Daniel:Protocols/FreezeCells

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Revision as of 20:04, 4 August 2014 by >Djacobse
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Freeze Cells for Storage

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Protocol

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