Mouse cd4 cd25 regulatory t cell isolation kit

For the suppression assay, T_eff and T_regs were cultured on feeder cells derived from RAG1^−/− mice. These cells were treated with Low-Tox^®-M Rabbit Complement (Cedarlane) and an anti-mouse CD90.2 antibody (30-H12; BioLegend). Afterwards, 5 × 10⁴ feeder cells were seeded into 96-well roundbottom plates (BD Falcon). CD4⁺CD25⁻ Teff and CD4⁺CD25⁺ Tregs were isolated from naive Anp32b KO and wild type mice or EAE treated KO an wild type animals (day 60) using the mouse CD4⁺CD25⁺ Regulatory T cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Purity of sorted cells was assessed by FACS directly after isolation and revealed ≥92%. T_eff were labeled with 0.75 µM CellTraceTM Violett (CellTraceTM Violett Cell Proliferation Kit; molecular probes by life technologiesTM) per 10⁶ cells and 5 × 10⁴ labeled T_eff per well were added to the RAG1^−/− feeder cells. Finally, T_regs were plated onto these cells in titrated numbers and co-cultures were stimulated using 0.25 mg/ml of an anti-mouse CD3ε antibody (145-2C11; BioLegend) and incubated for 72 h before percentage of proliferating cells was determined by FACS. Results were analyzed using FCS Express Flow Cytometry Software (De Novo).

For human T cells, PBMCs were isolated by centrifugation of blood over a Ficoll (Biotest, Dreieich, Germany) layer. Overall, 10 ml of Ficoll was loaded under 20 ml blood diluted with 20 ml phosphate-buffered saline (PBS) and centrifuged at 400 × g for 20 min at room temperature. PBMCs were harvested and washed once with PBS. Total CD4⁺ T cells, naive CD4⁺ T cells, CD25^- CD4⁺ T cells and CD25⁺ CD4⁺ T cells were purified from human PBMCs by magnetic cell separation (MACS) using the Human CD4⁺ T Cell or CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec).
For murine T cells, spleens were homogenized into a single-cell suspension using gentleMACS™ C Tubes and a gentleMACS™ Dissociator (Miltenyi Biotec). Cell populations were purified from splenocytes either by MACS or by fluorescence-activated cell sorting (FACS) on a MoFlo (Beckman Coulter, Brea, CA). Total CD4⁺ T cells were purified using the Mouse CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). CD25⁺ CD4⁺ and CD25⁻ CD4⁺ T cells were purified using the Mouse CD4⁺ CD25⁺ Regulatory T Cell Isolation Kit (Miltenyi Biotec). The purity of the cell populations was evaluated by flow cytometry.