Britelite luciferase substrate

Viruses pseudotyped with 4-2.J41 Env were produced by co-transfection of envelope expressing plasmid (pSVIII-env) with env-deleted HIV-1 backbone plasmid (pSG3ΔEnv) into 293T cells in 6-well tissue culture plates using FuGENE6 Transfection Reagent (Promega Inc). Cell supernatants containing pseudotyped viruses were harvested 48 hours post-transfection and then stored at -80°C until further use. The infectivity assays were done in Tzm-bl cells (1 X 10⁵ cells/ml) containing DEAE-Dextran (25 μg/ml) in 96-well microtiter plates and infectivity titers were determined by measuring luciferase activity using Britelite luciferase substrate (Perkin Elmer) with a Victor X2 Luminometer (Perkin Elmer).
Viruses pseudotyped with 4-2.J41 Env were assessed for their neutralization sensitivity against neutralizing and non-neutralizing antibodies in a Tzm-bl neutralization assay. Briefly, pseudoviruses (1 X 10⁵ RLU/well) were incubated with serial dilutions (2-fold) of monoclonal antibodies in duplicate wells of a 96-well flat-bottom culture plate. After 1 hour of incubation at 37°C, 1 X 10⁵ TZM-bl cells containing 25 μg/ml DEAE-Dextran were added to each well. After 48 hours, luciferase activity was measured using the Britelite luciferase substrate (Perkin Elmer). The IC₅₀ values were defined as antibody concentrations that caused a 50% reduction in RLU compared to the virus control.

HIV-1 Env-pseudotyped viruses were prepared as described previously [32 (link)]. Briefly, 293T cells were co-transfected with envelope-expressing plasmid and an env-deleted HIV-1 backbone plasmid (pSG3ΔEnv) using a FuGENE6 transfection kit (Promega Inc.). Cell supernatants containing pseudotyped viruses were harvested 48 h post transfection and used for infection in TZM-bl cells using DEAE-dextran (25 μg/ml) in 96-well microtiter plates. The virus infectivity titers were determined by measuring the luciferase activity using Britelite luciferase substrate (PerkinElmer Inc.) in a luminometer (Victor X2, PerkinElmer Inc.).

Pseudotyped viruses were prepared by the co-transfection of env-expressing plasmid DNA along with the plasmid DNA expressing HIV-1 genes with premature stop codon for env (pSG3ΔEnv) into 293 T cells in 6-well tissue culture plates using FuGENE6 transfection reagent kit (Promega Inc.). Cell supernatants containing pseudotyped viruses were harvested at 48 h post transfection and subsequently stored at -80° C until use. The virus infectivity was measured using TZM-bl reporter cells by addition of pseudoviruses containing DEAE-dextran (25 µg/ml) in 96-well microtiter plates, and the viral titers were determined by measuring the luciferase activity using Britelite luciferase substrate (PerkinElmer Inc.) with a Victor X2 luminometer (PerkinElmer Inc.).