TissueCulture:Thawing cells

Overview

This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).

Materials

List reagents, supplies and equipment necessary to perform the protocol here.

• 10-50 ml centrifuge tube
• Cell culture medium (depends on cell line/cells)
• Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
• Basic TC centriguge (0 - 2500 rpm)

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

   Keep frozen cells on dry ice until ready for thawing.

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 (80-100 x g) 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

    Removes remaining DMSO

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

Notes

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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.

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== See also

Jacobs:Protocol_Freezing_and_Thawing_Cells

WangLab:Thawing_Cells

Marek:_Freeze-down/Thaw

Wittrup:_Thawing_Protocol

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.