3.6. Single-Round Infection Assay

The details of the single-round HIV-1 infection assay have been published previously [15,18,19]. Briefly, U87.CD4.CCR5/CXCR4 (1.2 × 10⁴ cells/well) target cells were seeded in 96-well luminometer-compatible tissue culture plates (Greiner Bio-One, Monroe, NC, USA). After 24 h, compound, DMSO (vehicle control for compounds, Sigma) was mixed with pseudotyped viruses (normalized to p24 content), and 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 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).