Galacto star β galactosidase reporter gene assay system for mammalian cells

The anti-HIV activity of the compounds was evaluated according to the previously reported procedure.^{11 (link), 19 (link)–21 (link)} Compound anti-HIV activity was evaluated in single-round (MAGI) infection assays using X4 (IIIB) and R5 (BaL) HIV-1 and P4R5 cells expressing CD4 and coreceptors. In summary, P4R5MAGI cells were cultured at a density of 1.2 × 10⁴ cells/well in a 96 well plate approximately 18 h prior to infection. Cells were incubated for 2 h at 37 °C with purified, cell-free HIV-1 laboratory strains IIIB or BaL (Advanced Biotechnologies, Inc., Columbia, MD) in the absence or presence of each agent. After 2 h, cells were washed, cultured for an additional 46 h, and subsequently assayed for HIV-1 infection using the Galacto-Star β-Galactosidase Reporter Gene Assay System for Mammalian Cells (Applied Biosystems, Bedford, MA). Reductions in infection were calculated as a percentage relative to the level of infection in the absence of agents, and 50% inhibitory concentrations (EC₅₀) were derived from regression analysis. Each compound concentration was tested in triplicate wells. Cell toxicity was evaluated using the same experimental design but without the addition of virus. The impact of compounds on cell viability was assessed using an MTT (reduction of tetrazolium salts) assay (Invitrogen, Carlsbad, CA).