Software House.
A. Univariate Analysis of Cellular DNA Content
Progression through S phase and completion of mitosis (cytokinesis) result in changes in cellular DNA content. The cells position in the major phases (G0/1 vs S vs G2/M) of the cycle, therefore, can be estimated based on DNA content measurement. A variety of fluorochromes and numerous methods are used for DNA content analysis. A simple protocol, which can be modified to accommodate different dyes, has been developed and applied to numerous cell types.
1. Cell Staining with DAPI
Staining solution: Phosphate-buffered saline (PBS) containing 0.1% (v/v) Triton X-100 and 1Bg/ml DAPI (final concentrations)
2. Staining with Propidium Iodide
If excitation with UV light is not possible, the procedure given earlier for DAPI can be modified to apply PI as the DNA fluorochrome. Thus, instead of DAPI, PI is included into the staining solution at a concentration of 10µg/ml. Because PI also stains doublestranded RNA, RNA is removed enzymatically during the staining reaction. This is accomplished by the addition of RNase A into the staining solution.
Staining solution: PBS containing 0.1% (v/v) Triton X-100, 10µg/ml of PI, and 100µ/ml of DNase-free RNase A.
All cells in G1 have a uniform DNA content, as do cells in G2 and M; the latter have twice as much DNA as G1 cells. Under ideal conditions of DNA staining, the fluorescence intensities of all G1 or G2/M cells are expected to be uniform and, after analog to digital conversion of the electronic signal from the photomultiplier (representing their fluorescence intensity), to have uniform numerical values, respectively. In practice, however, G1 and G2 cell populations are represented on frequency histograms by peaks of various width. The coefficient of variation (cv) of the mean value of DNA-associated fluorescence of the G1 population (width of the peak) is a reflection of an accuracy of DNA content measurement and should not exceed 8%. Improper staining conditions, instrument missadjustment, and the presence of a large number of dead or broken cells all result in high cv of the G1 cell populations.
Apoptotic cells often have fractional DNA content due to the fact that the fragmented (low MW) DNA undergoes extraction during the staining procedure. Some cells may also lose DNA (chromatin) by shedding apoptotic bodies. Only a fraction of DNA thus remains within apoptotic cells. They are represented then on the DNA content frequency histograms by the "sub-G1l" peak (Fig.l). Commercially available software packages to deconvolute DNA histograms are able to identify and quantity the "sub-G1" cell population.
If the length of the cell cycle (or cell doubling time) is known, the duration of each of the phases can be estimated from the percentage (fraction) of cells in that phase. For example, during the exponential phase of cell growth, the duration of G1 (TG1) can be calculated from
B. Multiparameter Analysis
Multiparameter analysis of other attributes of the cell, in addition to DNA content, allows one not only to distinguish cells in G1 vs S vs G2/M, but also to identify quiescent (G0) or mitotic cells. Thus, bivariate analysis of cell population with respect to their cellular DNA and RNA content discriminates between G0 and G1 cells. Bivariate analysis of DNA content and proliferation-associated proteins, particularly cyclins, provides another means to distinguish between proliferating and quiescent cells and yields additional information about the proliferative potential of cell populations (Darzynkiewicz et al., 1996). Immunocytochemical detection of histone H3 phosphorylation combined with DNA content analysis offers a convenient approach to distinguish M from G2 cells and to quantify the mitotic index in the cell population (Juan et al., 2001). This section presents examples of these methods.
