Soluble anti cd28 37.51

Sort purified naive (CD62L⁺ CD44^lo CD25⁻) CD4 or CD8 T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Thermofisher) and Gluta-MAX in the presence of plate-bound anti-CD3 (145-2C11; Biolegend) and soluble anti-CD28 (37.51; Bio X Cell) at concentrations of 0.1 μg/ml and 0.5 μg/ml, respectively, unless specified otherwise, in multiwell tissue culture plates coated with rabbit antibody to hamster IgG (0855395; MP Biomedicals). For T_H1 polarization 10 ng/mL of IL-12 (R&D Systems) and 10 μg/mL of anti-IL-4 (11B11) were added to culture media. For T_H2 polarization 10 ng/mL IL-4 (eBioscience), 10 μg/mL anti-IL-12 (BioXCell), and 10 μg/mL anti-IFN-γ (Biolegend) were added to culture media. Following 3 days of anti-CD3 and anti-CD28 stimulation cells were removed from stimulation and rested for 3 days in conditioned medium at concentration of 0.5–1 x 10⁶ cells/mL. For CD8 T cell cultures, cells were cultured in 40 U/mL IL-2 during the resting period.
Day 6 CD4 T cells were harvested, washed in fresh media, and restimulated using 50 ng/mL PMA (Sigma) and 1 μM Ionomycin (Sigma) or plate-bound anti-CD3 (Biolegend) and soluble anti-CD28 (BioXcell) as described above for the indicated times. For intracellular cytokine analysis, Brefeldin A (Biolegend) was added to the cultures 2 hours before harvest. Supernatant was collected 24 hours post-stimulation.