Cell Cycle Analysis by Flow Cytometry


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.



Begg AC (1995) The clinical status of Tpot as a predictor? Or why no tempest in the Tpot!. International Journal of Radiation Oncology, Biology, Physics 32: 1539–1541.

Begg AC, McNally NJ, Shrieve DC and Karcher H (1985) A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6: 620–626.

Bruno S and Darzynkiewicz Z (1992) Cell cycle dependent expression and stability of the nuclear protein detected by Ki‐67 antibody in HL‐60 cells. Cell Proliferation 25: 31–40.

Celis JE, Madsen P, Nielsen S and Celis A (1986) Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S‐phase in cultured cells – some applications of PCNA antibodies. Leukemia Research 10: 237–249.

Crissman HA and Tobey RA (1974) Cell cycle analysis in 20 min. Science 184: 1297–1298.

Darzynkiewicz Z (1990) Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine orange. Methods in Cell Biology 33: 285–298.

Darzynkiewicz Z (2011) Critical aspects in analysis of cellular DNA content. Current Protocols in Cytometry, chap. 7. 52: 7.2.1–7.2.8

Darzynkiewicz Z, Crissman HA and Jacobberger JW (2004a) Cytometry of the cell cycle. Cycling through history. Cytometry 58A: 21–32.

Darzynkiewicz Z, Gong J, Juan G, Ardelt B and Traganos F (1996) Cytometry of cyclin proteins. Cytometry 25: 1–13.

Darzynkiewicz Z, Juan G and Srour EF (2004b) Differential staining of DNA and RNA. Current Protocols in Cytometry, chap. 7. 30: 7.3.1–7.3.16.

Darzynkiewicz Z, Robinson JP and Crissman HA (1994) Flow Cytometry: Part A. 2nd edn. San Diego, CA: Academic Press.

Darzynkiewicz Z, Sharpless T, Staiano‐Coico L and Melamed MR (1980) Subcompartments of the G1 phase of the cell cycle detected by flow cytometry. Proceedings of the National Academy of Sciences of the USA 77: 6696–6700.

Darzynkiewicz Z, Traganos F and Kimmel M (1987) Assay of cell cycle kinetics by multivariate flow cytometry using the principle of stathmokinesis. In: Gray JW and Darzynkiewicz Z (eds) Techniques in Cell Cycle Analysis, pp. 291–336. Clifton, NJ: Humana Press.

Diermeier‐Daucher S, Clarke ST, Hill D et al. (2009) Cell type specific applicability of 5‐ethynyl‐2′‐deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry A 75A: 535–546.

Dolbeare F, Kuo WL Beisker W, Vanderlaan M and Gray JW (1990) Using monoclonal antibodies in bromodeoxyuridine‐DNA analysis. Methods in Cell Biology 33: 207–216.

Gerdes J, Baisch H, Wacker HH, Schwab U and Stein H (1984) Cell cycle analysis of a cell proliferation‐associated human nuclear antigen defined by the monoclonal antibody Ki‐67. Journal of Immunology 133: 1710–1715.

Gong JP, Ardelt B, Traganos F and Darzynkiewicz Z (1994) Unscheduled expression of cyclin B1 and cyclin E in several leukemic and solid tumor cell lines. Cancer Research 54: 4285–4288.

Gratzner HG (1982) Monoclonal antibody to 5‐bromo and 5‐iododeoxyuridine: a new reagent for detection of DNA replication. Science 218: 474–475.

Hartwell LH and Kasten MB (1994) Cell cycle control and cancer. Science 5192: 1821–1828.

Huang X, Kurose A, Tanaka T et al. (2006) Sequential phosphorylation of Ser‐10 on histone H3 and Ser‐139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell‐cycle and apoptosis. Cytometry A 69A: 222–229.

Juan G and Darzynkiewicz Z (1998) Phosphorylation of pRb in individual HL‐60 cells during their proliferation and differentiation. Experimental Cell Research 244: 83–92.