1. Differential Staining of Cellular DNA and RNA
Quiescent (G0) cells are characterized by a manyfold lower RNA content compared to their cycling G1 counterparts (Darzynkiewicz et al.,1976). Simultaneous staining of RNA and DNA, therefore, allows one to distinguish G0 from G1 cells (based on differences in RNA content), as well as to identify cells in S and G2/M. Differential staining of cellular DNA and RNA can be accomplished with the metachromatic fluorochrome acridine orange (AO). At appropriate concentrations and ionic conditions AO intercalates into dsDNA and fluoresces green, while its interactions with RNA result in red fluorescence (Darzynkiewicz et al., 1994). Prior to staining with AO the cells are permeabilized with Triton X-100 in the presence of 0.08 M HCl and serum proteins. Such treatment makes cells permeable to AO, yet they are not lysed and their DNA and RNA content is preserved. Alternatively, the cells may be prefixed in 70% ethanol, as described previously for univariate DNA content analysis. Apoptotic cells stained under these conditions are characterized by markedly diminished DNA associated (green) AO fluorescence.
A characteristic distribution of G0, G1, S, G2/M, and apoptotic cells, differing in RNA and DNA content, is shown in Fig. 2. The differences in RNA content enable G0 cells to be discriminated from G1 cells. However, the differences in DNA content provide the basis to identify apoptotic and nonapoptotic cells and, among the latter, to distinguish G0/1, S, and G2/M cell subpopulations.
The major advantages of this assay are its simplicity, applicability to different instruments that use either laser or mercury lamp as a light source for fluorescence excitation, and the possibility it offers to distinguish G0 from G1 cells. Differential stainability of DNA vs RNA, however, requires very stringent conditions of cell staining in terms of dye concentration and ionic composition of the medium. AO also has a propensity to attach to sample flow lines of flow cytometers and therefore requires careful rinsing of the instrument with a bleaching solution (~15 min) to lower the background fluorescence for the subsequent analysis of weakly fluorescent samples. Further details of the AO methodology are presented elsewhere (Darzynkiewicz et al., 1994).
2. Cellular DNA Content and Expression of Proliferation-Associated Proteins
The expression of proliferation-associated proteins often varies during the cell cycle, as well as is different in cycling and quiescent cells. Their immunocytochemical detection, therefore, provides information on the proliferative status of the cell. The most common markers of proliferating cells are the proliferating cell nuclear antigen (PCNA) (Larsen et al., 2001), the antigen detected by the Ki-67 antibody (Endl et al., 2001) and certain cyclins (Darzynkiewicz et al., 1996).
Methods for detection of the proliferation associated proteins, particularly the choice of optimal fixative, may vary depending on the particular antigen (Jacobberger, 2001). The following method is applicable not only to cyclins (Darzynkiewicz et al., 1994, 1996), but also other intracellular antigens.
The critical steps for immunocytochemical detection of intracellular proteins are cell fixation and permeabilization. The fixative is expected to stabilize the antigen in situ and preserve its epitope in a state where it remains reactive with the Ab. The cells have to be permeable to allow the access of the Ab to the epitope. The choice of optimal fixative and permeabilizing agent varies, primarily depending on the intracellular antigen, less on the cell type. General strategies of cell fixation, permeabilization, and stoichiometry of antigen detection are discussed by Jacobberger (2001). Cold methanol appears to be optimal for the detection of D-type cyclins. For cyclins E, A, and B1, 70% cold ethanol is equally good.
Also critical is choice of a proper Ab. Often, the Ab applicable to immunoblotting fails in immunocytochemical application, and vice versa. This may be due to differences in the in situ accessibility of the epitope or differences in degree of denaturation of the antigen on the immunoblots compared to that within the cell. Some epitopes may not be accessible in situ at all, whereas the accessibility of others may vary depending on their functional state, e.g., due to phosphorylation or steric hindrance. Because there is strong homology between different cyclin types, crossreactivity may also be a problem. Because commercially available Abs may differ in specificity, degree of cross-reactivity, and so on, it is essential to provide detailed information (vendor and the hybridoma clone number) of the reagent used in the study.
