Adcc reporter bioassay kit

Our ADCC reporter system consisted of an Fc region of an antibody that binds to the FcγRIIIa receptor expressed on an engineered, immortalized Jurkat T-lymphocyte cell line. This binding can activate gene transcription through the nuclear factor of activated T cells pathway, inducing the expression of firefly luciferase.^{38 (link), 39 (link)} This FcγRIIIa stimulation assay can be used to evaluate the ability of an antibody to activate the ADCC reporter gene. Here we performed the FcγRIIIa stimulation assay using an ADCC Reporter Bioassay kit from Promega according to the manufacturer's instructions. In brief, H9/IIIB or 293 T cells expressing HIV-1 Env (2.5 × 10⁴ cells in 25 μL) as the target cells were seeded in each well of a 96-well flat-bottomed culture plate. A measure of 50 μL of serially diluted antibody was added to the culture plates. As the effector cells, engineered, immortalized Jurkat T lymphocytes from Promega expressing the FcγRIIIa receptor (1.5 × 10⁵ cells in 25 μL) were co-cultured with the antibody-treated target cells at 37 °C for 24 h, and the luciferase activity was then measured using luciferase assay regents (Promega) and a luminescence counter (Infinite M200 Pro).

ADCC was measured using the ADCC Reporter Bioassay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. ADCP was measured using the FcyRIIa-H Reporter Kit (Promega). Luminescence was measured immediately using a Synergy Hybrid Reader (BioTek Instruments, Inc., Winooski, VT, USA).

The ADCC Reporter Bioassay Kit (Promega Life Sciences) was used to measure the induction of ADCC by serum antibodies. The 293T cells were seeded on poly-D lysine coated, flat bottom white 96-well tissue culture plates (Costar) at 50,000 cells per well the day before transfection. Twenty-four hours later, cells were transfected with pCAGGS PR8 M2 (200 ng/well). The next day, transfected cells were washed with 100 µL of PBS and supplemented with 25 µL of Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific). Pooled mouse sera were diluted 1:3 (from a starting dilution of 1:30) in RPMI medium and added (25 µL per well) to the transfected cells in triplicates. ADCC mouse effector cells (Promega Life Sciences) were added at a concentration of 75,000 cells per well and incubated for 6 h at 37 °C. At the end of the incubation, 75 µL of Bio-Glo luciferase assay substrate (Promega Life Sciences) was added to each well and incubated at RT for 5 min. Luminescence was read using a Synergy 4 microplate reader (BioTek) and Gen5 2.09 software. Fold induction over baseline was calculated and graphed using Prism 7.0.