Freezing cells in liquid nitrogen

 

1. Take off Media
2. Trypsinate with 1ml x2 Dulbecco A trypsin
3. Add 7ml Media
4. Pipette up and down to distribute cells throughout media (i.e. not clumped together)
5. Add media to sterile falcon tube (15ml = 1 flask, 50ml = 5 flasks)
6. Spin down 1-2K, 5 mins
7. Take off media
8. Resuspend pellet in 9ml FCS and 1ml DMSO
9. Distribute in 1ml aliquots (10 cryovials)
10. Move cells to -84^o C overnight wrapped in cotton wool in a polystyrene box
11. Finally freeze cells in liquid N₂

Unthawing

1. Warm DMEM in waterbath
2. Thaw cryovial at 37^o C quickly until cells become molten.

Aliquot the 1ml of cells using a disposable pipette into 10ml fresh media in a TC flask