||Limitations of the Fluorescent Probe Viability Assay.
Massaro, E. J. ;
Elstein, K. H. ;
Bair, K. W. ;
Zucker, R. M. ;
||Health Effects Research Lab., Research Triangle Park, NC. ;ManTech Environmental Technology, Inc., Research Triangle Park, NC.
Cell survival ;
Fluorescent dyes ;
Toxic substances ;
Flow cytometry ;
Acute erythroblastic leukemia ;
Antineoplastic agents ;
Cell division ;
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||Cell viability commonly is determined flow cytometrically by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. CFDA is taken up by the viable cell and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF). CF fluorescence intensity is considered to be an index of cellular vigor. It is generally accepted that the viable cell excludes PI. PI uptake is indicative of irreversible cellular injury and presages cell death. The authors observe that, following incubation for 4 hr with 0.5 - 1.0 microMolar tributyltin (TBT), murine erythroleukemic cells (MELC) exhibit supranormal CF fluorescence and exclude PI. Apparent cell volume is unaltered. However, 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. Furthermore, at higher TBT concentrations (>1.0, <50.0 microMolar), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. These observations indicate that, by and of itself, CF fluorescence is neither a reliable indicator of cell viability nor vigor and suggest that at least in the case of perturbed cells, viability/growth assays based on intrinsic enzyme activities potentially are unreliable and inaccurate.
||Pub. in Molecular Toxicology, v2 p271-284 Oct 89. Prepared in cooperation with ManTech Environmental Technology, Inc., Research Triangle Park, NC.
|NTIS Title Notes
||Reprint: Limitations of the Fluorescent Probe Viability Assay.
||PC A03/MF A01