Search

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.

Biohazardous aerosols generated during cell sorting have been of increased concern recently because of interest in sorting specimens containing human immunodeficiency virus type 1 (HIV-1). Current flow cytometers have features designed to contain such aerosols within the sorting chamber, but the efficacy of these features has not been established. Therefore, we tested aerosol containment by two ELITE flow cytometers (Coulter Cytometry, Inc., Hialeah, FL) during sorting of specimens containing high titers of bacteriophage. Agar plates confluent with susceptible Escherichia coli were used to detect infectious units released from the sorting chamber. Under recommended operating conditions very few infectious units were released from the sorting chambers. Release increased when the center stream was not optimally collected in a vacuum-exhausted tube or the chamber door was not completely closed. Failure of the negative pressure and high efficiency particle air (HEPA) filtration features had less of an effect. The data indicate that these standard safety features provide a rational expectation of safety for the flow cytometry operator. © 1995 Wiley-Liss, Inc.

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16+PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1½ year period, 78 shared peripheral blood specimens Were prepared and analyzed in each laboratory. The CD45brightCD14− percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were