Victor light 2030 luminometer

Two HIV-1 pseudoviruses, tier 1 and tier 2 virus strains, respectively with a higher and lower level of neutralization susceptibility were employed in the TZM-bl assay: SF162 (Clade B, Tier 1 virus), QH0692 (Clade B, Tier 2). Viral stocks were generated and titered. In particular, 293 T cell-derived stocks of pseudoviruses were generated by proviral DNA transfection using FuGENE HD, according to the manufacturer’s protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post transfection and clarified at 1800 rpm for 20 min. The virus stocks were analysed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of viruses were added to 96 flat-bottomed plate wells containing 10⁴ TZM-bl cells per well, in DMEM supplemented with 10% FBS and 7.5 μg/ml DEAE-dextran in a final volume of 200 μl. After 48 h of incubation at 37°C, 100 μL of culture medium were removed from each well and replaced with 100 μL of Bright Glo luciferase reagent (Promega). After 2 min of incubation, 100 μL of the cell lysate were transferred to a 96 well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Neutralization titers were expressed as ID50 values, defined as the virus dilution required to achieve half maximal infection (Reed–Muench calculation).

293 T cell-derived stocks of pseudoviruses were generated by proviral DNA transfection using FuGENE 6, according to the manufacturer's protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post transfection, clarified at 1800 rpm for 20 min, and frozen at –70 °C. The virus stocks were further analyzed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of viruses were added to 96 flat-bottomed plate wells containing 10⁴ TZM-bl cells per well, in 10% D-MEM growth medium with 7.5 μg ml^–1 DEAE-dextran (Sigma) in a final volume of 200 μl. After 48 h of incubation at 37 °C, 100 μL of culture medium was removed from each well and replaced with 100 μL of Bright-Glo luciferase reagent (Promega). After 2 min of incubation, 150 μL of the cell lysate was transferred to a 96-well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Fifty percent infectious dose (ID50) titers were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed–Muench calculation).

293 T cell-derived stocks of pseudoviruses and replication-competent IMCs were generated by proviral DNA transfection using FuGENE 6, according to the manufacturer’s protocol (Promega, Madison, WI). Viral supernatants were harvested 72 h post-transfection, clarified at 1800 rpm for 20 min, and frozen at −70°C. The virus stocks were further analyzed for firefly luciferase expression in the TZM-bl cell line. Four replicates of five-fold dilutions of virus were added to 96 flat-bottomed plate wells containing 1 × 10⁴ TZM-bl cells per well, in 10% D-MEM growth medium with 7.5ug/ml of DEAE-dextran (Sigma) in a final volume of 200ul. After 48 h incubation at 37°C, 100uL of culture medium were removed from each well and replaced with 100 uL of Bright-Glo luciferase reagent (Promega). After 2 min incubation, 150 uL of the cell lysate was transferred to a 96-well white solid plate and luminescence was measured using a Victor Light 2030 luminometer (Perkin Elmer). Fifty percent infectious dose (ID50) titers were defined as the reciprocal of the virus dilution yielding 50% positive wells (Reed-Muench calculation).