Marek: Freeze-down/Thaw

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
• 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 90% 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)

1. Centrifuge at 1000-1200 rpm for 4-5 min at RT.
2. Suck out the medium.
3. Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
4. Transfer cells into freeze-down box and place 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)

1. Wash cells with PBS.
2. Add thin layer of Trypsin-EDTA on cells (approx. 1ml for 90 mm Petri dish)
3. Observe cells under microscope until these are rounded.
4. Add serum-containing medium and resuspend cells. Transfer into centrifuge tubes.
5. Centrifuge at 1000-1200 rpm for 4-5 min at RT.
6. Suck out the medium.
7. Resuspend cells in appropriate volume of freezing medium (e.g. 1.5 ml for 1.8ml Cryo-tube).
8. Transfer cells into freeze-down box and place at -80 gr.C. (Comments as for suspension cells).

Thawing

1. Keep frozen cells on dry ice until ready for thawing.
2. Warm-up cells by shaking/incubation in 37gr.C water bath until little ice cube visible.
3. Spray tube with 70% ethanol to sterilize.
4. Aspirate cells from cryo-tube into pre-half-filled pipette with warm culture medium.
5. Resuspend cell in culture medium: 10 ml total volume.
6. Centrifuge for 5 min at 1000 rpm (RT).
7. Resuspend pelleted cells in appropriate volume of culture medium.

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.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.