Generation of HIV-1 Viral Stocks in 293T Cells

Viral stocks of HIV-1_JR-CSF were generated by transfecting HIV-1_JR-CSF DNA^{50 (link)–54 (link)} into 293T cells (American Tissue Culture Collection, catalog number CRL-3216, passage <10) using Lipofectamine 2000 (Invitrogen). 293T cells were cultured at 37 °C, 10% CO₂ in Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum, 25 mM HEPES, 500 units/ml penicillin, 50 µg/ml streptomycin and 2 mM L-glutamine (Cellgro), cells were regularly checked for morphology by microscopy. Viral particles were collected in tissue culture medium that was centrifugated at 2000×g for 20 min at 4 °C to remove cell debris. Tissue culture infectious units (TCID)/ml of HIV were determined by titration using TZM-bl cells (NIH AIDS Research and Reference Reagent Program, catalog number 8129, passage number 2–6). Infected TZM-bl cells were stained using a solution containing 4 µM potassium ferrocyanide, 4 µM potassium ferricyanide, 2 µM magnesium chloride, 0.4 mg/ml X-gal^{55 (link),56 (link)} and stained cells counted. TZM-bl cells were cultured at 37 °C 10% CO₂ in TZM-bl medium (Dulbecco’s Modified Eagle Medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, 25 mM HEPES, 500 units/ml penicillin, 50 µg/ml streptomycin and 2 mM L-glutamine (Cellgro)), and regularly checked for morphology by microscope.