Cells were grown at 37 °C in 5% CO2 incubators at a density of 0.5–1.5 × 10^6 cells/mL and passaged at 0.1 × 10^6 cell/mL 24-h before electroporation. For electroporation, cells were harvested by centrifugation (200 × g, RT, 5 min) and resuspended at 10 × 10^6 cells/mL (2 × 10^5 cells/20 µL) in supplemented SF nucleofection buffer (Lonza). Cell culture media supernatant was periodically tested for mycoplasma contamination using the MycoAlert PLUS mycoplasma detection kit (Lonza).
4D Nucleofector with Shuttle unit (V4SC-2960 Nucleocuvette Strips) was used for electroporation, following the manufacturer’s instructions. Jurkat cells were electroporated using the SF Cell Line Nucleofector X Kit (Lonza), CA-137 program, with 2 × 10^5 cells in 20 µL SF buffer for each nucleofection reaction. The cell suspension was mixed with RNPs, immediately transferred to the nucleocuvette, and subjected to nucleofection in the 96-well Shuttle device. Cells were immediately re-suspended in the cultivation medium and plated on 96-well, flat-bottom, non-cell culture treated plates (Falcon).