The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant...

Area of Science:

• Pharmacology
• Cell Biology
• Biochemistry

Background:

• The MTT assay is a common method for assessing cell viability and cytotoxicity.
• Plant extracts are frequently evaluated for their cytotoxic potential using such assays.
• Potential interference between plant compounds and assay reagents can compromise results.

Purpose of the Study:

• To investigate the reliability of the MTT assay when used with methanol plant extracts.
• To compare MTT assay results with a more sensitive ATP assay and morphological assessments.
• To identify potential interferences affecting MTT assay outcomes in plant-based cytotoxicity studies.

Main Methods:

• Methanol extracts of three plants (Hypericum adenotrichum, Salvia kronenburgii, Pelargonium quercetorum) were tested on MCF-7 and MDA-MB-231 breast cancer cell lines.
• Cell viability was assessed using the MTT assay and compared with the ATP assay.
• Cell density and morphology were confirmed using phase-contrast microscopy and Hoechst 33342 fluorescence staining.

Main Results:

• The MTT assay consistently yielded significantly higher viability percentages compared to the ATP assay.
• False increases in viability were observed even when complete cell death was expected.
• Morphological evaluations using microscopy and Hoechst staining correlated well with ATP assay results, not MTT.
• Plant extracts likely interfere with the MTT assay, possibly due to reducing compounds interacting with MTT tetrazolium.

Conclusions:

• The MTT assay may not be suitable for cytotoxicity testing of certain plant extracts due to significant interference.
• Plant compounds can artificially convert MTT tetrazolium to formazan, leading to overestimated viability.
• Researchers should exercise caution and consider alternative, interference-free assays like the ATP assay when evaluating plant extracts.