Penicillin and streptomycin antibiotics

Sorted naive αβ CD8⁺ T cells were cultured for 2 h in RPMI 1640 containing 10% FCS, 50 μM 2-ME, and penicillin and streptomycin antibiotics (Sigma-Aldrich). For stimulation, 1 × 10⁶ cells were spun (1500 rpm for 5 min) onto αCD3-coated (10 μg/ml) and αCD28-coated (10 μg/ml) 48-well plates washed in PBS and incubated at 37°C 10% CO₂ before analysis. For CD8⁺ T effector differentiation cultures, cells were washed three times after stimulation and then cultured with 20 ng/ml recombinant IL-2 (ImmunoTools). For fetal thymic organ culture (FTOC), E15.5 fetal thymic lobes were cultured on Medicell membrane floated on IMDM/10% FCS, then disaggregated in accutase (PAA Laboratories) before FACS analysis. For intracellular cytokine staining, cells were stimulated with phorbol 12,13-dibutyrate (PDBu; 50 ng/ml) and ionomycin (1 μg/ml) (both Sigma-Aldrich) in the presence of Brefeldin A (5 μg/ml) (Sigma-Aldrich) for 5 h at 37°C before staining. For IL-4 intracellular staining, thymocyte suspensions were cultured for 1 h with 1.5 μM ionomycin and 50 ng/ml PMA (Sigma-Aldrich), then cultured for 2 h with the addition of Brefeldin A (5 μg/ml).