6.2.1 Methyl Thiazolyl Tetrazolium (MTT) assay
The widely accepted and utilised colorimetric methods for assessing cell viability or cytotoxicity is the (3-(4,5-dimethylthiazol-2-yl)-2–5-diphenyltetrazolium bromide) MTT assay. By assessing the activity of succinate dehydrogenase in cells and evaluating mitochondrial function, this test primarily assesses cell viability(Lippman, 1983). In this test, NADH reduces MTT to a purple formazan, which is solubilized in DMSO and measured by light absorbance at 570 nm. The cytotoxicity of the test substances is calculated in comparison with untreated cell control(Stone et al., 2009). Since it is simple, safe, and reproducible, this method outperforms the dye exclusion methods listed above.
The inability of formazan to dissolve in water, which results in the formation of purple needle-shaped crystals inside the cells, is one of the main issues with the MTT assay. The crystals are solubilized with an organic solvent like dimethyl sulfoxide (DMSO) or isopropanol before an absorbance reading is taken. Due to the cytotoxicity of formazan, it is challenging to remove cell culture fluid from platform wells when there are floating cells with formazan crystals, which can lead to serious errors. Additional control tests should be performed in order to prevent false positive or false negative results from background interference brought on by particle inclusion. The vitality of the cell might be overestimated as a result of this effect.