Cobas ampliprep taqman system

After 3 days of co-culture, plates were spun at 500 ×g for 5 min, culture media discarded and the cell pellets were resuspended in RLT buffer containing B2M and were stored at -80°C until RNA isolation. Total RNA was isolated using the RNeasy 96 kit (Qiagen) and cell-associated vRNA levels in these samples were analyzed by a robotic COBAS Ampliprep/Taqman system (Roche Diagnostics), which extracts total nucleic acid and quantifies HIV RNA in copies per milliliter using the HIV Test, v2.0 kit (Roche Diagnostics). RNA from three individual wells was tested for each experimental condition and for testing purpose each RNA sample was diluted 1 to 2,000 in molecular grade water in order get vRNA levels within the detection range of the Cobas system. Maximum vRNA copies/mL detected in these samples ranged from 30,000 to 60,000 for IN and BaL infected samples respectively. Final data is represented as % reduction in vRNA levels in DART treated co-cultures with comparison to No DART treated co cultures.

HIV-infected participants were selected based on sustained plasma viral load suppression (<50 copies/ml for >12 months), CD4 counts (>350 cells/μL), and absence of co-infection with hepatitis B or C virus. Clinical laboratory results were reconfirmed 2 weeks before leukapheresis or blood draw. Leukapheresis was conducted for 3–4 hours, and samples were processed within 2 hours after collection. The leukapheresis product was diluted 1:1 with PBS and layered over Ficoll for isolation of PBMCs. PBMCs were treated with red blood cell lysis buffer (eBioscience) and rested overnight (1 x 10⁷ cells/ml) in RPMI-10. To assess HIV activation, 15 million PBMCs were plated in 6-well plates in 5 ml of media, supplemented with antiretrovirals (ARVs) (100 nM elvitegravir and 100–300 nM efavirenz) for the entire duration of culture incubation. ALT-803 was added at 1 nM for 7 days. To measure HIV RNA levels, 1 ml of culture supernatant was analyzed by a robotic COBAS AmpliPrep/TaqMan system (Roche Diagnostics), which extracts total nucleic acid and quantifies HIV RNA in copies per milliliter using the HIV-1 Test, v2.0 kit (Roche Diagnostics).