L 90 ultracentrifuge

Viruses were produced by co-transfecting 293T-cells with HIV-1 molecular clones using polyethylenimine (PEI) [20 (link)]. PEI stocks were prepared at 1 mg/mL by dissolving PEI in water, adjusting the pH to 7.0, followed by filtration with a 0.2 μm sterile syringe filter. 2.5 × 10⁶ 293T-cells were seeded into 100 mm² tissue culture plates 24 h prior to transfection. Transfection mix was prepared by adding 3 μg total HIV-1 proviral DNA to 500 μL of serum-free DMEM and 27 μL of PEI, vortexed and incubated for 5 min before addition to cells. 12 h post infection, cells were washed twice in DPBS- and the medium was replaced with fresh DMEM. Supernatants were collected 36 h post transfection and clarified by centrifugation for 30 min at 1462 × g at 4 °C to remove cellular debris. Clarified supernatant was then further purified by sequential filtration through 0.8 μm and 0.45 μm sterile syringe filter. Purified virus was then concentrated by ultracentrifugation through a 20% sucrose cushion using an L-90 ultracentrifuge (Beckman Coulter, Sudney, NSW, Australia) at 100,000 × g for 1 h at 4 °C. Pellets were resuspended in DMEM and virus quantified using the Vironostika HIV-1 p24 antigen enzyme-linked immunosorbant assay (ELISA) (BioMérieux, Marcy I’Etoile, France), according to the manufacturer’s instructions.