Day 10+1: Splitting Day 10 NPCs
Some lines may survive 1:2, but it is advisable to split cells 2:3, as there will be quite some cell death and the cells need to be fairly confluent to maintain viability and cell identity. If split too harshly, they may differentiate into other cell types.
4.3.1. Aspirate media and wash each well with 1mL of PBS.
4.3.2. Immediately aspirate PBS and add 1mL of Accutase.
4.3.3. Incubate at 37⁰C for 5 minutes.
4.3.4. Gently collect cells using a 1000 μL pipette and place in pre-warmed spinning falcon.
4.3.5. Spin cells for 5 minutes at 350g.
4.3.6. While cells are spinning, aspirate Geltrex and replace with 3mL of pre-warmed D10 media (step 3.12)+ ROCKi (1:1000).
4.3.7. Aspirate media from pelleted cells, re-suspend pellet in 1mL of D10 media (step 3.12) + ROCKi and add dropwise to well of 6-well plate.
4.3.8. Gently swirl to distribute cells evenly around dish.