NPC medium was aspirated and 1 ml/well of Accutase was added to NPCs for 5–10 min at 37 °C. Following NPC detachment, 2 ml of DMEM was added to the well to dilute the Accutase. NPCs were collected by pipette, transferred to the 15 ml tube containing DMEM and centrifuged (5 min × 1000g). After aspirating the DMEM, the cell pellet was gently resuspended by pipetting. NPCs were counted with a hemocytometer and 2.5–3 × 106 cells were plated per well of a 6-well plate. The NPCs were then evenly distributed and put back into the incubator. NPCs were typically split 1:3 every 7 days.
