Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.
Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.
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发表时间：
1988-09
期刊：
影响因子：
11.2
通讯作者：
D. Scudiero;R. Shoemaker;K. Paull;A. Monks;Siobhan Tierney;Thomas H. Nofziger;M. Currens;D. Seniff-D.-Senif
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作者：
D. Scudiero;R. Shoemaker;K. Paull;A. Monks;Siobhan Tierney;Thomas H. Nofziger;M. Currens;D. Seniff-D.-Senif
我们先前已经描述了自动微量培养四唑测定（MTA）的应用，其涉及细胞产生的3-（4，5-二甲基噻唑-2-基）-2，5-二苯基溴化四唑（MTT）-甲瓒的二甲亚砜增溶，以体外评估药物对细胞生长的影响（M.C. Alley等，美国药典癌症研究协会，27：389，1986; M.C. Alley等，48：589-601，1988）。该测定存在几个固有缺点，包括人员暴露于大量二甲基亚砜的安全危害、该溶剂对实验室设备的有害影响以及某些人细胞系对MTT的代谢效率低下。认识到这些局限性促使开发了可能的替代MTA，其利用不同的四唑鎓试剂2，3-双（2-甲氧基-4-硝基-5-磺基苯基）-5-[（苯基氨基）羰基] -2H-氢氧化四唑鎓（XTT），其在活细胞中代谢还原为水溶性甲瓒产物。该试剂允许直接读取吸光度，因此消除了增溶步骤并缩短了微量培养物生长测定程序。大多数人肿瘤细胞系检查代谢XTT的效率低于MTT，然而，吩嗪硫酸甲酯（PMS）的加入显着增强细胞减少XTT。在存在PMS的情况下，XTT试剂产生的吸光度值可用于各种细胞系的生长和药物敏感性评价。根据给定细胞系的代谢还原能力，4小时XTT孵育试验的最佳条件为每孔50微克XTT和0.15至0.4微克PMS。利用XTT-PMS方法，用代表性人肿瘤细胞系获得的几种标准化合物的药物谱与用MTT获得的谱相似。PMS的加入对MTT的代谢影响不大。因此，新的XTT试剂提供了一种简化的体外细胞生长测定，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仍然具有MTT或其他基于四唑的测定的许多局限性和潜在缺陷。
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.