CHEMOSENSITIVITY TESTING OF PRIMARY HUMAN RENAL-CELL CARCINOMA BY A TETRAZOLIUM BASED MICROCULTURE ASSAY (MTT)
CHEMOSENSITIVITY TESTING OF PRIMARY HUMAN RENAL-CELL CARCINOMA BY A TETRAZOLIUM BASED MICROCULTURE ASSAY (MTT)
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DOI：
10.1007/bf00302474
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发表时间：
1990-03-01
影响因子：
--
通讯作者：
ALKEN, P
中科院分区：
文献类型：
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作者：
MICKISCH, G;FAJTA, S;ALKEN, P
MTT染色程序已用于人和其他来源以及人白血病的已建立细胞系的化学敏感性测试，但目前关于其在原发性实体人类肿瘤中的应用的信息有限。我们已经评估了MTT染色在原发性人肾细胞癌（RCC），研究了各种因素干扰的最佳使用，并最终将其应用于随后的化疗敏感性测试。该方法依赖于水溶性四唑盐（MTT）转化为紫色甲瓒沉淀，这是一种仅在活细胞中活性的酶作用的反应。通过酶促分散或机械解离获得RCC的单细胞悬浮液，通过纱布过滤，并通过Ficoll密度离心纯化。在96孔微量培养板中进行试验。每孔104个活肿瘤细胞与20 μ g MTT/100 μ l总培养基体积孵育4小时产生最佳结果。 用DMSO溶解甲瓒晶体，并立即在微量培养板读数器上在540 nm处测量板。在这些标准下，可以证明系统的线性。对于化学敏感性测试，在MTT染色程序之前将细胞连续暴露于多种药物。通过在烧瓶中额外培养RCC，在1周、2周和4周后将其重新提交至MTT测定来评估和确认结果的再现性。我们的结论是，半自动MTT法提供了一个有效的，快速，可靠和简单的方法来确定在原代人RCC的耐药程度。
MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 104 viable tumor cells per well at 4 h incubation time with 20 .mu.g MTT/100 .mu.l total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.