Daniel:Notebook/HiResChrPaint/2013-9-24

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Revision as of 00:26, 25 September 2013 by >Djacobse (Created page with "=Freezing Fibroblast Cells= Back to Calendar ===Protocol=== from cell freezing protocol 1. Check cells...")
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Freezing Fibroblast Cells

Back to Calendar

Protocol

from cell freezing 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); add 10 uL cell suspension and 90 uL DMEM to petri dish
6. Centrifuge 3 minutes at 1000 rpm
7. Aspirate media
           **Do the next steps fast!
8. 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
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)