Cyclins are key components of the cell cycle progression machinery (Sheer, 2000). During unperturbed growth of normal cells the timing of expression of several cyclins, particularly cyclins D, E, A, and B, is discontinuous, occurring as discrete and well-defined periods of the cell cycle (Fig. 3). This periodicity in cyclins expression provides new cell cycle landmarks that can be used to subdivide the cell cycle into several subcompartments, additional to the subdivision of into four major phases (Darzynkiewicz et al., 1996). Furthermore, bivariate analysis of cyclins expression vs DNA content makes it possible to discriminate between cells having the same DNA content but residing in different phases of the cycle, such as between G2 and M cells (based on differences in cyclin A content), or between G2 diploid and G1 tetraploid cells (based on differences in expression of cyclins E and/or B1). Likewise, G0 cells lacking expression of D-type cyclins or cyclin E can be distinguished from cells that entered cell cycle and become cyclins D, and subsequently cyclin E, positive. Strategies for the use of cyclins as additional markers of the cell cycle position are discussed elsewhere (Darzynkiewicz et al., 1996). It should be noted, however, that some tumor cell lines, or normal cells when their cell cycle progression is perturbed, show unscheduled expression of cyclins D, E, and B1; namely G1 cyclins (e.g., cyclin E) are expressed during G2/M and the G2/M cyclins (cyclin B1) during G1 (Darzynkiewicz et al., 1996).
3. Identification of Mitotic Cells by Cytometry
There is often a need to estimate mitotic index, e.g., to assess effectiveness of the drugs that disrupt microtubules or in stathmokinetic experiment (Darzynkiewicz et al., 1987) to reveal the rate of cell entrance to mitosis. The cytometric methods used to identify mitotic cells are reviewed by Juan et al. (2001). The most convenient immunocytochemical method appears to be the one that utilizes Ab that is specific to histone H3 phosphorylated on Ser-l0 (H3-P), the event that occurs during mitosis (Juan et al., 2001). Because histone H3 is phosphorylated during prophase and is dephosphorylated late in telophase, the "time window" of detection of mitosis by this Ab spans these two mitotic stages. Histone H3-P-specific Abs are offered by Sigma Chemical Co. (monoclonal) and Upstate Biotechnology, Inc. (polyclonal). The methodology of cell staining and fluorescence measurement is similar to that described earlier for analysis of DNA content and proliferation-associated proteins. Optimal cell fixation, however, requires a brief (15min) pretreatment with 1% formaldehyde (in PBS, on ice) followed by postfixation in 70% ethanol. The results of fluorescence measurement are shown in Fig. 4.
C. Analysis of DNA Replication and Cell Cycle Kinetics
1. Stathmokinetic Approach
In a classical stathmokinetic experiment, the agent arresting cells in mitosis (e.g., colcemide or vinblastine) is added into the culture during the exponential phase of cell growth and the proportion of cells in mitosis is estimated as a function of the time of arrest. The slope of the plot representing an increase in the percentage of M cells during stathmokinesis reveals the rate of cell entry to M ("mitotic rate"; "cell birth rate").
Flow cytometric analysis of the stathmokinetic experiment can be based either on quantification of the increased proportions of cells in G2 + M, represented by the G2/M peak on the DNA content frequency histograms (by DNA content measurement followed by univariate data analysis), or by enumeration of cells in M (by selective staining of M cells, e.g., as shown in Fig. 4, followed by multivariate analysis of such data). Depletion of cells from the G1 compartment (G1 exit rate), as well as the rate of cell progression through S phase can also be measured during stathmokinesis (Darzynkiewicz et al., 1987). This section describes the scheme of a simple stathmokinetic in vitro experiment.
Depending on the method used to stain DNA (Fig. 1) or detect mitotic cells (Fig. 4), appropriate solutions, as described earlier in the article, should be applied.
A major drawback of the methods based on single time point measurement is lack of kinetic information. These methods, thus, cannot distinguish whether cell cycle progression is accelerated, slowed down, or even halted, e.g., during drug treatment, if G1, S, and G2/M phases are affected proportionally to each other. The stathmokinetic approach can be used in such instances to reveal cell kinetics. The alternative method, namely cell synchronization followed by observation of the cycle progression of the synchronized cells cohort, is more complex and time-consuming.
