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 -84o C overnight wrapped in cotton wool in a polystyrene box Finally freeze cells in liquid N2

Unthawing

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

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