Daniel:Protocols/FreezeCells
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Freeze Cells for Storage
Protocol
- Check cells for confluency
- Aspirate, wash with PBS
- Add 500 uL trypsin, incubate 3 minutes
- Add 7.5 of media and pool as you go (for five dishes you’ll end up with 10 mL)
- Take 200 uL and plate it (in 10 mL media)
- Centrifuge 3 minutes at 1000 rpm
- Aspirate media
- Do the next steps fast!
- Prepare a fresh tube with 6 mL of 5%DMSO in DMEM;
- try to find DMSO in Alan’s box or next to tryp LE
- Resuspend the cells in 5 mL and aliquot 1 mL into each vial
- Initials, Date, Cell Line, Passage; top and side
- Put into Mr. Frosty; Transfer to -80
- Next day transfer to liquid nitrogen tank for long-term storage (ask Chris about a box, we use rack 1)