Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the GI and Gz phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90" light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90" light scatter and protein content. M-phase nuclei can be distinguished from Gzphase nuclei on cytograms of 90" light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent W "~O ) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTAfTris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/Gz region of the single-parameter histogram.Key terms: Propidium iodide, FITC, cell cycle, nucleic acids, detergent lysis, NPl" A common use of flow cytometry is to analyze the DNA distribution in cell populations. On the basis of DNA content, the percentage of cells residing in the different stages of the cell cycle can be determined (11). Increased resolution of the stages of the cycle can be obtained by incorporating a second parameter into the analysis and displaying the data as a cytogram. Secondparameter measurements include forward light scatter, protein content (fluorescein isothiocyanate), RNA content (acridine orange, pyronin y), 5-bromodeoxyuridine (BudR) incorporation, and nuclear andor surf'ace antigens (2,4,6-8,10,12-16,18,23). The acridine orange (AO) fluorescence spectrum is different for single-stranded (red) and double-stranded (green) nucleic acids (7). Also, the nucleus exhibits structural differences in different stages of the cell cycle as reflected in its varying resistance to denaturation by acid or heat. In conjunction with A 0 staining, these structural differences allow resolution of subclasses of the GI and G2 phases, as well as the M phase (6-8). In addition, the subclasses can be differentiated using two fluorochromes: propidium iodide (PI) to measure DNA content and fluorescein isothiocyanate (FITC) to measure protein content. Both DNA and protein increase in cells during progression through the cell cycle (4,15,16,18) and mitotic cells bind less FITC and PI than G2 cells (15,16,18). Differentiation and quantification of the proliferating fraction of S-phase cells can be achieved by measurement of the incorporation of BudR into DNA, employing monoclonal antibodies to BudR (10,141. Mitotic nuclei prepared by nonionic detergent lysis and fixed in formaldehyde can be discriminated from G2-phase nuclei by staining with mithramycin. Compared w...

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