Daniel:Protocols/FreezeCells
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Freeze Cells for Storage[edit]
Protocol[edit]
- Check cells for confluency
- Aspirate, wash with PBS
- Add 0.1X the culture volume (0.5 mL for a t25 with 5 mL media volume) trypsin
- Incubate 3-5 minutes at 37C
- Add 0.9X of media to flask
- Plate with 0.1X culture volume into flask/dish with 0.9X media (Plate with 0.5 mL cells in 4.5 mL media for 5 mL total)
- Add the rest of the media to a 15 mL tube
- Centrifuge 3 minutes at 1000 rpm
- Aspirate media
- Do the next steps fast!
- Prepare a fresh tube with 6 mL of 5%DMSO in media;
- try to find DMSO in Alan’s box or next to tryp LE
- Resuspend the cells in media such that cells are approximately 1E6 cells/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
- Be sure to add the cells to (this spreadsheet)