This short protocol describes how to freeze down and thaw (or bring up) mammalian cells (e.g. HeLa, KEK293, CHO, Jurkat T cells).
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)
- Setup two vials of cold (4 C) media (10 ml) in a 15 ml tube
- Warm remaining media (~5-8 ml) in a 15 ml tube to 37C
- When ready to thaw, remove vial of cells from liquid nitrogen
- Keep frozen cells on dry ice until ready for thawing.
- Swirl the vial in the water bath (37C) until a small ice chip is left in the tube (~1 min)
- Place the vial in the hood and clean it with 70% ethanol
- Immediately remove the contents of the vial and place into the cold media
- Rinse the tube down with some of the cold media from the second vial
- Spin down cells at 2000 rpm (80-100 x g) for 5 min
- Aspirate off the cold media and resuspend the cells in the warm media
- Transfer the cells and the media into a petri dish
- Count cells
- Place in incubator
- Change the media once the cells have attached to the plate
- Removes remaining DMSO
- Allow cells to grow to 80-90% confluency
- Split cells (1:3) into petri dishes
- Continue to split cells and freeze down as needed
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
- 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
- Who has experience with this protocol?
or instead, discuss this protocol.