Fitc conjugated anti ifn γ antibody

PBMCs from patients exhibiting more than 3% of PD-1-positive NK cells were used for functional assays. In brief, PBMCs were incubated (5 × 10⁵ per U-bottom well) in the presence or the absence of K562 target cells (5 × 10⁴ per U-bottom well), plate-bound anti-NKp30 or anti-NKp46 mAbs (10 μg/ml, R&D Systems) or respective isotype control, or PMA (50 ng/ml) plus ionomycin (1 μg/ml) for 5 hr. FITC-conjugated anti-CD107a (BD Biosciences) was added directly at the beginning of the experiment. After 1 hr at 37°C in 5% CO2, brefeldin A (1 μg/ml) and monensin (6 μg/ml, Sigma) were added for additional 4 hrs. Cells were then harvested, washed, and stained with APC-conjugated anti-PD-1 antibody, followed by Pacific Blue-conjugated anti-CD3, PE-Cy7-conjugated anti-CD56, and 7-AAD. For intracellular IFNγ analysis, cells were fixed following surface staining, permeabilized with 0.2% saponin and stained with FITC-conjugated anti-IFN-γ antibody (BD Biosciences) for an additional 30 min. Where indicated, cells were preactivated overnight with IL-2 (100 U/ml) or IL-15 (50 ng/ml) before stimulation. For mAb-induced redirected degranulation experiments, PBMCs were incubated for 5 hours with untransfected P815 or P815.PD-L1 cells preincubated with anti-CD16 mAb (10 μg/ml, BD Biosciences) or control isotype (effector:target ratio 10:1), and processed as above.