Victor 3 luminometer

Neutralization titers of monoclonal Abs were determined using a luciferase-based assay in TZM.bl cells as previously described.50 (link), 51 (link) Briefly, mAb samples were tested using a primary concentration of 25 μg/mL with 5-fold serial dilutions against a panel of 10 HIV-1 Env pseudoviruses that were selected for being either 3BNC117 sensitive/PGT121 resistant or 3BNC117 resistant/PGT121 sensitive. This allowed for the measurement of neutralizing activity of single Abs in samples containing a mixture of both 3BNC117 and PGT121. Antibody titrations were incubated with HIV-1 Env pseudoviruses for 1 h at 37°C, and TZM.bl cells were then added in growth media containing DEAE-dextran at a final concentration of 11 μg/mL. Assay plates were incubated for 48 h at 37°C, 5% CO₂, and luciferase reporter gene expression was measured using Bright-Glo luciferase reagent (Promega) and a Victor 3 luminometer (Perkin Elmer). Neutralization titers (50% and 80% inhibitory concentrations, IC50 and IC80, respectively) were calculated as the mAb concentration at which relative luciferase units (RLU) were reduced by 50% or 80% compared to RLU in virus control wells after subtraction of background RLU in cell control wells. All assays were performed in a laboratory meeting good current laboratory practice standards.

The control and DMD myoblasts cell lines were plated in 96-well culture plates at a density of 10⁴ cells/well. The day after transfection, proteasome activities (caspase-, trypsin- and chymotrypsin-like activities) were measured using cell-based Proteasome-Glo™ assay (Promega) as previously described [42 (link)]. The proteasome inhibitors MG132 and lactacystin (10 µM, 2 h) were used as negative controls. Luminescence was measured using a Victor3 Luminometer (Perkin Elmer). The relative light units produced were reported to 50 µg of total proteins.

The metabolic activity of TIGK cells was assessed by measuring total ATP levels using the CellTiter-Glo reagent (Promega, Madison WI), as described by the manufacturer. TIGK cells were seeded at a density of 6 × 10⁴ cells in 1 mL media per well and incubated at 37°C, 5% CO₂ for 24 h in a 12-well flat bottom plate. Cells were then incubated with BAR or 10:90 PLGA-PEO BAR-EFs (1.3 or 3.4 μM) for 24 h at 37°C in 5% CO₂. Cells were then lysed with 500 μL of 0.1% Triton X-100 for 30 min at 37°C. The lysates were collected and centrifuged at 1,000 × g for 10 min at 4°C, and 50 μL of supernatant was mixed with 50 μL of CellTiter-Glo reagent. Samples were incubated at ambient temperature for 10 min in a black 96-well plate in the dark. Total luminescence was measured with a Victor 3 luminometer (Perkin-Elmer, Inc.). Cells incubated with 1 ng of staurosporine or with medium-only served as positive and negative controls for cell death, respectively (Mahmoud et al., 2019 (link)).