Daniel:Protocols/FreezeCells

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Freeze Cells for Storage[edit]

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Protocol[edit]

  1. Check cells for confluency
  2. Aspirate, wash with PBS
  3. Add 0.1X the culture volume (0.5 mL for a t25 with 5 mL media volume) trypsin
  4. Incubate 3-5 minutes at 37C
  5. Add 0.9X of media to flask
  6. 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)
  7. Add the rest of the media to a 15 mL tube
  8. Centrifuge 3 minutes at 1000 rpm
  9. Aspirate media
    1. Do the next steps fast!
  10. Prepare a fresh tube with 6 mL of 5%DMSO in media;
    1. try to find DMSO in Alan’s box or next to tryp LE
  11. Resuspend the cells in media such that cells are approximately 1E6 cells/mL and aliquot 1 mL into each vial
  12. Initials, Date, Cell Line, Passage; top and side
  13. Put into Mr. Frosty; Transfer to -80
  14. Next day transfer to liquid nitrogen tank for long-term storage
    1. Be sure to add the cells to (this spreadsheet)

Useful Numbers for Cell Culture (From Thermo)[edit]

File:Cell Culture Tables.png