Low 4 igg serum

ADCC activity was measured using a reporter bioassay (Promega; ADCC Reporter Bioassay, #G7010). The ADCC bioassay was carried out according to the manufacturer’s protocol. Briefly, cells were seeded at 5,000 cells per well in a 96-well clear bottom black tissue culture plates (Corning; #3904) in low 4% IgG-serum (Promega; #G711A) containing media. Serial dilutions of primary antibody were incubated in triplicate wells for approximately 15 min at 37 °C, 5% CO₂. Following incubation, engineered effector cells were added to the wells at approximately 150,000 cells per well. After more than 5 h (or as indicated in results), Bio-Glo Luciferase Assay Substrate (Promega; #G719A and #G720A) was added to the wells and luminescence was measured using the Infinite 200 microplate reader (Tecan). Experiment was carried out twice (2 biological repeats) with 3 technical replicates for each condition.

ADCC activity was measured using the ADCC Reporter Bioassay (Promega). Briefly, cells were seeded at 5,000 cells per well in a 96-well clear bottom black tissue culture plates (Corning) in low 4% IgG-serum (Promega) media. Serial dilutions of primary antibody were incubated in triplicate wells for approximately 15 min at 37° C, 5% CO2. Following incubation, engineered effector cells were added to the wells at approximately 150,000 cells per well. After 5 to 16 h (as indicated in results), Bio-Glo™ Luciferase Assay Substrate (Promega) was added to the wells and luminescence was measured using the Infinite^® 200 microplate reader (Tecan). Estimated EC₅₀ values were calculated by linear regression analysis using GraphPad Prism 6 (GraphPad).