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Freezing cells in liquid nitrogen

Standard Protocols

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 -84oC 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 37oC quickly until cells become molten.

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

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