Anti cd4 monoclonal antibody mab

CD4 + T cells and CD11c + dendritic cells (DCs) in the draining LNs were isolated by positive selection using the magnetic activated cell sorting (MACS) system with anti-CD4 monoclonal antibody (mAb) and anti-CD11c mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) respectively. The prepared cells were 98.8 ± 0.15% pure CD4 + T cells and 97.4 ± 0.35% pure CD11c + DCs.
1 × 10⁵ CD4 + T cells and 2 × 10⁴ CD11c + DCs were cultured in the presence of 100 μg/mL of denatured CII for 96 h. Culture medium was RPMI 1640 medium (Sigma-Aldrich, MO, USA) containing 10% fetal bovine serum (FBS), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 50 μM 2-mercaptoethanol.

Spleen CD4⁺ cells were isolated by positive selection using the magnetic affinity cell sorting (MACS) system with anti-CD4 monoclonal antibody (mAb) (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4⁺ T cells (5 × 10⁵ cells/well) were cultured for 48 h in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA) containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 μg/ml of streptomycin with 1 μg/ml soluble anti-CD3ε mAb (eBioscience) and 1 μg/ml soluble anti-CD28 mAb (BioLegend).