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Toxicol In Vitro. 2008 Jun;22(4):1099-106. doi: 10.1016/j.tiv.2008.02.013. Epub 2008 Mar 4.

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

Author information

1
NIH Chemical Genomics Center, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892-3370, USA.

Abstract

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 dehydrogenase (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.

PMID:
18400464
PMCID:
PMC2386563
DOI:
10.1016/j.tiv.2008.02.013
[Indexed for MEDLINE]
Free PMC Article

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