Lentiviral Transduction of Jurkat T Cells

A suspension of Jurkat T cells was prepared in fresh RPMI medium with 10% FBS at a concentration of 2 × 10⁶ cells/mL containing 16 µg/mL of polybrene (Sigma Aldrich, St. Louis, MO, USA, H9268) and plated in 24-well plates at 500 µL/well. Serial dilutions of lentiviral supernatant were prepared in fresh medium and added to each well at 500 µL/well, resulting in a final concentration of polybrene of 8 µg/mL [38 (link)]. Cells were incubated at 37 °C; 24 h later, another 1000 µL of fresh medium was added to each well. At 48 h post-transduction, half of the cells were analyzed for transduction efficiency based on GFP and CAR expression at the cell surface as measured by flow cytometry. The remaining cells were recovered by centrifugation and resuspended in fresh medium containing 200 µg/mL of zeocin (Invivogen, San Diego, CA, USA, #ant-zn) at a concentration of 0.2 × 10⁶ cells/mL. Cells were kept under zeocin selection for 3 to 4 weeks with regular medium exchange and cell dilution, until reaching 1 × 10⁶ cells/mL. Thereafter, CAR-Jurkat T cells were reanalyzed for CAR expression by flow cytometry and Western blotting.