Scudiero D A, Shoemaker R H, Paull K D, Monks A, Tierney S, Nofziger T H, Currens M J, Seniff D, Boyd M R
Program Resources Inc., National Cancer Institute-Frederick Cancer Research Facility, Maryland 21701.
Cancer Res. 1988 Sep 1;48(17):4827-33.
We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.
我们之前描述了一种自动微量培养四氮唑检测法（MTA）的应用，该方法涉及将细胞产生的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）-甲臜用二甲基亚砜溶解，用于体外评估药物对细胞生长的影响（M.C. Alley等人，《美国癌症研究协会会刊》，27:389，1986；M.C. Alley等人，《癌症研究》48:589 - 601，1988）。该检测法存在几个固有的缺点，包括人员大量接触二甲基亚砜的安全风险、这种溶剂对实验室设备的有害影响，以及一些人类细胞系对MTT的代谢效率低下。认识到这些局限性促使人们开发可能的替代MTA，利用一种不同的四氮唑试剂，即2,3-双(2-甲氧基-4-硝基-5-磺基苯基)-5-[(苯基氨基)羰基]-2H-四氮唑氢氧化物（XTT），它在活细胞中代谢还原为水溶性甲臜产物。这种试剂允许直接读取吸光度，因此省去了溶解步骤并缩短了微量培养生长检测程序。大多数检测的人类肿瘤细胞系代谢XTT的效率低于MTT；然而，加入吩嗪硫酸甲酯（PMS）显著增强了细胞对XTT的还原作用。在PMS存在的情况下，XTT试剂产生了可用于多种细胞系生长和药物敏感性评估的吸光度值。根据给定细胞系的代谢还原能力，4小时XTT孵育检测的最佳条件是每孔50微克XTT和0.15至0.4微克PMS。利用XTT - PMS方法对几种标准化合物用代表性人类肿瘤细胞系获得的药物谱与用MTT获得的谱相似。加入PMS似乎对MTT的代谢影响很小。因此，新的XTT试剂提供了一种简化的体外细胞生长检测法，可能适用于细胞药理学和生物学中的各种问题。然而，使用XTT试剂的MTA仍然存在MTT或其他基于四氮唑的检测法的许多局限性和潜在陷阱。
