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#MTT assay

2 public questions tagged with this topic.

Animal Tissue Culture 3

The purple color in MTT assay is due to formation of:

Purple color appearing in wells after MTT incubation arises from formation of formazan product 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan. Enzymatic reduction transfers electrons from succinate via succinate dehydrogenase, NADH and NADPH to tetrazolium ring breaking positively charged quaternary nitrogen and producing neutral formazan with extensive conjugated double bond system delocalizing electrons, absorbing green-yellow wavelengths around 570 nm and appearing purple. Crystals observable microscopically as dark needle-like precipitates clustered around nucleus and in cytoplasm before dissolution. Reaction occurs intracellularly, formazan insoluble in water precipitates. Solubilization with dimethyl sulfoxide disrupts cell membranes releasing formazan into solution yielding purple color intensity proportional to dehydrogenase activity reflecting mitochondrial integrity and overall metabolic fitness. NADH alone yellow, cytochrome c reddish, ATP colorless do not generate purple. Therefore quantification of formazan provides surrogate for viable cell number. Care required as redox-active compounds ascorbic acid and plant polyphenols can directly reduce MTT causing false positive without involving cellular enzymes. Solubilization step must avoid bubbles ensuring accurate absorbance reading and linear correlation with cell number. This knowledge strengthens laboratory safety, protocol reproducibility, and regulatory compliance critical for translational research and clinical applications, ensuring reliable data and workforce protection.

Ref: Berridge et al Arch Biochem Biophys 2005 MTT formazan formation mechanism; Abcam MTT assay protocol formazan purple product succinate dehydrogenase.

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Animal Tissue Culture 3

MTT assay measures:

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.

Ref: Mosmann T J Immunol Methods 1983 MTT viability assay; ATCC MTT Cell Proliferation Assay mitochondrial dehydrogenase reduction protocol.

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