NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.
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 non-fluorescent 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 leukemia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 microM and 500 microM. The inhibition of mitochondrial 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.
Huet
O
O
Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Petit
J M
JM
Ratinaud
M H
MH
Julien
R
R
D000818:Animals; D002701:Chloramphenicol; D005434:Flow Cytometry / Q000379:methods; D006019:Glycolysis; D007939:Leukemia L1210 / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D013696:Temperature; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000378:metabolism / Q000473:pathology
D013778:Tetrazolium Salts; D013844:Thiazoles; D002701:Chloramphenicol; D009245:NADH Dehydrogenase; C022616:thiazolyl blue