2. BrdUrd Incorporation
Incubation of cells in medium containing BrdUrd results in its incorporation during DNA replication (S phase). The incorporated BrdUrd can be detected either cytochemically, by virtue of its propensity to quench the fluorescence of several DNA fluorochromes such as Hoechst 33358 or AO, or immunocytochemically using poly- or monoclonal Abs against this precursor.
Continuous or pulse-chase cell labeling with BrdUrd, followed by detection of BrdUrd simultaneously with measurement of cellular DNA content and bivariate data analysis (Dolbeare et al., 1983; Terry and White, 2001), allows one to estimate a variety of cell cycle parameters. The protocol of Dolbeare et al., (1983), with more recent modifications (Gray et al., 1990), is given here. DNA denaturation by acid (HCl) gives more satisfactory results in some cell types.
a. Thermal Denaturation of DNA
b. Denaturation of DNA by HCl
Diluting buffer: same as for thermal denaturation of DNA
The critical step in this procedure is induction of partial DNA denaturation. This step often results in cell damage and leads to significant cell loss. Use of silinized tubes during centrifugations may decrease cell loss. Also, there are differences in sensitivity of DNA to denaturation between cell types, depending on their chromatin structure. Thus, while induction of DNA denaturation by acid may prove to be satisfactory with one cell type, it may fail with another. Some cell types require higher acid concentration (4M) for optimal results.
The alternative approach is based on selective photolysis of DNA that contains the incorporated BrdUrd followed by DNA strand break labeling (Li et al., 1996). Because no heat or acid treatment is required, the latter procedure is applicable in combination with immunocytochemical analysis.
The scope of this article makes it impossible to describe all possibilities of analysis of the cell cycle based on BrdUrd incorporation, either after the pulsechase or continuous cell labeling. Readers are advised to consult Dolbeare and Selden (1994) and Terry and White (2001) for a more detailed description of these methods.
IV. CELL ANALYSIS BY LASER.SCANNING CYTOMETER
All the methods described earlier can be adapted to stain cells mounted on microscope slides, to be analyzed by the multiparameter (three-laser excitation, four-color fluorescence detection) LSC (Darzynkiewicz et al., 1999; Kamentsky, 2001). To be analyzed by LSC the cells are attached to the slides electrostatically or by cytospinning, fixed, rinsed and then subjected to the procedures as presented earlier. To attach cells by cytospinning, 300µl of cell suspension in tissue culture medium (with serum) containing approximately 20,000 is added to a cytospin chamber. The cells are then cytocentrifuged at 1000 rpm for 6 min and are submerged in the respective fixative in Coplin jars.
Small volumes of the respective buffers, rinses, or staining solutions as described for each of the methods in this article, are carefully layered on the cytospin area of the horizontally placed slides. At appropriate times these solutions are removed with a Pasteur pipette (or vacuum suction pipette). Small pieces (1 × 1 cm) of thin polyethylene or Parafilm foil may be layered on slides atop of the drops of the solutions used for cell incubations, over the cytospins, to prevent drying. The incubations should be carried out in a moist atmosphere.
At the final step of each particular staining procedure the cells are mounted in a drop of the respective staining solution, made identical as for flow cytometry, under the coverslip. The coverslips may be sealed with melted paraffin or a gelatin-based sealer. The cell fluorescence is measured by LSC, and the choice of the fluorescence excitation wavelength and emission filters is the same as described earlier for flow cytometers.
V. LIMITATIONS AND PITFALLS
Supported by NCI RO1 96 704. Dr. Gloria Juan is currently at the Sloan-Kettering Cancer Institute, New York. Dr. P. Pozarowski is on leave from the Department of Immunology, School of Medicine, Lublin, Poland.
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