McManusLab

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1. Quickly transfer one vial of ES cells (2 X 10⁶ cells/vial), stored in the gas phase of liquid nitrogen into a 37˚C water bath. 
2. Take the vial out of water bath BEFORE the cells have been completely thawed. You should be able to see a small ice clump floating on top.
3. Carefully, transfer the cells into a 15ml Falcon tube that contains pre-warmed medium using 1ml pipette. Rinse out the cells with medium to make sure all the cells have been transferred. 
4. Pellet the cells by spinning for 5 minutes at 1000 rpm.
5. Aspirate off medium and gently resuspend cells in 5ml of pre-warmed medium.
6. Transfer the cell suspension to a 10cm gelatinized dish with 5ml of medium (total volume of ~10ml) and grow in a 37˚C humidified 5% CO₂ incubator.
7. Change medium daily until 80% confluent (approx. 10-12 million cells). Usually, it takes 2-3 days.
8. When confluent, split 1:3 to 1:6 depending on its confluency.

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