Peripheral blood mononuclear cells were cultured with anti-CD107a PE (Biolegend), monensin, brefeldin A, phorbol 12-myristate 13-acetate (PMA), and ionomycin (Sigma) for 4 h, then stained with anti-IFNγ FITC, anti-TNF APC, and anti-IL-2 APC-Cy7 (Biolegend or eBioscience), and analyzed by flow cytometry.
To evaluate the effect of TCR-dependent activation, PBMCs were cultured with anti-CD3 (1 µg/mL) and anti-CD28 (0.5 mg/mL). After 72 h, PBMCs were cultured with monensin, brefeldin A, and anti-CD107a PE for 6 h and then stained with anti-human CD8 APCCy7, anti-NT rabbit and anti-rabbit Alexa 647, anti-IL-2 PECy7, anti-TNF PerCPCy5.5, or anti-IFNγ PerCPCy5.5.
To evaluate the effect of TCR-dependent activation, PBMCs were cultured with anti-CD3 (1 µg/mL) and anti-CD28 (0.5 mg/mL). After 72 h, PBMCs were cultured with monensin, brefeldin A, and anti-CD107a PE for 6 h and then stained with anti-human CD8 APCCy7, anti-NT rabbit and anti-rabbit Alexa 647, anti-IL-2 PECy7, anti-TNF PerCPCy5.5, or anti-IFNγ PerCPCy5.5.