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Flow Cytometric Quantification of Mutant T Cells with Altered Expression of the T-Cell Receptor: Detecting Somatic Mutants in Humans and Mice

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

Abstract

Spontaneously generated mutant T cells defective in T-cell receptor (TCR) gene expression are detectable at the frequency of 2×10−4 in vivo, and the mutant fractions are dose dependently increased by exposure to genotoxic agents such as ionizing radiation. Mutant cells with altered expression of TCRα or -β among CD4+ T cells can be detected as CD3−/CD4+ cells by two-color flow cytometry using anti-CD3 and anti-CD4 monoclonal antibodies labeled with different fluorescent dyes, because incomplete TCRαβ/CD3 complexes cannot be transported to the cellular membrane. This flow cytometric mutation assay can be applied to CD4+ T cells from human peripheral blood and mouse spleen. Methods for both preparation of target cells and detection of the mutant cells are described.

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Acknowledgements

The authors would like to acknowledge M. Yamaoka and K. Koyama for excellent technical help and C. A. Waldren for valuable suggestions.

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Authors and Affiliations

  1. Laboratory of Immunology, Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami Ward, Hiroshima, 732-0815, Japan

    Seishi Kyoizumi

  2. Laboratory of Immunology, Department of Radiobiology, Radiation Effects Research Foundation, Hiroshima, Japan

    Yoichiro Kusunoki & Tomonori Hayashi

Authors
  1. Seishi Kyoizumi
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  2. Yoichiro Kusunoki
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  3. Tomonori Hayashi
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Corresponding author

Correspondence to Seishi Kyoizumi .

Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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© 2014 Humana Press

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Kyoizumi, S., Kusunoki, Y., Hayashi, T. (2014). Flow Cytometric Quantification of Mutant T Cells with Altered Expression of the T-Cell Receptor: Detecting Somatic Mutants in Humans and Mice. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_19

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_19

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

  • eBook Packages: Springer Protocols

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