Luciferase assay regents

HIV-1 Env pseudoviruses were prepared as described previously.^{35 (link), 36 (link)} In brief, 293 T cells (5 × 10⁶ cells in 15 mL growth medium in a T-75 culture flask) were transfected with 10 μg of rev/env expression plasmid and 20 μg of an env-deficient HIV-1 backbone plasmid, pSG3^Δenv, using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Pseudoviruses containing culture supernatants were collected 2 days after transfection, filtered (0.45 μm) and stored in 1-mL aliquots at −80 °C or in the vapor phase of liquid nitrogen until use. Neutralization was measured using HIV-1 Env-containing pseudovirus to infect TZM-bl cells, as described previously.^{37 (link)} In brief, the 50% tissue culture infectious dose (TCID₅₀) of a single thawed aliquot of each pseudovirus batch was examined in TZM-bl cells. The test antibodies were serially diluted in a fourfold stepwise manner, mixed with 50 μL of 100 TCID₅₀ of the pseudovirus in duplicate wells of a 96-well flat-bottomed culture plate, and incubated for 30 min at 37 °C. The same medium was used in the control wells. The mixture was transferred to U87 cells (10⁴ per well) and incubated at 37 °C for 48 h. Luciferase activity was measured using luciferase assay regents (Promega, Madison, WI, USA) and a luminescence counter (Infinite M200 Pro) according to the manufacturer's instructions.

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).