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LIMITATIONS OF THE FLUORESCENT PROBE VIABILITY ASSAY
Citation:
Massaro, E., K. Elstein, K. Bair, AND R. Zucker. LIMITATIONS OF THE FLUORESCENT PROBE VIABILITY ASSAY. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-89/551 (NTIS PB92113166), 1989.
Description:
Cell viability commonly is determined flow cytometrically by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. FDA is taken up by the viable cell and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF). F fluorescence intensity is considered to be an index of cellular vigor. t is generally accepted that the viable cell excludes PI. i uptake is indicative of irreversible cellular injury and presages cell death. e observe that, following incubation for 4 hr with 0.5 - 1.0 tributyltin (TBT), murine erythroleukemic cells (MELC) exhibit supranormal CF fluorescence and exclude PI. pparent cell volume is unaltered. owever, the rate of growth (cell duplication) of these cells is depressed, suggesting that supranormal CF fluorescence, even in the absence of PI uptake, is indicative of cellular perturbation. urthermore, at higher TBT concentrations (>1.0, <50.0 uM), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. EL exposed to the cancer chemotherapeutic agents adriamycin, m-AMSA or crisnatol (770842) also exhibit increased mean cellular CF fluorescence. owever, rate of growth is decreased and apparent cell volume increased. he latter, measured either as a function of electrical resistance (Coulter Counter) or axial light loss, could account for the increase in mean CF fluorescence. hese observations indicate that, by and of itself, CF fluorescence is neither a reliable indicator of cell viability nor vigor and suggest that in the case of perturbed cells, viability/growth assays based on intrinsic enzyme activities potentially are unreliable and inaccurate.