0.45 μm pore filter

To generate lentivirus, HEK293FT or Lenti-X cells were plated in 10 cm dishes at a density of 5×10 6 cells/dish (6.25×10 5 cells/mL). 6-12 h later for HEK293FT or 24 h later for Lenti-X, cells were transfected with 10 μg of viral vector, 8 μg psPAX2, and 3 μg pMD2.G via calcium phosphate transfection as described above. Medium was changed 12-16 h later. 28 h post media change, lentivirus was harvested from the conditioned medium. Medium was centrifuged at 500 g for 2 min to clear cells, and the supernatant was filtered through a 0.45 μm pore filter (VWR). Lentivirus was concentrated from the filtered supernatant by ultracentrifugation in Ultra Clear tubes (Beckman Coulter 344059) at 100,420 g at 4°C in a Beckman Coulter Optima L-80 XP ultracentrifuge using an SW41Ti rotor. Supernatant was aspirated, leaving virus in ~100 μL final volume, and concentrated lentivirus was left on ice for at least 30 min prior to resuspension, then used to transduce ~1×10 5 cells, either plated at the time of transduction or the day before. When appropriate, drug selection began 2 d post transduction, using antibiotic concentrations of 1 μg/mL puromycin (Invitrogen ant-pr) and 10 μg/mL blasticidin (Alfa Aesar J61883) on HEK293FT cells or 0.2 μg/mL puromycin and 2 μg/mL blasticidin on Jurkat cells. Cells were kept in antibiotics for at least two weeks with subculturing every one to two days.