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Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting

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Immunological Tolerance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1899))

Abstract

Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4+CD25+CD127lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25+ PBMC, which reduces the duration of the subsequent FACS isolation.

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Abbreviations

cGMP:

Current good manufacturing practice

FACS:

Fluorescence-activated cell sorting

PBMC:

Peripheral blood mononuclear cell

Treg:

Regulatory T cell

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Acknowledgements

KM holds a British Heart Foundation Research Studentship. The work from the authors’ own laboratory was supported by grants from The Wellcome Trust and European Union FP7 project The ONE Study.

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Authors and Affiliations

  1. Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK

    Kate Milward, Joanna Hester & Kathryn J. Wood

Authors
  1. Kate Milward
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  2. Joanna Hester
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  3. Kathryn J. Wood
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Corresponding author

Correspondence to Kathryn J. Wood .

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Editors and Affiliations

  1. Associate Professor in Stem Cell Biology and Regenerative Medicine, Research Department of Surgical Biotechnology, Division of Surgery and Interventional Science, University College London (UCL), Royal Free Hospital, London, UK

    Ashleigh S. Boyd

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Milward, K., Hester, J., Wood, K.J. (2019). Isolation of Human Regulatory T Lymphocytes by Fluorescence-Activated Cell Sorting. In: Boyd, A. (eds) Immunological Tolerance. Methods in Molecular Biology, vol 1899. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8938-6_4

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  • DOI: https://doi.org/10.1007/978-1-4939-8938-6_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8936-2

  • Online ISBN: 978-1-4939-8938-6

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