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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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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