A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker

被引：65

作者：

Cho, Ming-Hsuang ^{[1
]}

Niles, Andrew ^{[2
]}

Huang, Ruili ^{[1
]}

Inglese, James ^{[1
]}

Austin, Christopher P. ^{[1
]}

Riss, Terry ^{[2
]}

Xia, Menghang ^{[1
]}

机构：

[1] NIH, Chem Genom Ctr, Bethesda, MD 20892 USA

[2] Promega Corp, Res & Dev, Madison, WI USA

来源：

TOXICOLOGY IN VITRO | 2008年 / 22卷 / 04期

关键词：

1536-well; NTP 1408 compound library; membrane integrity; cytotoxicity assay; protease release assay; qHTS Renal mesangial cells; HEK293; cells;

D O I：

10.1016/j.tiv.2008.02.013

中图分类号：

R99 [毒物学（毒理学）];

学科分类号：

100405 ;

摘要：

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.

引用

页码：1099 / 1106

页数：8

共 23 条

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