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Dead cells represent a si@cant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining.To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells Grom each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-a-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specitic staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufticiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its speciticity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe. o 1995 Wiley-Liss, hc.

No abstract

A recently described mitochondrial membrane protein-specific monoclonal antibody, APO2.7, was examined for monitoring early apoptotic responses in anti-CD95 (7C11)-induced Jurkat cells. Jurkat cells were harvested at 1.5, 3, 4.5, 6, 12, and 18 h after induction of apoptosis, and APO2.7 antibody monitored in unprocessed (no permeabilization agent used prior to staining) and processed (permeabilized prior to staining) cells. Light-scatter changes (decreased forward-scatter and increased side-scatter) by flow cytometry were observed after 3 h, and detection of cell permeability in unprocessed cells, as measured by light microscopic examination of Trypan blue-stained cells and flow cytometric detection of tubulin, showed little change until after 6 h. In addition, unprocessed cells stained with APO2.7 antibody showed little increase in staining until after 6 h following induction of apoptosis, when DNA fragmentation was demonstrated by flow cytometry and gel electrophoresis; however, processed cells stained with APO2.7 antibody showed significant increase in staining after 1.5 h. Detection, using annexin V and flow cytometry, of phospholipid membrane asymmetry from exposure of phosphatidylserine showed greater, apparent nonspecific staining in noninduced cells as compared to the other markers of apoptosis, but nearly paralleled the results of APO2.7 staining in processed cells from 3-18 h following CD95 induction of apoptosis. The data presented herein indicate that the mitochondrial membrane protein-specific antibody, APO2.7, is useful as a marker for the detection of apoptotic cells.

Treatment of exponentially growing Chinese hamster ovary cells with bleomycin causes a dose-dependent decrease in cell survival due to DNA damage. This lethal effect can be potentiated by the addition of a nonlethal dose of the anticalmodulin drug N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide ( W13 ) but not its inactive analog N-(4-aminobutyl)-2-naphthalenesulfonamide ( W12 ). By preventing the repair of damaged DNA, W13 also inhibits recovery from potentially lethal damage induced by bleomycin. These data suggest a role for calmodulin in the DNA repair pathway.