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.