Kir2dl2 3 apc

Cells were labeled with the following fluorescently conjugated human antibodies: CD56-peridinin-chlorophyll-protein complex-Cy5.5 (Bio-Legend), CD3-APC-Cy7 (BioLegend), KIR2DL1-fluorescein isothiocyanate (R&D Systems), KIR2DL2/3-APC (Miltenyi Biotec), and KIR3DL1phycoerythrin (Beckman Coulter). Cells were stained with antibodies at concentrations according to the manufacturers' guidelines and titrated when necessary. Cells were sorted with a FACSAria IIu sorter (BD Biosciences).

NK cells were labeled with fluorescently conjugated human antibodies to determine licensed and unlicensed NK cell populations: CD56-peridininchlorophyll-protein complex-Cy5.5 (BioLegend, San Diego, Calif), CD3-allophycocyanin (APC)-Cy7 (BioLegend), KIR2DL1-fluorescein isothiocyanate (R&D Systems, Minneapolis, Minn), KIR2DL2/3-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), and KIR3DL1-phycoerythrin (Beckman Coulter, Fullerton, Calif). Cells were stained with antibodies at concentrations according to the manufacturers' guidelines and titrated when necessary. For intracellular staining, cells were washed with staining buffer containing PBS and 5% FBS. Cells were fixed in 2% formaldehyde for 10 minutes and then washed and permeabilized with methanol on ice for 10 minutes. They were then washed with staining buffer, followed by staining for p38 mitogen-activated protein kinase (MAPK; D13E1) antibody (Cell Signaling Technologies, Danvers, Mass). Flow cytometry was performed on an LSR Fortessa (BD Biosciences, San Jose, Calif).