Arichard:Protocols/thawing mammalian cells from LN2

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Thawing mammalian cells from LN2[edit]

Andrew Richards 7/21/2016

Prep[edit]

Prepare everything before thawing cells to minimize thaw time:

  • 15 ml tube with 9 ml of complete media, pre-warmed to 37 C in water bath
  • Additional 10 ml of complete media, pre-warmed to 37 C in water bath
  • T75 with 5 ml complete media, pre-warmed to 37 C in incubator
  • 15 ml balance tube with 10 ml H2O
  • 1 ml micropipette with pipette tips

Protocol[edit]

  1. Freeze slow, thaw fast --> To thaw from LN2, put cryotube directly into 37 C water bath and let thaw until ice is almost completely gone.
  2. Using 1 ml pipette, add cells dropwise to 10 ml pre-warmed media.
  3. Spin down at minimum rcf (1000 rpm on the small round bucket rotor in CERC) for 3 min.
  4. Tap the tube to loosen pellet and resuspend in 10 ml pre-warmed media.
  5. Transfer to T75.