Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment
Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment
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DOI：
10.1002/em.21760
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发表时间：
2013-04-01
影响因子：
2.8
通讯作者：
Dertinger, Stephen D.
中科院分区：
文献类型：
--
作者：
Bryce, Steven M.;Avlasevich, Svetlana L.;Dertinger, Stephen D.
已经描述了体外微核 (MN) 评分的自动化方法，其中流式细胞术分析与单个 96 孔板中的化合物暴露、处理和采样相结合 (Bryce SM 等人 [2010]: Mutat Res 703:191-199)。当前的报告描述了方案优化以及检测的可转移性和再现性的实验室间评估。在培训阶段，该方法得到了完善，合作实验室通过反复测试三种化合物获得了资格。其次，在四个地点分别测试了由参考基因毒物和推定非基因毒物组成的 32 种化学物质。将 TK6 细胞暴露于 10 个紧密间隔的化合物浓度中，进行 1.5 至 2 个细胞群体倍增，然后进行染色和裂解以进行流式细胞术分析。通过评估每个重复孔 5,000 个细胞来确定 MN 频率，并获得一些细胞毒性指数。阳性结果的发生率根据用于表示基因毒性结果的 MN 倍增加以及用于定义细胞毒性限度的终点而变化。通过改变这些参数，检测灵敏度和特异性值分别为 82% 至 98% 和 86% 至 97%。在第三阶段，一个实验室进一步测试了六种基因毒性剂和五种非基因毒性细胞凋亡诱导剂。在这些实验中，当最高浓度选择基于两个细胞毒性终点相对存活率和单叠氮化乙锭阳性事件的定量时，测定特异性显着提高。总的来说，结果表明小型化测定可以跨实验室转移。与传统的体外 MN 测试方法相比，96 孔格式消耗的化合物要少得多，并且流式细胞术提供的高信息内容有助于防止因明显毒性而产生不相关的阳性结果。环境。摩尔。诱变剂。 54:180194, 2013。(c) 2013 Wiley 期刊公司。
An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpointsrelative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity. Environ. Mol. Mutagen. 54:180194, 2013. (c) 2013 Wiley Periodicals, Inc.