Rat anti cd28 mab

Isolated PBMCs were divided into six groups and cultured in different media at 1 × 10⁶ cells/ml: 10% control serum + 90% RPMI-1640 medium for the control, model, and hydrocortisone (HC) groups; 10% KJL containing serum + 90% RPMI-1640 medium for the KDH group; 5% KJL containing serum + 5% control serum + 90% RPMI-1640 medium for the KDM group; and 2.5% KJL containing serum + 7.5% control serum + 90% RPMI-1640 medium for the KDL group. Rat anti-CD3 mAb (BioLegend, Inc., USA) and rat anti-CD28 mAb (BD Pharmingen, USA) were used for activation of the T cell in the PBMCs. The 24-well culture plates for PBMC culture were precoated with 5 μg/ml anti-CD3 mAb at 4°C overnight. Anti-CD28 mAb was added into the medium at a final concentration of 2 μg/μl. Polarization towards Th17 cells was induced with 5 ng/ml transforming growth factor-β (TGF-β), 20 ng/ml IL-6, and 25 ng/ml IL-23 (all PeproTech, Inc., USA) in all groups except for the control group, to cause an imbalance between Th17 and Treg cells. For the HC group, 20 ng/ml HC was added into the medium. All groups of cells were cultured under conditions of 5% CO₂ and 37°C for 48 h prior to subsequent experiments.

Isolated PBMCs were divided into four groups and cultured with different media at 1 × 10⁶ cells/ml: 10% control serum + 90% RPMI-1640 medium for the control group; 10% KJL-containing serum + 90% RPMI-1640 medium for the high KJL dose (KDH) group; 5% KJL-containing serum + 5% control serum + 90% RPMI-1640 medium for the medium KJL dose (KDM) group; and 2.5% KJL-containing serum + 7.5% control serum + 90% RPMI-1640 medium for the low KJL dose (KDL) group. Rat anti-CD3 mAb (BioLegend, Inc., USA) and rat anti-CD28 mAb (BD Pharmingen, USA) were used for activation of the T cells in the PBMCs. The 96-well culture plates for PBMC culture were precoated with 5 μg/ml anti-CD3 mAb at 4°C overnight. Anti-CD28 mAb was added into the medium at a concentration of 2 μg/μl. The cell viability at 12, 24, and 48 h was analyzed using an MTT Cell Proliferation and Cytotoxicity Assay kit (Beijing Solarbio Science & Technology Co., Ltd., China).