Daniel:Protocols/FreezeCells: Difference between revisions

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(Created page with "=Freeze Cells for Storage= Back to Main ===Protocol=== 1. Check cells for confluency 2. Aspirate, wash with PBS 3. Add 500 uL trypsin, incubate 3 min...")
 
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===Protocol===
===Protocol===


1. Check cells for confluency
#Check cells for confluency
2. Aspirate, wash with PBS
#Aspirate, wash with PBS
3. Add 500 uL trypsin, incubate 3 minutes
#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)
#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)
#Take 200 uL and plate it (in 10 mL media)
6. Centrifuge 3 minutes at 1000 rpm
#Centrifuge 3 minutes at 1000 rpm
7. Aspirate media
#Aspirate media
            **Do the next steps fast!
##Do the next steps fast!
8. Prepare a fresh tube with 6 mL of 5%DMSO in DMEM;  
#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
##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
#Resuspend the cells in 5 mL and aliquot 1 mL into each vial
10. Initials, Date, Cell Line, Passage; top and side
#Initials, Date, Cell Line, Passage; top and side
11. Put into Mr. Frosty; Transfer to -80
#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)
#Next day transfer to liquid nitrogen tank for long-term storage (ask Chris about a box, we use rack 1)

Revision as of 20:04, 4 August 2014

Freeze Cells for Storage

Back to Main

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)