Freezing cells in liquid nitrogen

Take off Media

Trypsinate with 1ml x2 Dulbecco A trypsin

Add 7ml Media

Pipette up and down to distribute cells throughout media (i.e. not clumped together)

Add media to sterile falcon tube (15ml = 1 flask, 50ml = 5 flasks)

Spin down 1-2K, 5 mins

Take off media

Resuspend pellet in 9ml FCS and 1ml DMSO

Distribute in 1ml aliquots (10 cryovials)

Move cells to -84^oC overnight wrapped in cotton wool in a polystyrene box

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