Vistica D T, Skehan P, Scudiero D, Monks A, Pittman A, Boyd M R
Laboratory of Drug Discovery Research and Development, National Cancer Institute, Frederick, Maryland 21702-1201.
Cancer Res. 1991 May 15;51(10):2515-20.
The hydrogen acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) is commonly utilized to estimate cellular viability in drug screening protocols. The present investigation was prompted, in part, by observations that reduction of MTT to its colored reaction product, MTT formazan, varied between cell lines and with culture age. A correlation was established between the D-glucose concentration of the culture medium at the time of assay and the production of MTT formazan for cell lines representing seven tumor histologies. A decrease in the concentration of D-glucose from culture medium was accompanied by a decrease in MTT specific activity (MTT formazan/microgram cell protein) for a number of cell lines. Cells which extensively metabolized D-glucose exhibited the greatest reduction in MTT specific activity. Further evidence that the D-glucose concentration of the culture medium played an important role in MTT reduction was provided by experiments which demonstrated that transfer of cells to a glucose-free medium (L-15) was accompanied by an immediate decrease in MTT reduction which was pH independent. These studies suggested that cellular transport and constant metabolism of glucose were required for maximum MTT reduction. Decreases in the cellular concentration of the reduced pyridine nucleotides NADH and NADPH were accompanied by concomitant decreases in MTT formazan production. MTT formazan varied significantly among cell lines in both the kinetics of its formation and the degree of saturability exhibited. Apparent IC50 values for Adriamycin varied, in a cell line-specific manner, with MTT exposure time. These results indicate that MTT specific activity is significantly influenced by a number of parameters and suggest that assay conditions should be established which minimize their effects.
氢受体3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑(MTT)常用于药物筛选方案中评估细胞活力。本研究部分是由以下观察结果引发的：MTT还原为其显色反应产物MTT甲臜的过程在不同细胞系之间以及随着培养时间而有所不同。对于代表七种肿瘤组织学类型的细胞系，测定时培养基中D-葡萄糖浓度与MTT甲臜的产生之间建立了相关性。许多细胞系中，培养基中D-葡萄糖浓度的降低伴随着MTT比活性(MTT甲臜/微克细胞蛋白)的降低。大量代谢D-葡萄糖的细胞表现出MTT比活性的最大降低。将细胞转移至无葡萄糖培养基(L-15)时，MTT还原立即降低且与pH无关，这一实验进一步证明了培养基中D-葡萄糖浓度在MTT还原中起重要作用。这些研究表明，葡萄糖的细胞转运和持续代谢是MTT最大程度还原所必需的。还原型吡啶核苷酸NADH和NADPH细胞浓度的降低伴随着MTT甲臜产生的相应降低。MTT甲臜在其形成动力学和表现出的饱和程度方面在不同细胞系之间有显著差异。阿霉素的表观IC50值因细胞系而异，且与MTT暴露时间有关。这些结果表明，MTT比活性受多种参数的显著影响，并建议应建立能将其影响降至最低的测定条件。
