COMPARISON OF CELL VIABILITY PROBES COMPATIBLE WITH FIXATION AND PERMEABILIZATION FOR COMBINED SURFACE AND INTRACELLULAR STAINING IN FLOW-CYTOMETRY

Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptak e of probes, increased autofluorescence, and altered antigen expressio n and DNA content. Traditional methods of dead cell exclusion, based o n light scatter or uptake of dyes such as propidium iodide (PI) or flu orescein diacetate (FDA), are appropriate for the analysis of fresh, r elatively homogeneous samples, However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody p anel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, a nd most of the widely used viability probes are incompatible, due to w eak or reversible binding, with the use of permeabilizing agents for i ntracellular staining. To determine the best viability marker for incl usion in the solid tumor panel, we compared cultured tells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel pr ocedure (unprocessed cells from each day, stained with PI, were used a s standards), The viability probes included PI (in processed and unpro cessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye sty ryl (LDS)-751; ethidium monoazide (EMA);and actin, cytokeratin, and tu bulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC), The selection criteria for the best viability probe included broad cell ty pe specificity: low nonspecific staining of live cells, specific stain ing of dead cells strong enough to withstand the permeabilization proc edure, high signal-to-noise ratio throughout the time course, and comp atibility with the four other fluorescent probes making up the turner 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 sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every resp ect other than its specificity only for cells of epithelial origin, Tu bulin-SAM-FITC alone satisfied all the criteria and was selected for i nclusion in the monoclonal antibody panel as a viability probe. (C) 19 95 Wiley-Liss, Inc.