Figure 1. Relationship between the cell cycle position and DNA content. The content of DNA (DNA index, DI) doubles during S phase and therefore G₂ and M cells have twice as much DNA as G₁ cells (DI = 2.0). The cell's progression through S 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 (unit) bars. However, owing to the inaccuracy of DNA content measurements, their actual distributions are in the form of peaks 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. These typical histograms of the DNA content represent untreated cells (a) and cells treated with the antitumour drug fostriecin (b). The drug induced changes in the cell cycle distribution that resulted in a higher percentage of S and G₂/M cells, and was also cytotoxic inducing apoptosis. Because DNA undergoes fragmentation during apoptosis, apoptotic cells (Ap) can be identified on DNA frequency histograms as cells with a fractional DNA content (0.0>DI>1.0).

Figure 2. G₀ and G₁ cells can be discriminated by differences in RNA content. Bivariate distributions (cellular RNA versus DNA content; scatterplots) represent nonstimulated lymphocytes (G₀ cells; panel (a)) and mitogen-stimulated lymphocytes (b). Quiescent G₀ cells have a distinctly lower RNA content than cycling G₁ cells. The DNA content frequency histograms of these cells are shown in the insets. The dashed line indicates the threshold RNA level that discriminates G₀ from G₁ cells.

Figure 3. Identification of mitotic cells. The scatterplot shows that bivariate distribution (phosphorylated histone H3 immunofluorescence 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 characterized by strong H3^P+ immunofluorescence.

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 fluoresceinated 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 S during incubation with BrDU (which have strong anti-BrDU immunofluorescence).

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 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 S phase and its level drops during S. In contrast, expression of cyclin A progressively increases during S and is maximal in G₂. Cyclin B1 is detected in late S and is maximally expressed by G₂/M cells.