Huet O, Petit J M, Ratinaud M H, Julien R
Institut de Biotechnologie, Faculté des Sciences, Limoges, France.
Cytometry. 1992;13(5):532-9. doi: 10.1002/cyto.990130513.
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.
MTT还原通常通过比色法分析，以研究线粒体脱氢酶活性作为细胞毒性测试。这种酶促反应产生深蓝色的甲臜颗粒，增加细胞折光率。在这项工作中，我们定义了MTT在定量流式细胞术分析中的使用条件。MTT还原提供了一种该技术可用的非荧光染料，用于研究活细胞中依赖细胞内NADH的脱氢酶活性。我们观察到甲臜的产生随细胞浓度呈渐近增加，并且这种温度依赖性的米氏酶促还原主要由线粒体脱氢酶产生。在大鼠肝细胞的分离线粒体和整个L1210小鼠白血病细胞中，在存在呼吸底物的情况下观察到的米氏常数（KM）分别为10微摩尔和500微摩尔。氯霉素抑制线粒体蛋白质合成，由于糖酵解的相关刺激（巴斯德效应）导致MTT还原增加，这是MTT法作为细胞毒性测试的一个局限。
