- gelatin-coated plate
- E14 media
- Prepare 0.1% gelatin-coated plates by incubating plates completely covered with 0.1% gelatin in a 37 C incubator for at least one hour.
- Change media 2-4 hours prior to splitting. Cells must be >80% confluent and healthy.
- When cells are ready to be passaged, aspirate old media and wash with 150ul pre-warmed PBS. Add PBS gently to avoid disrupting the cells.
- After aspirating PBS, add 30ul of pre-warmed trypsin. Be sure to cover the entire plate by carefully swirling it.
- Incubate in a 37C incubator for 5 minutes and check under the microscope. If you see big clumps of cells, incubate additional 2-5 minutes. Do not over-trypsinize cells.
- While cells are incubating, remove gelatin from the pre-treated plates by quickly flicking gelatin off the plates. Carefully label the colony numbers on all three plates identically. Two of the new plates will be frozen, and the other one will be used to extract DNA to genotype.
- Once majority of the cells are uniformly dispersed into small clumps or single cells, inactivate trypsin by adding 150ul of medium. Gently pipette up and down using P200 Rainin multichannel pipette 10-15 times without making bubbles.
- Transfer 50-60ul of cell suspension to each plate. Add 150ul of additional medium to bring the final volume to 200ul.
- Change media everyday until the cells are ready to be frozen. This will take 2-4 days depending on confluence of the original master plate.