Treg cell isolation kit

Allogeneic bone marrow (BM) transplantations were performed as previously described (39 (link)). In brief, female, 6- to 8-wk-old BALB/c recipients were lethally irradiated with total body irradiation (TBI; 2 × 4.5 Gy). Directly after the second irradiation step mice received 5 × 10⁶ T cell– depleted C57BL/6 wild type (WT) BM cells for reconstitution. T cell depletion of freshly isolated BM cells was performed using CD90.2 microbeads (Miltenyi Biotec). In addition, some mice received 2 × 10⁵ CD8⁺ T cells and 2 × 10⁵ CD4⁺ CD25⁻ effector T cells with or without 6 × 10⁵ CD4⁺ CD25⁺ T_reg cells as indicated in the figure legends. All T cell subsets were isolated from complete splenocytes using the T_reg cell isolation kit (Miltenyi Biotec) and the CD8 T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions.

Treg cells were depleted in Foxp3^DTR mice by intraperitoneal injection of diphtheria toxin (DT, ip, 0.05 μg/g body weight) every three days from 3 days before SCI to 28 days after SCI. Spleens, inguinal, and axillary lymph nodes of healthy mice were collected to prepare a single-cell suspension. A Treg cell isolation kit (Miltenyi Biotec) was used to isolate CD4 + CD25 + Treg cells, and CD4 + cells were negative selected and CD25 + cells were positive selected. To transfer Treg cells into vivo, transfer was accomplished by injecting 3 × 10⁶ Treg cells into SCI mice via the tail vein of the mice. Control mice received an equal volume of PBS.