Cell Line Transfection.
A Lonza 4D-Nucleofector with Shuttle unit (V4SC-2960 Nucleocuvette Strips) was used for transfection, following the manufacturer’s instructions. For transfection, cells were harvested by centrifugation (200 × g, RT, 5 min) and re-suspended at 10 × 10^6 cells mL-1 (2 × 10^5 cells 20 µL-1 ) in the SF Cell Line Nucleofector X Kit buffer (Lonza), unless stated otherwise. The cell suspension was mixed with the RNPs, immediately transferred to the nucleocuvette, and transfected using the CA-137 Nucleofector program, except where indicated otherwise. After transfection, the cells were immediately re-suspended in the pre-warmed cultivation medium and plated onto 96-well, flat-bottom, non-treated plates (Falcon) and cultured at 37 °C in 5% CO2 incubators and maintained at a density of 0.5-1.0 × 10^6 cells mL-1.
Primary T-Cell Transfection.
Forty-eight hours after isolation, the cells were harvested by centrifugation (300 × g, RT, 5 min) and re-suspended at 50 × 10^6 cells mL-1 (1 × 10^6 cells 20 µL-1 ) in the supplemented P3 Primary Cell Nucleofector Kit buffer (Lonza). The cells were mixed with HDRT, and the suspension was transferred to the RNPs immediately before transfection (Nucleofection program EH-115). After transfection, 80 µL of pre-warmed cultivation medium without IL-2 was added to the electroporation cuvettes.