transfection.45Dissociate to single cells or small clusters of no more than ten cells (as viewed under the microscope) with Accutase and gentle resuspension.46Count the number of cells needed for nucleofection (200,000 cells per transfection) and spin down at 200g for 5 min at room temperature.47Remove the medium completely and resuspend it in 20 μl of S1-supplemented P3 nucleofection solution, per 2 × 105 cells.48Pipette the resuspended cells with added DNA (Steps 9 and 19) into electroporation cuvettes and electroporate according to the suggested program. For 2 × 105 cells, we typically use 1 μg of total DNA.49Gently plate the electroporated cells onto coated 100-mm plates supplemented with 10 μM ROCK inhibitor.50Check transfection success (Steps 11 and 28) and refeed the cells daily with regular mTeSR1 medium beginning 24 h after nucleofection. Puromycin selection can be applied at a concentration of 0.5 μg ml–1 (may vary depending on the cell line). Typically, we observe >70% transfection efficiency with Amaxa nucleofection.
