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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


  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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