Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

Background: Combined analysis of DNA content and immunofluorescence on sing le cells by flow cytometry provides information on the proliferative respon se of subpopulations to stimuli in mixed cell preparations; however, in low -viability cell preparations, dead cells interfere with accurate flow cytom etric data analysis because of nonspecific binding of antibodies and altere d DNA-staining profiles. Light scatter differences between nonviable and vi able cells are unreliable, particularly after the cell permeabilization ste p that is necessary for DNA staining. We developed a method for identificat ion of nonviable cells by fluorescence in cell preparations that are staine d simultaneously for cell surface or intracellular immunofluorescence and D NA content. Materials and Methods: Nonviable cells that have lost membrane integrity ar e identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilizati on is prevented by blocking DNA binding with nonfluorescent actinomycin D ( AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY call be separated from the green fluorescein isothiocyanate (FITC) emission and the fed emission of I-AAD, respectively. Results: Application of the method to the analysis of the T-cell leukemia c ell line Molt-4f and of cultured human peripheral blood mononuclear cells i s presented. In both cell preparations, 7-AAD staining permitted reliable d ead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expres sion levels of cell surface CD4 and of intracellular CD3, showed a higher p roportion of cells in the GI phase of the cell cycle, and showed a lower co efficient of variation of the G(1) peak compared with data obtained from al l the cells in the preparation. Live, CD8(+) lymphocytes from OKT3-stimulat ed cultures of human peripheral blood mononuclear cells showed a specific p roliferative response as measured by DNA content analysis. Conclusions: The results show that cells stained with FITC-labeled antibodi es can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exc lusion of dead cells from the analysis of cell preparations with low viabil ity to obtain reliable data on immunofluorescence and cell-cycle distributi ons. Cytometry 35: 64-74, 1999. (C) 1999 Wiley-Liss, Inc.