HIV-1ADA isolate was kindly provided by Dr. Howard Gendelman (University of Nebraska Medical Center). HIV–1ADA was originally isolated from peripheral blood mononuclear cells of an AIDS patient with Kaposi’s sarcoma and propagated in MDM obtained from HIV-1-seronegative donors after ultracentrifugation as previously described [23] (link). Viral preparations were screened for endotoxin (10 pg/ml) (Associates of Cape Cod, Woods Hole, Mass.) and Mycoplasma (Gen-Probe II; Gen-Probe, San Diego, Calif.). Viral titer was determined on PHA-blasts as 103 TCID50/ml.
After 7 days in culture, MDM were infected with HIV-1ADA at a multiplicity of infection (MOI) of 0.1 or with serum-free media only (uninfected controls) [24] (link). After overnight incubation, virus was thoroughly washed away and fresh medium was added. Infected MDM were maintained in culture for up to 12 days. Culture supernatants were collected at different days post infection (dpi) depending on the analysis, centrifuged, and stored at −80°C. Infection efficiency was determined in MDM supernatants at 3, 5, 7, 9 and 12 dpi by HIVp24 antigen ELISA, following the manufacturer’s instructions (Express BioTech, Maryland, USA). Protein expression, function, and apoptotic activity were determined in supernatants collected at 3, 6, 9 and 12 dpi. Cells were harvested at the same time points for quantitative intracellular messenger RNA analysis.
After 7 days in culture, MDM were infected with HIV-1ADA at a multiplicity of infection (MOI) of 0.1 or with serum-free media only (uninfected controls) [24] (link). After overnight incubation, virus was thoroughly washed away and fresh medium was added. Infected MDM were maintained in culture for up to 12 days. Culture supernatants were collected at different days post infection (dpi) depending on the analysis, centrifuged, and stored at −80°C. Infection efficiency was determined in MDM supernatants at 3, 5, 7, 9 and 12 dpi by HIVp24 antigen ELISA, following the manufacturer’s instructions (Express BioTech, Maryland, USA). Protein expression, function, and apoptotic activity were determined in supernatants collected at 3, 6, 9 and 12 dpi. Cells were harvested at the same time points for quantitative intracellular messenger RNA analysis.
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