ADCC assay.

NK cells were isolated according to the manufacturer’s protocol (Miltenyi Biotec, 130-092-657). PMNs were isolated as previously described (76). Enriched neutrophils were isolated using the EasySep human neutrophil isolation kit (Stemcell Technologies, 17957). Killing assays were performed at an effector to target ratio of 5:1 for NK cells and 50:1 for PMNs and enriched neutrophils using A431 or B16F10gp75 as target cells. Cell survival was determined using the CellTiter-Blue (CTB) Cell Viability Assay (Promega, G8080). Target cells (8000 cells/well in a flat-bottom 96-well plate) were cultured for 24 hours. The next day, culture medium was refreshed with 50 μL/well complete RPMI 1640 in the presence of antibodies (10 μL/well, 11 times the test concentration in RPMI 1640 medium) in various concentrations, i.e., 0.1, 1, and 10 μg/mL. Next, 50 μL/well freshly isolated PMNs or enriched neutrophils (8 × 10⁶ cells/mL in RPMI 1640 medium) or NK cells (8 × 10⁵ cells/mL in RPMI 1640 medium) were cocultured with the target cells for either 4 or 24 hours at 37°C and 5% CO₂. Target cells alone, effector cells alone, RPMI 1640 medium alone, and no antibody were used as controls. Flat-bottom 96-well plates were washed twice with 200 μL/well PBS, blotted dry, and incubated with 100 μL/well CTB reagent (CTB stock concentration was diluted 1:6 in complete RPMI 1640) for 1 hour at 37°C and 5% CO₂. Incubation plates were shaken at 400 rpm for 5 minutes and fluorescence of formed resorufin, proportional to the amount of living cells in the wells, was recorded by a FLUOstar Galaxy Microplate Reader (MXT Lab Systems). Resorufin was excited using a 560/10 nm excitation filter and emission was acquired using a 590/12 nm filter. Gain of the detector was set to 115 (gain 0–255). The percentage of cellular cytotoxicity was calculated in Microsoft Excel using the equation % cytotoxicity = (1 – [experimental value – medium control]/[no antibody – medium control]) × 100. Data were plotted and analyzed using GraphPad Prism 7.0.