of mTeSR1 medium and pellet at 200g for 5 min at room temperature.35Aspirate the GelTrex coating (Step 32) and seed ~1 × 106 cells with 10 ml of mTeSR1 medium containing ROCK inhibitor from Step 31.36Replace with mTeSR1 medium without ROCK inhibitor after 24 h and refeed daily.37Passaging cells. Passage the cells before they reach 70% confluency.38Aspirate the mTeSR1 medium and wash the cells once with DPBS.39Dissociate the cells by adding 2 ml of Accutase and incubating them at 37 °C for 3–5 min.40Add 10 ml of mTeSR1 medium to the detached cells, transfer the mixture to a 15-ml Falcon tube and resuspend gently.41Replate the cells onto GelTrex-coated plates in mTeSR1 medium with 10 μM ROCK inhibitor.42Replace with normal mTeSR1 medium 24 h after plating.43Transfection. We recommend culturing cells for at least 1 week after thawing and before transfecting by using the Amaxa P3 primary cell 4D Nucleofector kit.44Refeed log-phase growing cells (50–70% confluency) with fresh medium 2 h before 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
