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Concentration of Rat Splenic CD4+CD25+ Regulatory T Cell by Magnetic Cell Sorting System in vitro

【Abstract】ObjectiveTo find a stable and efficient method for ex vivo concentration of CD4+CD25+ regulatory T cells in rats. MethodsCD4+CD25+ regulatory T cells were separated from the rat splenic cells in two steps by magnetic cell sorting (MACS) system. CD4+ T cells were first negatively sorted by cocktail antibodies and antiIgG magnetic microbeads. And then CD4+CD 25+T cells were positively sorted by antiCD25 PE and antiPE magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing respectively, and the immunosuppressive action of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was also assessed by in vitro cell proliferation assay. ResultsThe purity of negatively sorted CD4+ T cells were (83.6±2.5)% (79%~87%), and the purity of positively sorted CD4+CD25+ T cells was (90.2±1.8)% (86%~93%) with the survival rate of (92.8±3.4)% (92%~95%). These concentrated cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture （CD25+/CD25- versus CD25-, P＜0.01）. ConclusionWe created a twostep procedure of magnetic cell sorting system for CD4+CD25+ regulatory T cells sorting, which insures the cells to be satisfactorily purified and well functioned.