Cell Cycle Analysis by Flow Cytometry

Abstract

Flow cytometry, the methodology that provides a way to measure physical and chemical attributes of individual cells rapidly and with high accuracy, is widely used in cell cycle analysis. The most common assays are: univariate analysis of cellular deoxyribonucleic acid (DNA) content discloses frequencies of cells in G0/1, S and G2/M phases; bivariate analysis of DNA versus ribonucleic acid (RNA) content reveals frequencies of G0, G1, S and G2M cells, of DNA content versus histone H3 phosphorylated on Ser10 defines frequencies of G0/1, S, G2 and M cells. Detection of cells that incorporated DNA precursor, such as 5‐bromo‐2′‐deoxyuridine (BrdU) or 5‐ethynyl‐2′‐deoxyuridine (EdU) identifies cells that replicated DNA. Immunocytochemical detection of cyclin D1, cyclin E, Cyclin A and cyclin B1 concurrent with DNA content reveals the proteins that are essential for driving the cell through the cell cycle. The stathmokinetic approach makes it possible to assess the kinetics of progression through the cell cycle.

Key Concepts:

  • Cellular DNA content identifies cell position in the cell cycle.

  • Fractional cellular DNA content (‘sub‐G1’ cells) is a marker of apoptotic cells.

  • Low cellular RNA content is a biomarker of G0 (quiescent) cells.

  • Incorporation of BrdU or EdU identifies DNA replication cells.

  • Phosphorylation of histone H3 on Ser10 is a marker of mitotic cells.

  • Detection of cyclin proteins is a marker of cell progression machinery.

  • Stathmokinetic approach based on cell arrest in metaphase makes it possible to assess the entry of cells to mitosis (‘mitotic rate’, ‘cell birth rate’) and reveals kinetics of cell progression through the cycle.

Keywords: cell cycle; flow cytometry; DNA replication; DNA content; cell cycle phases; mitosis; apoptosis; stathmokinesis; cell cycle kinetics; cyclin proteins

Figure 1.

(A) Schematic representation of changes in cellular DNA content during the cell cycle. As DNA replication is restricted to the S phase, the content of DNA (in flow cytometry defined by DNA index, DI) doubles during that phase and therefore G2 and M cells have twice as much DNA as G1 cells (DI=2.0). The cell's progression through the S phase can be estimated based on the amount of replicated DNA (the increase in DI from 1.0 to 2.0). G1 and G2/M cells have a uniform DNA content (DI=1.0 and 2.0, respectively) so that if their DNA could be measured with absolute accuracy these cell populations would be represented on the DNA content frequency histograms by single‐channel (single unit) bars. However, owing to the inaccuracy of DNA content measurements, their actual plots are in the form of peaks resembling Gaussian distribution, whose width reflects the inaccuracy. Percentages of cells in G1, S and G2/M phases are estimated by deconvoluting the histograms using special computer programs. (B) Actual DNA content histograms representing untreated cells (a) and cells treated with the antitumour drug fostriecin (b). As is evident the drug‐induced changes in the cell cycle distribution resulted in a higher percentage of S and G2/M cells. The drug was also cytotoxic inducing apoptosis. As during apoptosis DNA undergoes fragmentation, the apoptotic cells (Ap) are identified on DNA frequency histograms as cells with a fractional DNA content (0.0>DI>1.0). They often are defined as the ‘sub‐G1’ cell population (Wlodkowic et al., ). The dashed lines represent the populations of cells in the respective cell cycle phases based on deconvolution of raw data.

Figure 2.

Differential staining of DNA versus RNA is able to distinguish G0 from G1 cells and also to identify cells in S and G2/M as well as dead (apoptotic) cells. (a) The metachromatic fluorochrome AO differentially stains double‐stranded (ds) nucleic acids (green fluorescence; F530) and single‐stranded (ss, red fluorescence; F>600) nucleic acids, as shown. The methodology of differential staining of DNA versus RNA is based on the selective denaturation of RNA, which then becomes ssRNA, while DNA still remains in ds conformation (Darzynkiewicz et al., ). (b) The panels show bivariate distributions (scatterplots) of DNA versus RNA of quiescent, nonstimulated human lymphocytes and lymphocytes stimulated with the polyvalent mitogen phytohaemagglutinin. As is evident, such staining makes it possible to identify cells in G0, G1, S, G2M as well as dead (apoptotic) cells that have fractional DNA content (Wlodkowic et al., ).

Figure 3.

Identification of mitotic cells. The scatterplot shows that bivariate distribution (phosphorylated histone H3 immunofluorescence (H3P+) versus DNA content) of cultured leukaemic cells enriched for M phase by growing in the presence of the mitotic blocker, vinblastine. Mitotic cells (M) are characterised by strong H3P+ immunofluorescence and the same DNA content as G2 cells.

Figure 4.

Distinguishing cells replicating their DNA. Cultured leukaemic cells were incubated for 30 min in the presence of BrdU. Bivariate analysis of BrdU incorporation (detected immunocytochemically with fluorescein‐tagged anti‐BrdU Ab) versus DNA content of these cells allows G1 and G2/M cells (which did not incorporate the precursor) to be distinguished from the cells that progressed through the S phase during incubation with BrdU (which have distinct anti‐BrdU immunofluorescence). The dashed line presents the threshold separating the cells that incorporated BrdU (S) from the unlabelled cells (G1 and G2M).

Figure 5.

Bivariate distributions of cyclins D, E, A and B1 versus DNA content. The scatterplots represent the characteristic pattern of expression of individual cyclins vis‐à‐vis the cell cycle position as identified by the cellular DNA content, in normal, nontumour cells. Note that cyclin D1 is detected only in a fraction of G1 cells; the cells in S and most G2/M cells are cyclin D1 negative. Cyclin E is maximally expressed in cells entering the S phase and its level drops during the S phase. In contrast, expression of cyclin A progressively increases during the S phase and is maximal in G2. Cyclin B1 is detected in late S phase and is maximally expressed by G2/M cells.

Figure 6.

Schematic representation of the principle of stathmokinesis. Cells in exponentially growing culture are arrested in mitosis (M) by adding a mitotic blocker such as nocodazole or colcemid. (a) The frequency of mitotic cells is then estimated from samples of the culture taken sequentially at different time points of incubation. The number of mitotic cells determined, for example, as in Figure is then plotted on the scale (log 1+fM) as shown, where fM is the fraction of mitotic cells in cell population. Alternatively, or in parallel, the frequency of G2M cells can be estimated by univariate DNA content analysis (Figure ) and plotted as log (1+fG2M). The slope of the M or G2M plots, expected to be straight line if cells grow exponentially, is representative of the cell kinetics (cell ‘birth rate’). The scale of log (1+fM) covers the range between 0 and 0.301 for the full cell cycle and its duration can be measured by interpolation of the plot on the time axis (Darzynkiewicz et al., ). The length of G2 can be estimated from the time difference between the M and G2 plots. (b) The rate of cell exit from G1 (transition from G1 to S) is revealed by the G1 plot reporting the number of cells in G1 as a function of time.

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Darzynkiewicz, Zbigniew, and Zhao, Hong(Feb 2014) Cell Cycle Analysis by Flow Cytometry. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002571.pub2]