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Limitations of the fluorescent probe viability assay
E J Massaro1, K H Elstein, R M Zucker
1Developmental Toxicology Division, U.S. Environment Protection Agency, Research Triangle Park, North Carolina.
Molecular Toxicology
|October 1, 1989
Summary
Chemical exposure can alter cell viability assays, making fluorescent probes unreliable indicators of cellular health. Studies show increased fluorescence does not always mean healthy cells, impacting toxicant and chemotherapy assessments.
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- Accurate in vitro assessment of toxicant effects on cell viability and growth is crucial for drug development and environmental safety.
- Fluorescent probes offer rapid measurement of cell viability, but their reliability under chemical exposure is questioned.
- Common viability assays, like carboxyfluorescein diacetate (CFDA)/propidium iodide (PI), rely on metabolic activity and membrane integrity.
Purpose of the Study:
- To investigate the impact of chemical exposure on the interpretation of cell viability data obtained through fluorescent probes.
- To evaluate the validity of using CFDA fluorescence as a reliable index of cellular vigor in the presence of toxicants.
- To assess the effects of environmental toxicants and chemotherapeutic agents on cell viability and growth.
Main Methods:
- Murine erythroleukemic cells (MELC) were exposed to tributyltin (TBT), adriamycin, m-AMSA, and crisnatol.
- Cell viability was assessed using the CFDA/PI assay via flow cytometry (FCM).
- Cell growth rate and apparent cell volume (ACV) were measured over 48 hours.
Main Results:
- TBT exposure at low concentrations (0.5-1.0 microM) resulted in supranormal CF fluorescence in PI-excluding cells, indicating cellular perturbation, not vigor.
- Higher TBT concentrations (>1.0 microM) led to increased CF and PI fluorescence, coupled with growth inhibition.
- Chemotherapeutic agents also induced increased CF fluorescence but were associated with decreased growth rate and increased ACV.
Conclusions:
- Increased CF fluorescence, even in viable-excluding cells, is not a reliable indicator of cellular vigor when cells are exposed to certain chemicals.
- Chemicals can interfere with fluorescent viability assays, potentially leading to misinterpretation of cellular responses.
- Alternative or complementary methods are needed to accurately assess cell viability and perturbation under toxicant exposure.