Tissue Culture - Thawing Cells from Liquid Nitrogen

1. 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.
   
   
2. Get vial from Liquid Nitrogen - use gloves and face shield. *Make sure the cover is replaced properly!!
   
3. QUICK THAW" in 37ºC H₂O bath by shaking vial rapidly in water till approximately 3/4 thawed, with a small pea sized portion still frozen.
   
4. Remove from H₂O bath, but continue to shake vial until it has thawed completely.
   
5. Rinse vial with Ethanol.
   
6. Open vial carefully, pipet up contents and 'layer' it onto cold media (in the test tube you have chilling)
   
7. Spin at approx. 1000 rpm for 5 min.
   
8. Wash 1x with 10 - 15 mls of media, 1000 rpm 5 min (to wash out all DMSO). Gently resuspend the pellet.
   
9. 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.
   
10. NEXT DAY: Change media if growing adherent cells
    to get rid of excess dead cells
    
11. Check non-adherent cells to see how soon they will need fresh media and/or need to be split.
    
12. 1 - 4 DAYS: Passage cells when required in usual fashion.