Jacobs:Protocol Freezing and Thawing Cells

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Materials for Freezing

  • Trypsin
  • Trypan Blue Solution (0.4%)
  • Hemocytometer
  • Media for frozen cells
  • DMSO
  • Gloves, 70% ethanol, etc.


  1. Check cell culture log and be sure there is room for frozen vials
  2. Label vials with cell type, date, passage number, etc.
  3. Collect cell suspension by trypsinizing (Take out media and put in 3 mL of trypsin)
  4. Combine all flasks into 50 ml tube
  5. Count cells
    1. 1 million cells per vial (for MC=4 vials/flasks, for UMR=6 vials per flask.)
    2. 1-2 million cells per vial for MLO-A5
  6. Make fresh freezing down media
    1. General (including FAK cells): 95% FBS + 5% DMSO
    2. MLOs: 50% α-MEM, 40% FBS, 5%DMSO
  7. Spin down the cell suspension and pellet the cells (1500rpm, 7-10 min)
  8. Aspirate media
  9. Resuspend cell pellet in freezing media
  10. Aliquot 1ml freezing media per 2ml tube
  11. Place tubes in round isopropanol box in -80C freezer OR
  12. Transfer to the -20C for 2-3 hours
  13. Transfer to the -80C for at least overnight
  14. Store permanently in liquid nitrogen within 1-2 days
  15. Be sure to record in the cell culture log where the cells are permanently stored

Materials for Thawing

  • Media for cells
  • Petri dishes for cells
  • Trypan Blue Solution (0.4%)
  • Hemocytometer


  1. Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
  2. Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
  3. When ready to thaw, remove vial of cells from liquid nitrogen
  4. Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
  5. Place the vial in the hood and clean it with 70% ethanol
  6. Immediately remove the contents of the vial and place into the cold media
  7. Rinse the tube down with some of the cold media from the second vial
  8. Spin down cells at 2000 rpm for 5 min
  9. Aspirate off the cold media and resuspend the cells in the warm media
  10. Transfer the cells and the media into a petri dish
  11. Count cells
  12. Place in incubator
  13. Change the media once the cells have attached to the plate
  14. Allow cells to grow to 80-90% confluency
  15. Split cells (1:3) into petri dishes
  16. Continue to split cells and freeze down as needed

Alternative Thawing

  • From step 5 above, using a 1 ml pipet add 1 ml of the cold media from the 15 ml tube drop by drop to the vial (about 1 drop every 10 seconds).
  • Plate immediately with the cold media and change the media once cells have attached (12 - 24 hours later) to remove DMSO that was in the freezing media. Usually a 1:3 ratio is good for MC3T3s and a 1:4 ratio for MLOY4s.



  • History: CMBL – CRJ/JJR, last updated 2/17/11 by AMN

or instead, discuss this protocol.