Cd107a pe h4a3

PBMC samples were thawed and washed with RPMI-1640 (Corning Cellgro) containing 10% fetal bovine serum (heat-inactivated), 2mM L-Glutamine and antibiotics (penicillin [100 units/ml] and streptomycin [100mg/ml]) (Gibco BRL/Life technologies) and incubated at 37°C with 5% CO₂ for 4 hours. Four to eight million PBMC were stained for mass cytometry analyses, which were performed as described (24 ) using the 39 antibodies listed in Table S1 as well as cisplatin (used as a cell viability reagent). NK cells were identified with a serial gating strategy as described (36 (link)) (Figure S7). The data were acquired with a CyTOF 2^® instrument (Fluidigm, Inc.) and analyzed using FlowJo software v9.4.8 (Treestar, Inc) and Cytobank (Cytobank, Inc). Spanning-tree progression analysis of density normalized events (SPADE) analyses were performed on 20,000 CD94:NKG2A⁺ NK cells from each donor using Cytobank and were restricted to a limit of 1,000 phenotypes. For functional assays, NK cells were gated on using anti-human antibodies (BD Biosciences): CD3 FITC (UCHT1), CD56 PE-Cy7 (NCAM 16.2). NK cell function was measured by using antibodies (BD Biosciences): CD107a PE (H4A3) and IFN-γ APC (4S.B3). Samples were acquired on a BD Accuri flow cytometer (BD Biosciences).