Bright glo kit

HeLa13^{63 (link)} cells stably expressing the AR and a luciferase reporter gene construct were used for this assay as previously described.^{37 (link),47 (link)} Briefly, cells were cultured in phenol red free media supplemented with 5% CDFBS (CSS media) for 48 h. The cells were then harvested and plated in a 96-well plate for 6–7 h, after which fresh CSS media (see below) containing 1 nM 5α-DHT in the presence of inhibitor was added. After 20 h incubation, the media was removed and the luciferase activity in the cells was measured using the Bright Glo kit (Promega) according to the manufacturer’s instructions. Fold induction of luciferase was evaluated relative to untreated cells.

One day prior to infection, 1.7 × 10⁴ TZM-bl cells per well were seeded on a 96-well plate in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% FCS, penicillin and streptomycin (both at 100 U/ml), and incubated at 37°C in an atmosphere containing 5% CO₂. A fixed amount of virus (2.5 ng/ml of p24-antigen equivalent) was incubated for 30 min at room temperature with serial 3-fold dilutions of each test mAb (Bontjer et al., 2009 (link); Eggink et al., 2009 (link); Sanders et al., 2013 (link)). This mixture was added to the cells and 40 μg/ml DEAE, in a total volume of 200 μl. Three days later, the medium was removed. The cells were washed once with PBS (150 mM NaCl, 50 mM sodium phosphate, pH 7.0) and lysed in Lysis Buffer, pH 7.8 (25 mM Glycylglycine (Gly-Gly), 15 mM MgSO₄, 4 mM EGTA tetrasodium, 10% Triton-X). Luciferase activity was measured using a Bright-Glo kit (Promega, Madison, WI) and a Glomax Luminometer according to the manufacturer’s instructions (Turner BioSystems, Sunnyvale, CA). All infection measurements were performed in quadruple. Uninfected cells were used to correct for background luciferase activity. The infectivity of each mutant without inhibitor was set at 100%. Nonlinear regression curves were determined and 50% inhibitory concentrations (IC₅₀) were calculated using a sigmoid function in Prism software version 8.