Anti cd4 l3t4 microbeads

To assess CD4⁺ T helper (Th) cell subsets from splenocytes, we measured cytokine production by enzyme-linked immunosorbent assay (ELISA). First, the splenocytes at a concentration of 1 × 10⁷ cells/ml were prepared from mice. Then, splenic CD4⁺ T cells were isolated by positive selection using anti-CD4 (L3T4) MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purified splenic CD4⁺ T cells were stimulated for 12 h with 50 ng/ml of phorbol myristate acetate (Sigma-Aldrich, St Louis, MO, USA) and 1,000 ng/ml of ionomycin (Sigma-Aldrich, St Louis, MO, USA). The supernatants were taken and the levels of IFN-γ, IL-4, and IL-17A were measured from these supernatants by ELISA kits (BD Biosciences, San Jose, CA, USA) according to the supplier’s instructions.

To isolate CD4⁺ cells, the suspension of splenocytes (1 × 10⁷ cells) was labeled with 10 μl of anti-CD4 (L3T4) Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 10 min. Thereafter, the labeled cells were separated into cell fractions that were positive or negative for CD4 by using MACS column, according to the manufacturer’s protocol (Miltenyi Biotec). We obtained 3 × 10⁵ CD4⁺ cells from 1 × 10⁷ of splenocytes. The isolated cells were further diluted 1:10 in PEB buffer [PBS, pH 7.2, 0.5% bovine serum albumin, and 2 mM EDTA; prepared by diluting MACS BSA Stock Solution 1:20 with autoMACS^® Rinsing Solution (Miltenyi Biotec)] and used for intravenous injection into naive mice.