Funk Dorothee, Schrenk Hans-Hermann, Frei Eva
German Cancer Research Center, Heidelberg, Germany.
Biotechniques. 2007 Aug;43(2):178, 180, 182 passim. doi: 10.2144/000112528.
Tetrazolium salts like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) or sodium 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) that form formazans after reduction are widely used to investigate cell viability. Besides cellular enzymes, some constituents of cell media and other substances reduce tetrazolium salts, thereby interfering with these assays. We describe here that different preparations of serum albumin from bovine or human origin can lead to a concentration-dependent increase in the signals of the XTT assay; therefore leading to an overestimation of cell numbers and to an underestimation of potential cytotoxic effects of compounds to be tested. The same effect was seen in the MTT assay with human serum albumin (HSA). We demonstrate that this reductive activity cannot be inactivated by proteolytic digestion, but that it is due to the free cysteine residue in albumin, and is also observed when cysteine or glutathione (GSH) are used. Binding of N-ethylmaleimide (NEM) to the free cysteine residue leads to a decrease of the albumin interference in the XTT assay.
像3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐（MTT）或2,3-双-(2-甲氧基-4-硝基-5-磺基苯基)-2H-四唑-5-羧基苯胺钠盐（XTT）这样在还原后形成甲臜的四唑盐被广泛用于研究细胞活力。除了细胞内的酶之外，细胞培养基的一些成分以及其他物质也会还原四唑盐，从而干扰这些检测。我们在此描述，来自牛源或人源的不同血清白蛋白制剂可导致XTT检测信号呈浓度依赖性增加；因此导致对细胞数量的高估以及对受试化合物潜在细胞毒性作用的低估。在使用人血清白蛋白（HSA）的MTT检测中也观察到了同样的效应。我们证明这种还原活性不能通过蛋白水解消化使其失活，而是由于白蛋白中的游离半胱氨酸残基，并且当使用半胱氨酸或谷胱甘肽（GSH）时也会观察到这种现象。N-乙基马来酰亚胺（NEM）与游离半胱氨酸残基结合会导致白蛋白对XTT检测的干扰降低。
