Jacobs:Protocol Freezing and Thawing Cells

Procedure

Freezing

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
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: 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

Thawing

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 to a T25 (or T75 depending on the number of cells)
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 T75 flasks
16. Continue to split cells and freeze down as needed

Contact

• History: CMBL – CRJ/JJR, last updated 8/1/07

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