Practice question

MTT assay developed by Tim Mosmann in 1983 measures cell viability and proliferation based on reduction capacity of living cells. Principle uses yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide which enters cells via endocytosis and is reduced by mitochondrial succinate dehydrogenase of complex II and cytosolic NAD(P)H-dependent oxidoreductases cleaving tetrazolium ring with electrons from NADH and NADPH producing insoluble purple formazan crystals accumulating intracellularly in mitochondria and lipid droplets. Metabolically incompetent dead cells fail to reduce MTT. After incubation 1-4 hours, medium removed and crystals solubilized in dimethyl sulfoxide or acidified isopropanol producing homogeneous purple solution quantified spectrophotometrically at 570 nm with reference 630 nm. Absorbance directly proportional to number of viable cells within linear range. Assay does not measure migration requiring Boyden chamber, DNA synthesis requiring BrdU incorporation, or specific protein expression requiring immunoassay. Applications include cytotoxicity screening, IC50 determination, chemosensitivity testing, and biocompatibility evaluation requiring validation with orthogonal methods. Proper controls including cell-free blanks and vehicle treatments correct for nonspecific dye reduction artifacts. This knowledge strengthens laboratory safety, protocol reproducibility, and regulatory compliance critical for translational research and clinical applications, ensuring reliable data and workforce protection.