96 well luminometer compatible tissue culture plates

The single-round HIV-1 infection assay details have been published previously [55 (link),57 (link),58 (link)]. Briefly, U87.CD4.CXCR4 (1.2 × 10⁴ cells/well) target cells were seeded in 96-well luminometer-compatible tissue culture plates (Greiner Bio-one). After 24 h compound, DMSO (vehicle control for compounds, Sigma) was mixed with pseudotyped viruses (normalized to p24 content). The mixture was added to the target cells and incubated for 48 h at 37 °C. Following this, the media was removed from each well, and the cells were lysed by the addition of 50 μL/well of luciferase lysis buffer (Promega) and one freeze-thaw cycle. A GloMax 96 microplate luminometer (Promega) was used to measure the luciferase activity of each well after the addition of 50 μL/well of luciferase assay substrate (Promega).

The details of the single-round HIV-1 infection assay for detecting viral infectivity have been published previously [17 (link),20 (link),21 (link)]. Briefly, U87.CD4.CCR5 (1.2 × 10⁴ cells/well) target cells were seeded in 96-well luminometer-compatible tissue culture plates (Greiner bio-one). After 24 hours incubation at 37°C, a compound or DMSO (vehicle control for the compound, Sigma) were mixed with pseudotyped viruses (normalized to p24 content) and the mixture was added to the target cells and incubated for 48 hours at 37°C. Subsequently, the media was removed from each well, and the cells were lysed by the addition of 50 μl of luciferase lysis buffer (Promega) and one freeze-thaw cycle. A GloMax 96 microplate luminometer (Promega) was used to measure the luciferase activity of each well after the addition of 50 μl of luciferase assay substrate (Promega). For the timeof-addition assay, DMSO or compounds were added at indicated time points post-infection and the resulting luciferase signal was measured after 48 hours.

The details of the single-round HIV-1 infection assay for detecting viral infectivity have been published previously [51 (link), 54 (link), 55 (link)]. Briefly, U87.CD4.CCR5 (1.2 × 104 cells/well) target cells were seeded in 96-well luminometer-compatible tissue culture plates (Greiner bio-one). After a 24-hour incubation at 37°C, the compound or a DMSO vehicle control (Sigma) was mixed with pseudotyped virus and the mixture was added to the target cells. After a 48-hour 37°C incubation, the media was removed from each well and the cells were lysed by adding 50 μl/well of luciferase lysis buffer (Promega). Following one freeze-thaw cycle, 50ul/well of luciferase assay substrate (Promega) was added and a GloMax 96 microplate luminometer (Promega) was used to measure the luciferase activity of each well. Luciferase activity of virus produced with compound were normalized to the luciferase activity of virus produced from DMSO vehicle control treated cells. The compound-induced effects are manifested as a decrease in normalized luciferase activity which indicates a decrease in infectivity in the target cells when the compound was present.