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

  • Kate Milward
  • Joanna Hester
  • Kathryn J. WoodEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1899)


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.

Key words

Regulatory T cells Treg FACS Fluorescence-activated cell sorting Cell isolation 



Current good manufacturing practice


Fluorescence-activated cell sorting


Peripheral blood mononuclear cell


Regulatory T cell



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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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe HospitalUniversity of OxfordOxfordUK

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