NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.
NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.
复制标题
通过流式细胞术分析 3-(4,5-二甲基噻唑基-2-基)-2,5-二苯基溴化四唑 (MTT) 还原来评估 NADH 依赖性脱氢酶活性。
DOI：
--
复制
发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Raymond Julien
中科院分区：
文献类型：
--
作者：
Olivier Huet;Jean;M. Ratinaud;Raymond Julien
MTT还原通常通过比色测定法分析，以研究线粒体脱氢酶活性作为细胞毒性试验。这种酶促反应产生深蓝色的甲瓒颗粒，增加细胞粘附性。在这项工作中，我们定义了MTT用于定量流式细胞术分析的条件。MTT还原提供了可通过该技术用于研究活细胞中的细胞内NADH依赖性脱氢酶活性的非荧光染料。我们观察到，甲瓒的生产与细胞浓度的渐近增加，这种温度依赖性米氏酶的减少基本上是由线粒体酶。在大鼠肝细胞和整个L1210小鼠白血病细胞分离的线粒体中，在呼吸底物存在下观察到的米氏常数（KM）分别为10 μ M和500 μ M。氯霉素对线粒体蛋白质合成的抑制，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测定法作为细胞毒性试验的限制。
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.
影响因子：
11.2
作者：
Douglas D. Ross;Christopher C. Joneckis;José V. Ordóñez;Allison M. Sisk;Richard K. Wu;Anne W. Hamburger;Richard E. Nora
通讯作者：
Douglas D. Ross;Christopher C. Joneckis;José V. Ordóñez;Allison M. Sisk;Richard K. Wu;Anne W. Hamburger;Richard E. Nora
影响因子：
2.2
作者：
GREEN, LM;READE, JL;WARE, CF
通讯作者：
WARE, CF
DOI：
10.1073/pnas.78.4.2383
发表时间：
1981-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DARZYNKIEWICZ, Z;STAIANOCOICO, L;MELAMED, MR
通讯作者：
MELAMED, MR