Background information
CD4+ CD25+ immunoregulatory T cells have been shown to actively suppress immune responses against autologous and foreign antigens in vivo and in vitro . CD25, the IL-2Rα chain, is also expressed on activated CD4+ T cells, CD8+ T cells, dendritic cells, and B cells.
Detailed separation procedure
The isolation is performed in a two-step procedure. First, non-CD4+ are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies against CD8, CD11b, CD45R, CD49b, Ter-119 and Anti-Biotin MicroBeads. The labeled cells are subsequently depleted over a MACS® Column. In the second step, the flow-through fraction of pre-enriched CD4+ T cells is labeled with CD25 MicroBeads for subsequent positive selection of CD4+ CD25+ regulatory T cells (Tregs). Use our optimized protocol to save 30 minutes of handling time.
Downstream applications
Regulatory T cells isolated from mouse lymph nodes with the CD4+ CD25+ Regulatory T Cell Isolation Kit were used in coculture experiments with dendritic cells to study priming of dendritic cells for tolerance induction in vitro and after adoptive transfer of primed dendritic cells in vivo. Furthermore, they were isolated and used in various models for adoptive transfer experiments, e.g. in an experimental autoimmune thyroiditis mouse model, and a melanoma mouse model where they prevented tumor immunity. In addition, in vitro suppression assays were performed with isolated regulatory T cells.
Columns
For the first magnetic separation (depletion): LD or autoMACS® Columns. For the second magnetic separation (positive selection): MS or autoMACS Columns.
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