Raveh D, Huberman E
Mutat Res. 1983 Oct;113(6):499-506. doi: 10.1016/0165-1161(83)90004-3.
6-Thioguanine-resistant mutants can be efficiently recovered from Chinese hamster V79 cells incubated at high cell densities in microtiter plates (10(3)-10(4) cells/0.2 ml growth medium/0.4 cm2) when selected with 30 microM 6-thioguanine and 0.1 microgram/ml phorbol-12-myristate-13-acetate, an inhibitor of metabolic cooperation among V79 cells. Mutant frequencies in the microtiter plates were calculated from a direct count of mutant colonies. After treatment of the V79 cells with the carcinogen benzo[a]pyrene in a fibroblast-mediated assay, the mutation frequencies determined with the microtiter assay system were quantitatively similar to those obtained with a conventional procedure in which selection with 6-thioguanine was performed in petri dishes. The mutagenic activities of 3 polycyclic aromatic hydrocarbons (activated in the cell-mediated assay) were assessed with the microtiter plate selection procedure. The active carcinogen benzo[a]pyrene at 1 microgram/ml yielded about 100 mutants per 10(5) colony-forming cells. The same dose of a less active carcinogen, cyclopenta[c,d]pyrene, yielded about 20 mutants per 10(5) colony-forming cells, and benz[a]anthracene, not an active carcinogen, was inactive as a mutagen at all doses tested. Because of the small requirements for growth medium and tissue culture vessels compared with other assays, this microtiter plate assay can serve as an inexpensive system for detecting the mutagenic activity of environmental chemicals in mammalian cells.
当用30微摩尔6-硫鸟嘌呤和0.1微克/毫升佛波醇-12-肉豆蔻酸酯-13-乙酸盐（V79细胞间代谢协同作用的抑制剂）进行筛选时，在微量滴定板中以高细胞密度（10³-10⁴个细胞/0.2毫升生长培养基/0.4平方厘米）培养的中国仓鼠V79细胞中，可以高效回收6-硫鸟嘌呤抗性突变体。微量滴定板中的突变频率通过直接计数突变菌落来计算。在用致癌物苯并[a]芘在成纤维细胞介导的试验中处理V79细胞后，用微量滴定试验系统测定的突变频率在数量上与在培养皿中用6-硫鸟嘌呤进行筛选的传统方法所获得的结果相似。用微量滴定板筛选程序评估了3种多环芳烃（在细胞介导的试验中被激活）的诱变活性。1微克/毫升的活性致癌物苯并[a]芘每10⁵个集落形成细胞产生约100个突变体。相同剂量的活性较低的致癌物环戊[c,d]芘每10⁵个集落形成细胞产生约20个突变体，而苯并[a]蒽不是活性致癌物，在所有测试剂量下均无诱变活性。由于与其他试验相比，对生长培养基和组织培养容器的需求较小，这种微量滴定板试验可以作为一种廉价的系统，用于检测环境化学物质在哺乳动物细胞中的诱变活性。
