Tissue Culture - Thawing Cells from Liquid Nitrogen
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Tissue Culture - Thawing Cells from Liquid Nitrogen
METHOD:
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Prepare before thawing:
Fill a test tube of 10 - 15 mls of cold media (appropriate for your cell line) with 10 - 20% fetal calf serum (use 2x the % you use for growth supplement).
Place in a beaker of crushed ice to CHILL . (DMSO is toxic at room temperature.)
Have everything in the hood that you will need: 70% alcohol, paper towels, pipets, etc.
- Get vial from Liquid Nitrogen - use gloves and face shield.
- QUICK THAW" in 37ºC H2 O bath by shaking vial rapidly in water till approximately 3/4 thawed, with a small pea sized portion still frozen.
- Remove from H2 O bath, but continue to shake vial until it has thawed completely.
- Rinse vial with Ethanol.
- Open vial carefully, pipet up contents and 'layer' it onto cold media (in the test tube you have chilling)
- Spin at approx. 1000 rpm for 5 min.
- Wash 1x with 10 - 15 mls of media, 1000 rpm 5 min (to wash out all DMSO). Gently resuspend the pellet.
- Seed into a small flask T25 or T75 with appropriate volume of media containing 2x the regular amount of FCS, and the regular amount of glutamine and antibiotic.
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NEXT DAY: Change media if growing adherent cells
to get rid of excess dead cells - Check non-adherent cells to see how soon they will need fresh media and/or need to be split.
- 1 - 4 DAYS: Passage cells when required in usual fashion.