The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC
The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC
复制标题
DOI：
10.1006/abbi.2000.1907
复制
发表时间：
2000-08-01
影响因子：
3.9
通讯作者：
Dobrucki, JW
中科院分区：
文献类型：
--
作者：
Bernas, T;Dobrucki, JW
尽管广泛使用的各种四唑测定，这些化合物的生物还原的机制尚未完全阐明。我们研究了四唑盐穿透完整细胞质膜的能力。氯化5-氰基-2，3-二甲苯基四唑鎓（CTC）和溴化3-（4，5-二甲基噻唑-2-基）-2，5二苯基四唑鎓（MTT）四唑鎓盐似乎代表通过不同机制还原的物质的实例。我们提供的证据表明MTT很容易穿过完整的质膜并在细胞内减少。MTT似乎减少质膜和细胞内还原酶;还原细胞不受损害，并保持代谢活性至少45分钟。相比之下，CTC仍然是细胞外相对于活细胞，因此需要质膜渗透电子载体被有效地减少。然而，在电子载体存在下CTC的还原对质膜造成损害。四唑盐的细胞内与细胞外还原位点是根据甲瓒的沉积建立的。使用荧光或反向散射光共聚焦激光显微镜检测甲瓒晶体。我们假设四唑盐穿过完整质膜的能力构成了一个重要的实验变量，需要对其进行控制，以正确解释四唑测定的结果，该测定旨在测量细胞产生的氧自由基、线粒体、胞质或外膜还原酶的活性等（C）2000 AcademicPress。
Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc. (C)2000AcademicPress.