CD4<sup>+</sup>CD25<sup>+</sup> Regulatory T Cell Isolation Kit

CD4⁺CD25⁺ Regulatory T Cell Isolation Kit

Excellent tool to isolate both Treg cells and CD4⁺ T cells for functional analysis
Cells can be directly used for downstream assays
Outstanding purity and unmatched recovery

Description

The CD4⁺CD25⁺ Regulatory T Cell Isolation Kit was developed for the isolation of CD4⁺CD25⁺ regulatory mouse T cells from single-cell suspensions of spleen and lymph nodes. The isolation is performed in a two-step procedure.

Details

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 (Ly-2), CD11b (Mac-1), CD45R (B220), CD49b (DX5), 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.

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.¹ Furthermore, they were isolated and used in various models for adoptive transfer experiments², e.g. in an experimental autoimmune thyroiditis mouse model³, and a melanoma mouse model where they prevented tumor immunity⁴. In addition, in vitro suppression assays were performed with isolated regulatory T cells.^5,6

Columns

For the first magnetic separation (depletion): LD or autoMACS Columns. For the second magnetic separation (positive selection): MS or autoMACS Columns.

Figure 1

Isolation of CD4⁺CD25⁺ regulatory T cells from a mouse spleen cell suspension using the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit, an LD and two MS Columns, a MidiMACS™ Separator, and a MiniMACS™Separator.

Spleen cells before separation

CD4⁺ T cells after depletion of non-CD4⁺ T cells

CD4<sup>+</sup> T cells after depletion of non-CD4<sup>+</sup> T cells

Isolated CD4⁺CD25⁺ regulatory T cells

Isolated CD4<sup>+</sup>CD25<sup>+</sup> regulatory T cells

Figure 2

Working scheme of the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit.

CD4+CD25+ Regulatory T Cell Isolation Kit

CD4⁺CD25⁺ Regulatory T Cell Isolation Kit