Day 4: 1:2 splitting of Neuronal Progenitor Cells (NPCs)
3.5.1. Add 3 μL of 10 μM ROCKi directly into each well of the 6-well plate containing cells and incubate at 37⁰C for 1 hour.
3.5.2. Aspirate media and wash each well with 1 mL of PBS.
3.5.3. Immediately aspirate PBS and add 1 mL of Accutase.
3.5.4. Incubate at 37⁰C for 5 minutes.
3.5.5. Gently collect cells using a 1000 μL pipette and place in pre-warmed spinning falcon.
3.5.6. Spin cells for 5 minutes at 350g.
3.5.7. While cells are spinning, aspirate Geltrex and replace with 2 mL of pre-warmed D4 media + 10 μM ROCKi (1:1000).
3.5.8. Aspirate media from pelleted cells, re-suspend pellet in 1 mL of D4 media + 10 μM ROCKi (1:1000) and add dropwise to each well of 6-well plate.
3.5.9. Gently swirl to distribute cells evenly around dish.