Cells were treated with CD16/32 antibody (93: BioLegend, San Diego, CA, USA) to block non-specific mAb binding, and then stained with the following fluorochrome-conjugated antibodies: CD8α (53–6.7: BioLegend), α-PD-1 (29F.1A12: BioLegend), α-MHC I (H-2Kb) (AF6-88.5.5.3: eBioscience, San Diego, CA, USA), α-OVA257-264 (SIINFEKL) peptide bound to H-2Kb (eBio25-D1.16: eBioscience), CD117 (c-kit) (2B8: BioLegend), CD11b (M1/70: BioLegend), CD45.1 (A20: BioLegend), and CD45.2 (104: BioLegend). OVA-specific CTLs were stained with PE-conjugated H-2Kb OVA Tetramer-SIINFEKL (MBL, Nagoya, Japan) and Alexa647-CD8 (KT15: MBL) according to the manufacturer’s instructions. Cells were analyzed on FACS Aria II or FACS Canto II (BD Biosciences).
For the analysis of cytokine production, cells were cultured in the medium containing 10% FBS, 45% RPMI1640 medium, 45% AIM-V medium, and 5 μg/ml of Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) with or without 7.5 μg/ml of SIINFEKL peptide for 4 hours at 37°C. Cells were then fixed and stained for intracellular cytokines using the Cytofix/Cytoperm kit (BD Biosciences) and antibodies against IFN-γ (XMG1.2: BD Biosciences) and TNF-α (MP6-XT22: BioLegend).
For the analysis of cytokine production, cells were cultured in the medium containing 10% FBS, 45% RPMI1640 medium, 45% AIM-V medium, and 5 μg/ml of Brefeldin A (Sigma-Aldrich, St. Louis, MO, USA) with or without 7.5 μg/ml of SIINFEKL peptide for 4 hours at 37°C. Cells were then fixed and stained for intracellular cytokines using the Cytofix/Cytoperm kit (BD Biosciences) and antibodies against IFN-γ (XMG1.2: BD Biosciences) and TNF-α (MP6-XT22: BioLegend).