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

The MTT assay is one of the often used cell viability/cytotoxicity assays. However, when the methanol extracts of plantsare used to test their cytotoxic potential, interference may occur, resulting in false-positive viability results. Therefore, in this study,the reliability of the MTT assay was investigated in the case of plant use. The methanol extracts of three different plants ( Hypericumadenotrichum ,Salvia kronenburgii , andPelargonium quercetorum ) were tested in breast cancer cell lines (MCF-7 and MDA-MB-231)using the MTT assay and the results were compared to the ATP assay, which is a much more sensitive and reliable assay due to itsinterference-free feature. Additionally, decreased cell density was confirmed with phase-contrast microscopy and fluorescence staining(Hoechst 33342 dye). Although both of the viability/cytotoxicity assays are considered as metabolic assays, viabilities (in %) in the MTTassay were found to be strikingly higher when compared to the results with the ATP assay. Even in the case of total death, the MTTassay still produced artificial/false increases in viability. The morphology-based evaluation of viability/cytotoxicity by phase-contrastmicroscopy and Hoechst 33342 staining were greatly compatible with the ATP assay results. Overestimated (false) viabilities in the MTTassay suggests a serious interference between the MTT assay itself and the extracts used. Some ingredients of plants may have reducingactivity (like the dehydrogenase activity of the cells) that converts the MTT compound into the colored formazan that is the principleof the assay. Therefore, the MTT assay may not be a suitable assay for some plant extracts, urging great caution when plants are used.

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