- Collect cells and resuspend in fresh complete RPMI1640 medium supplemented with 10mM HEPES and 5 ug/ml polybrene at 1 × 106 cells/ml.
- Add 1-10 μl concentrated lentivirus into each well of a 24-well plate pre-aliquoted with 900 μl complete RPMI1640 medium supplemented with 10mM HEPES and 5 ug/ml polybrene (dilute the lentivirus first with the same medium if less than 1 μl needed).
- Aliquot 100 μl cell suspension from step 1 (1 × 105 cells) into each well of step 2, mix well, and spin inoculate at 35°C, 3000 rpm for 30 min before incubating at 37°C at 5% CO2.
- At 20 hours post-incubation, change medium by spinning down cells from each well and resuspend into 1 ml fresh complete RPMI1640 medium supplemented with 10mM HEPES, plate into each well of a 12-well plate, and return to 37°C at 5% CO2.
- At 72 hours post-transduction, run FACS analysis or proceed to drug selection.
Last modified:
3 June 2025
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