Anti mouse cd3ε antibody 145 2c11

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).