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
- cryo tubes
- DMSO, Tissue culture (TC)-tested (e.g. Sigma, D2650)
- Cell culture medium (depends on cell line/cells)
- Trypsin-EDTA (e.g. from PAA, L11-004) - for adeherent cells
- Freeze-down medium
- 6% DMSO (some people prefer 10%)
- Cell culture medium (I recommend to use at least 20% FCS and you can use 100% fetal calf serum (FCS) instead of cell culture medium)
- Basic TC centriguge (0 - 2500 rpm)
- Freeze-down box (this could be simply made from a small styropore box and cotton or from two styropore racks from 15-ml centrifuge tubes )
Freeze-down (suspension cells)
- Centrifuge at 80-100 x g for 4-5 min at RT.
- Suck out the medium.
- Resuspend 1-5 x 10*6 cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
- Transfer cryo-vials with cells into freeze-down box and store at -80 gr.C.
*Leave over-night at -80 gr.C. *You can store cells at -80 gr.C for 6-12 months. *Transfer cells into liquid nitrogen a day later for longer storage (1-20 years or so).
Freeze-down (adherent cells)
- Wash cells with PBS.
- Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
- Observe cells under microscope until these are rounded.
- Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
- Centrifuge at 1000-1200 rpm for 4-5 min at RT.
- Suck out the medium.
- Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
- Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).
- Keep frozen cells on dry ice until ready for thawing.
- Swirl a vial with frozen cells in 37gr.C water bath and remove just while a sliver of ice remains.
- Spray tube with 70% ethanol for sterilization.
- Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
- Fill centrifugation tube with culture medium: at least 10 ml total volume.
- Centrifuge for 5 min at 80-100 x g (RT).
- Resuspend pelleted cells in appropriate volume of culture medium and culture o/n in CO2 incubator.
- Optional: Exchange medium next day.
- 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.
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