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

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Abstract

The 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.

Quantitative analysis of low copy number mutant cells with cell growth kinetic curves. a Low copy number HPRT gene mutant cells are growning on electronic chip and the cell growth kinetic curve is monitored by a real-time cell electronic sensor (RT-CES) system. b When set up a threshold is set at certain level of cell index (CI), the samples with different initial mutant cell copies take different amounts of time in order for their growth (or CI accumulation) to cross this threshold. A mutant cell growth time threshold (MT) value of each sample can be calculated to predict the number of original mutant cells

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Acknowledgments

We would like to thank Alberta Health & Wellness for providing the funding for this study. We would also like to thank Mr. Scott MacKay for providing valuable input during the revision of the manuscript.

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Authors and Affiliations

  1. Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada

    James Z. Xing

  2. Department of Biomedical Engineering, University of Alberta, Edmonton, Alberta, T6G 2V2, Canada

    Min Huang & Jie Chen

  3. Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Alberta, T6G 2V4, Canada

    Jie Chen

  4. National Institute for Nanotechnology, Edmonton, Alberta, T6G 2M9, Canada

    Jie Chen

  5. Department of Chemical Material Engineering, University of Alberta, Edmonton, Alberta, T6G 2V4, Canada

    Biao Huang & Tianhong Pan

  6. Alberta Health and Wellness, Edmonton, Alberta, T5J 2N3, Canada

    Stephan Gabos

Authors
  1. James Z. Xing
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  2. Stephan Gabos
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  3. Biao Huang
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  4. Tianhong Pan
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  5. Min Huang
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Correspondence to James Z. Xing.

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Xing, J.Z., Gabos, S., Huang, B. et al. High-throughput quantitative analysis with cell growth kinetic curves for low copy number mutant cells. Anal Bioanal Chem 404, 2033–2041 (2012). https://doi.org/10.1007/s00216-012-6328-5

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  • DOI: https://doi.org/10.1007/s00216-012-6328-5

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