Simultaneous assessment of chromatin structure, DNA content, and antigen expression by dual wavelength excitation flow cytometry.

7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.