Juan G and Darzynkiewicz Z (2004) Detection of mitotic cells. Current Protocols in Cytometry, chap. 7. 28: 7.24.1–7.24.7.

Juan G, Traganos F, James WM et al. (1998) Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 32: 71–77.

Kastan MB and Bartek J (2004) Cell‐cycle checkpoints and cancer. Nature 432: 316–323.

Krueger SA and Wilson GD (2011) Flow cytometric DNA analysis of human cancers and cell lines. Methods in Molecular Biology 731: 359–370.

Li X, Melamed MR and Darzynkiewicz Z (1996) Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Experimental Cell Research 222: 28–37.

Martin RM, Leonhardt H and Cardoso MC (2005) DNA labeling in living cells. Cytometry A 67A: 45–52.

Qu D, Wang G, Wang Z et al. (2011) 5‐Ethynyl‐2′‐deoxycytidine as a new agent for DNA labeling: detection of proliferating cells. Analytical Biochemistry 417: 112–121.

Salic A and Mitchison TJ (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proceedings of the National Academy of Sciences of the USA 105: 2415–2420.

Shapiro HM (1981) Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y. Cytometry 2: 143–159.

Shapiro HM (2003) Practical Flow Cytometry, 4th edn. NY: Wiley. Available at: http://www.coulterflow.com/bciflow/practical/book/index.html (assessed on 15 December 2013).

Smith PJ, Blunt N, Wiltshire M et al. (2000) Characteristics of a novel deep red/infrared fluorescent cell‐permeant DNA probe, DRAQ5 in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy. Cytometry 40: 280–291.

Telford WG (2004) Analysis of UV‐excited fluorochromes by flow cytometry using near‐ultraviolet laser diodes. Cytometry A 61: 9–17.

Traganos F, Ardelt B, Halko NM, Bruno S and Darzynkiewicz Z (1992) Effect of Genistein, a tyrosine kinase inhibitor, on the growth and cell cycle progression of normal human lymphocytes and human leukemic MOLT‐4 and HL‐60 cells. Cancer Research 52: 6200–6208.

Vindeløv L and Christensen IJ (1990) An integrated set of methods for routine flow cytometric DNA analysis. Methods in Cell Biology 33: 127–137.

Wlodkowic D, Telford W, Skommer J and Darzynkiewicz Z (2011). Apoptosis and beyond: Cytometry in studies of programmed cell death. Methods in Cell Biology 103: 55–98.

Zhao H, Halicka HD, Li J et al. (2013) DNA damage signaling, impairment of cell cycle progression and apoptosis triggered by 5‐ethynyl‐2′deoxyuridine (EdU) incorporated into DNA. Cytometry A 83A: 979–988.

Zhao H, Traganos F, Dobrucki J, Wlodkowic D and Darzynkiewicz Z (2009) Induction of DNA damage response by the supravital probes of nucleic acids. Cytometry A 75A: 510–519.

Further Reading

Blagosklonny MV and Darzynkiewicz Z (2002) Cyclotherapy: protecting of normal cells and unshielding of cancer cells. Cell Cycle 1: 375–382.

Bloom J and Cross FR (2007) Multiple levels of cyclin specificity. Nature Reviews Molecular Cell Biology 8: 149–160.

Darzynkiewicz Z, Bedner E and Smolewski P (2001) Flow cytometry in analysis of cell cycle and apoptosis. Seminars in Hematology 38: 179–193.

Inz'e D and DeVeylder L (2006) Cell cycle regulation in plant development. Annual Review of Genetics 40: 77–106.

Malumbres M and Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nature Reviews Cancer 9: 153–166.

Massague J (2004) G1 cell‐cycle control and cancer. Nature 432: 298–306.

Murray AV (2004) Recycling the cell cycle: cyclins revisited. Cycle 116: 221–234.

Spencer SL, Steven D, Cappell SD et al. (2013) The proliferation‐quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell 255: 269–383.

Vermeulen K, Van Bockstaele DR and Berneman ZN (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Proliferation 36: 131–149.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
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]