Daniel:Protocols/FreezeCells: Difference between revisions
Jump to navigation
Jump to search
>Djacobse (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...") |
>Djacobse No edit summary |
||
Line 5: | Line 5: | ||
===Protocol=== | ===Protocol=== | ||
#Check cells for confluency | |||
#Aspirate, wash with PBS | |||
#Add 500 uL trypsin, incubate 3 minutes | |||
#Add 7.5 of media and pool as you go (for five dishes you’ll end up with 10 mL) | |||
#Take 200 uL and plate it (in 10 mL media) | |||
#Centrifuge 3 minutes at 1000 rpm | |||
#Aspirate media | |||
##Do the next steps fast! | |||
#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 | |||
#Resuspend the cells in 5 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 (ask Chris about a box, we use rack 1) |
Revision as of 20:04, 4 August 2014
Freeze Cells for Storage
Protocol
- Check cells for confluency
- Aspirate, wash with PBS
- Add 500 uL trypsin, incubate 3 minutes
- Add 7.5 of media and pool as you go (for five dishes you’ll end up with 10 mL)
- Take 200 uL and plate it (in 10 mL media)
- Centrifuge 3 minutes at 1000 rpm
- Aspirate media
- Do the next steps fast!
- 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
- Resuspend the cells in 5 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 (ask Chris about a box, we use rack 1)