H 2db

Only fresh cultured cells or tissue prepared into single cell suspensions were analyzed. The harvest and digestion of MOC2 tumors into single cell suspensions for analysis was performed as previously described [18 ]. Nonspecific surface staining was minimized by staining with CD16/32 (FcR) blocking antibodies for 10 min prior to the addition of primary antibodies when applicable. Anti-mouse NK1.1, CD3, CD16, NKG2D, CD44, CD27, PD-L1, PD-1, Tim-3, CTLA-4, H-2K^b, H-2D^b, pan-RAE, H60 and MULT-1 antibodies were from Biolegend. For some experiments, pre-treatment of cells with interferon γ (IFNγ) (20 ng/mL × 24 h) or YAC cells were used as positive controls. Primary antibodies were applied for 30–60 min at concentrations titrated for each antibody. Dead cells were excluded via 7AAD uptake and a “fluorescence-minus-one” technique was used to validate specific staining in all antibody combinations. Intracellular staining was performed with the eBioscience Intracellular Fixation and Permeabilization Buffer Set per manufacturer protocol. Granzyme B and IFNγ antibodies were from Biolegend. All analyses were performed on a BD FACSCanto analyzer running FACSDiva software and interpreted using FlowJo (vX10.0.7r2).