NADH‐dependent dehydrogenase activity estimation by flow cytometric analysis of 3‐(4,5‐dimethylthiazolyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction

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Abstract
MTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark-blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a nonfluorescent dye usable by this technique to study an intracellular NADH-dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature-dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemiia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 μM And 500 μM. The inhibition of mitochoAldrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.