Anti lag 3 11c3c65

Cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain kit (Invitrogen) and surface antibodies for 30 min at 4 °C. For intracellular cytokine staining, cells were treated with 50 nM phorbol-12-myristate-13-acetate (MilliporeSigma) and 250 nM ionomycin (MilliporeSigma) for 4 hours in the presence of Brefeldin A (BD Biosciences) before harvesting. Cells were washed and fixed with BD Cytofix™ Fixation Buffer (BD Biosciences) for 10 min at RT, then washed with PBS. Intracellular cytokines were stained in permeabilization buffer (eBioscience) for 30 min at 4 °C. The following antibodies were used: anti-LAG-3 (11C3C65, BioLegend), anti-PD-1 (EH12.1, BD Biosciences), anti-TIGIT (MBSA43, eBioscience), anti-Tim-3 (F38–2E2, Biolegend), anti-IFN-γ (4S.B3, eBioscience), and IL-10 (JES3–9D7, Biolegend). Cells were acquired on a BD Fortessa flow cytometer and data was analyzed with FlowJo software v10 (Threestar).