Isolation and identification of CD4^+CD25^+ regulatory T cells in rat

摘要

Objective:To establish a stable and high efficient method for collection of CD4+CD25+ regulatory T cells from rats in vitro.Methods:CD4+CD25+ regulatory T cells were isolated from the rat splenic cells through two steps by magic cell sorting(MACS)system.The first step was negative selection of CD4+T cells by cocktail antibodies and anti-IgG magic microbeads,and the second step was positive selection of CD25+T cells by anti-CD25 PE and anti-PE magic microbeads.The purity and viability of separated cells were measured by flow cytometry(FACS)and Trypan blue staining.The suppressive ability of seperated cells on the proliferation of CD4+CD25-T cells was assessed by cell proliferation assay.Results:The purity of negatively enriched CD4+ T cells was 79%-87%(83.6% ± 2.5%),and the purity of positively enriched CD4+CD25+ T cells was 86%-93%(90.2 ± 1.8%)with the viability of 92%-95%(92.8% ± 3.4%).The enriched cells significantly suppressed the proliferation of CD4+CD25-T cells in mixed lymphocyte culture(P < 0.05).Conclusion:An effective method can be established for enrichment of CD4+CD25+ regulatory T cells in two steps by MACS,with satisfied cell purity,viability and function.