Intracellular Cytokine Staining of PBMCs

This procedure was performed as described before [16 (link)]. PBMCs were incubated for one hour stimulated with anti-CD28/CD49d (1 mg/ml) in a 200μL final volume medium in round-bottom 96-well plates at 37 °C and 5% CO₂. Incubation with Brefeldin A (GolgiPlug, BD Biosciences) for 5 h was performed for further intracellular cytokines staining. Use PBS to wash cells twice and then surface marker staining was performed with with specific antibodies for 30 min at 4 °C in the dark. Fixation and Permeabilization Solution (Cytofix/Cytoperm; BD Biosciences) were added for 45 min at 4 °C in the dark. For intracellular cytokines staining, the following antibodies were used: anti-Granzyme A-PE (clone CB9; Biolegend), anti-Perforin-APC (clone dG9; Biolegend), anti-IFN-γ-PE (clone 4S.B3; Biolegend) and anti-TNF-α-APC (clone MAb11; Biolegend) according to the manufacturer's instructions. The stained cells were fixed with 2% paraformaldehyde and then analyzed by flow cytometry.