High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells

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DOIResolve DOI: http://doi.org/10.1007/s00216-012-6328-5
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TypeArticle
Journal titleAnalytical and Bioanalytical Chemistry
ISSN1618-2642
1618-2650
Volume404
Issue6-7
Pages20332041
SubjectRT-CES mutagenicity test; cell growth kinetic curve; high-throughput assay; low copy number mutant cell; baseline and threshold analyses
AbstractThe mutation rate in cells induced by environmental genotoxic hazards is very low and difficult to detect using traditional cell counting assays. The established genetic toxicity tests currently recognized by regulatory authorities, such as conventional Ames and hypoxanthine guanine phosphoribosyl-transferase (HPRT) assays, are not well suited for higher-throughput screening as they require large amounts of test compounds and are very time consuming. In this study, we developed a novel cell-based assay for quantitative analysis of low numbers of cell copies with HPRT mutation induced by an environmental mutagen. The HPRT gene mutant cells induced by the mutagen were selected by 6-thioguanine (6-TG) and the cell’s kinetic growth curve monitored by a real-time cell electronic sensor (RT-CES) system. When a threshold is set at a certain cell index (CI) level, samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. The more cells that are initially seeded in the test well, the faster the cell accumulation and therefore the shorter the time required to cross this threshold. Therefore, the culture time period required to cross the threshold of each sample corresponds to the original number of cells in the sample. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells. For mutagenesis determination, the RT-CES assay displayed an equal sensitivity (p > 0.05) and coefficients of variation values with good correlation to conventional HPRT mutagenic assays. Most importantly, the RT-CES mutation assay has a higher throughput than conventional cellular assays.
Publication date
LanguageEnglish
AffiliationSecurity and Disruptive Technologies; National Research Council Canada
Peer reviewedYes
Identifier6328
NPARC number21268288
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Record identifierdf529995-c1f5-44ec-a47b-e55c8112623f
Record created2013-06-13
Record modified2016-05-09
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