A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker
A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker
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DOI：
10.1016/j.tiv.2008.02.013
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发表时间：
2008-06-01
影响因子：
3.2
通讯作者：
Xia, Menghang
中科院分区：
文献类型：
--
作者：
Cho, Ming-Hsuang;Niles, Andrew;Xia, Menghang
细胞膜完整性的测量已被广泛用于评估化学品的细胞毒性。有几种测定法可用于确定细胞膜完整性，包括使用中性红和台盼蓝染料或荧光化合物（如碘化丙啶）的差异标记技术。用于评估细胞毒性的其他常用方法是酶“释放”测定，其测量培养基中乳酸脱氢酶（LDH）、腺苷酸激酶（AK）或甘油醛-3-磷酸脱氢酶（GAPDH）的细胞外活性。然而，所有这些测定都受到几个实际限制，包括多次试剂添加、可扩展性、低灵敏度、差线性或必需的洗涤和培养基交换。我们已经开发了一种新的细胞毒性试验，其测量由于细胞膜损伤而释放的细胞内蛋白酶的活性。它允许使用发光读数进行均质的一步添加测定。我们已经将该测定优化并小型化为1536孔格式，并通过使用HEK 293和人肾系膜细胞通过定量高通量筛选（qHTS）筛选来自国家毒理学计划（NTP）的已知化合物库对其进行验证。从文库中鉴定出几种已知的和新的膜破坏物，这表明该测定法是稳健的并且适合于大规模文库筛选。这种细胞毒性试验与qHTS平台相结合，使我们能够快速有效地评估与细胞膜完整性相关的化合物毒性。(c)2008爱思唯尔有限公司保留所有权利。
Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate clehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity. (c) 2008 Elsevier Ltd. All rights reserved.