Fitc labeled mouse anti human cd107a clone h4a3

Single cell suspensions of liver and spleen tissue were cocultured with K562 or Raji cells at a ratio of 10:1 in RPMI 1640 (GIBCO, Thermo Fisher Scientific) with 10% FCS in a humidified incubator at 37°C and 5% CO₂. Culture medium contained FITC-labeled mouse anti-human CD107a (clone H4A3, BD Bioscience) antibody and monensin was added after 1 hour of coculture. For the ADCC assay, Raji cells were incubated with Rituximab (MabThera, Roche) at a concentration of μg/ml for 10min at 37°C prior to the addition of effector cells. For the stimulation with IL-12 (20ng/ml, Biolegend) and IL-18 (10ng/ml, Biolegend), the culture medium was supplemented with metalloproteinase inhibitor TAPI-1 (5 μg/ml, Tocris), and monensin and Brefeldin A was added after 1 hour of stimulation. After a total of 5 hours, cells were washed, stained, and analyzed on a BD LSR II Fortessa or BD FACSymphony A5 flow cytometers as described above.