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

Abstract

Background: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low‐viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA‐staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. Materials and Methods: Nonviable cells that have lost membrane integrity are identified by uptake of 7‐amino‐actinomycin D (7‐AAD). Transfer of 7‐AAD from stained nonviable cells to unstained viable cells after permeabilization 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 can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7‐AAD, respectively. Results: Application of the method to the analysis of the T‐cell leukemia cell line Molt‐4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7‐AAD staining permitted reliable dead cell exclusion. Live, 7‐AAD–negative Molt‐4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3‐stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. Conclusions: The results show that cells stained with FITC–labeled antibodies can be analyzed by single‐laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell‐cycle distributions. Cytometry 35:64–74, 1999. © 1999 Wiley‐Liss, Inc.

Improved Survival in Rhesus Macaques Immunized with Modified Vaccinia Virus Ankara Recombinants Expressing Simian Immunodeficiency Virus Envelope Correlates with Reduction in Memory CD4+T-Cell Loss and Higher Titers of Neutralizing Antibody

“…3A. To exclude nonspecific staining and autofluorescence, dead lymphocytes were differentiated from live cells by staining with 7-amino-actinomycin D (89). As can be observed in the samples from H387, a macaque from the control group immunized with nonrecombinant MVA, significant depletion of effector and central memory CD4…”

Requirement of aggregation propensity of Alzheimer amyloid peptides for neuronal cell surface binding

“…We treated NGF-differentiated PC12 cells with each peptide and monitored TMR fluorescence on live cells as a function of time by flow cytometry analysis. Dead cells were readily distinguished from live cells by staining with 7-AAD, a DNA binding dye that is excluded by live cells with intact membranes [44,45]. Figure 4 shows the striking differences in the levels of TMR florescence on live PC12 cells within only one hour of peptide exposure.…”

Extensive early apoptosis in frozen–thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation

“…Importantly, these dyes detect only permeable, necrotic cells. 28,29 Using a new assay, 22 which detects early apoptosis, we established that 58% of the CD34 + cells found in thawed leucapheresis products was apoptotic and/or secondary necrotic. This percentage by far exceeded the percentages determined with 7-AAD.…”