Tropix galacto star system

Where indicated on the figures or in the figure legends or text, cells were treated with 1,000 U/ml IFN-γ (PHC4031; Thermo Fisher) for 24 h before virus infection. In all cases, 2 × 10⁵ activated CD4⁺ T cells were infected at an MOI of 0.05 to 0.1 as specified on the figures or in the figure legends or text (infectious titer determined via TZM-bl assay). At 8 to 12 h postinfection, medium was replaced. Supernatants were harvested when indicated on the figures or in the figure legends or text for up to 17 days postinfection, and infectious viral release at each time point was determined by infecting HeLa-TZM-bl indicator cells. At 48 h post TZM-bl infection, virus release was assayed by measuring chemiluminescent β-galactosidase activity using a Tropix Galacto-Star system (Applied Biosystems) according to the manufacturer's instructions.

U87/CD4/CXCR4⁺ or U87/CD4/CCR5 cells stably expressing IFITMs 1, 2, or 3 or mutants thereof were infected with the indicated HIV-1 molecular clone at an MOI of 0.05. Media were replaced 8 hr post-infection, and culture supernatants were harvested every 24 hr post-infection for a total of 120 hr. Infectious viral release was determined by infecting HeLa-TZMbl indicator cells and 48 hr post-infection assaying for virus release by measuring chemiluminescent β-galactosidase activity, using the Tropix Galacto-Star system (Applied Biosystems) according to the manufacturer’s instructions. For one-round virus release assays, cells were infected with the indicated HIV-1 molecular clone at an MOI of 0.5. Viral production was measured for supernatants harvested at 48 hr post-infection on HeLa-TZMbl indicator cells, as above. For env-pseudotyped viral vector entry assays, the same cells were infected with a fixed dose of HIV-1 viral vectors at an MOI of 0.2 for 48–72 hr prior to analysis for GFP expression by flow cytometry.
Activated CD4⁺ T cells, transduced with the appropriate shRNA lentiviral vectors, were infected at an MOI of 0.1; then 8–12 hr post-infection, media were replaced. Supernatants were harvested every 72, 120, and 168 hr post-infection, and virus particle production was assessed on HeLa-TZMbl cells as described previously.