Anti cd107a buv395 h4a3

NK cells were cultured overnight with 100 IU/ml of IL-2 (Proleukin, Novartis Pharmaceuticals) or non-stimulated (resting) in 0.1 million cells/well before addition of 0.1 million K562 cells/well. Anti-CD107a-BUV395 (H4A3, BD Biosciences) was added to measure the degranulation. After 4 h-incubation, cells were stained with anti-CD56-BV711 (NCAM 16.2, BD Biosciences), anti-NKG2A-PE (Z199), anti-KIR2DL1/S1-PE-Cy7 (EB6B), anti-KIR2DL2/L3/S2-PE-Cy5.5 (GL183; all Beckman Coulter) and anti-KIR3DL1-APC (DX9, Biolegend). Gating strategy is shown in Supplementary Fig. 1.
In intracellular cytokine assays, 50 000 NK cells/well were cultured and stimulated overnight, using same conditions as in degranulation assays. A five-hour co-culture with 50 000 K562 cells/well at 37 °C was performed, adding Brefeldin A (GolgiPlug, BD Biosciences) after one hour. The cells were stained with the same antibody panel as mentioned above, followed by fixation and permeabilization with BD Cytofix/Cytoperm (BD Biosciences). At the end, cells were stained with anti-IFNγ-BUV395 (B27) and anti-TNFα-AF488 (Mab 11; both BD Biosciences).
Stained samples were analyzed using a five laser BD LSR Fortessa SORP instrument. Data was analyzed using FACSDiva (v.8.0.1 or later) and FlowJo (v.10.8.1) software (BD Biosciences).