Arichard:Protocols/thawing mammalian cells from LN2: Difference between revisions
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>Andrew (Created page with "==Thawing mammalian cells from LN2== Andrew Richards 7/21/2016 ===Prep=== Prepare everything before thawing cells to minimize thaw time: * 15 ml tube with 9 ml of complete me...") |
>Andrew |
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===Protocol=== | ===Protocol=== | ||
# Freeze slow, thaw fast | # 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. | ||
# Using 1 ml pipette, add cells dropwise to 10 ml pre-warmed media. | # Using 1 ml pipette, add cells dropwise to 10 ml pre-warmed media. | ||
# Spin down at minimum rcf (1000 rpm on the small round bucket rotor in CERC) for 3 min. | # Spin down at minimum rcf (1000 rpm on the small round bucket rotor in CERC) for 3 min. | ||
# Tap the tube to loosen pellet and resuspend in 10 ml pre-warmed media. | # Tap the tube to loosen pellet and resuspend in 10 ml pre-warmed media. | ||
# Transfer to T75. | # Transfer to T75. |
Latest revision as of 21:07, 21 July 2016
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]
- 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.
- Using 1 ml pipette, add cells dropwise to 10 ml pre-warmed media.
- Spin down at minimum rcf (1000 rpm on the small round bucket rotor in CERC) for 3 min.
- Tap the tube to loosen pellet and resuspend in 10 ml pre-warmed media.
- Transfer to T75.