Freezing cells in liquid nitrogen

For T25 Flask

1. Take off supernatant
2. Trypsinization with 1ml 0.25% EDTA
3. Give a hard Shock to the flask to remove all attached cells
4. Add 10ml Media +10%FCS
5. Pipette up and down to distribute cells throughout media (i.e. not clumped together)
6. Add resuspended cells to sterile universal tube 
7. Spin down 1500rpm, 3 mins
8. Take off media
9. Resuspend pellet in 2ml FCS +10%DMSO
10. Distribute in 500ul aliquots (0.5 to 2 Million cells/ml)
11. Move cells to -80^oC overnight in Mr. frosty box (filled with ethenol)
12. Finally freeze cells in liquid N₂

Unthawing

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

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