Difference between revisions of "Generating DMSO Stocks for Cell Culture"
From Bridges Lab Protocols
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*Cells in 10cm dishes, at 90-95% confluence. | *Cells in 10cm dishes, at 90-95% confluence. | ||
*Cryopreservation Container (Nalgene 5100-0001) | *Cryopreservation Container (Nalgene 5100-0001) | ||
| − | *Cryopreservation Vials Corning | + | *Cryopreservation Vials (Corning 430487) |
*Sterile DMSO | *Sterile DMSO | ||
Revision as of 15:48, 13 August 2009
Materials
- Cells in 10cm dishes, at 90-95% confluence.
- Cryopreservation Container (Nalgene 5100-0001)
- Cryopreservation Vials (Corning 430487)
- Sterile DMSO
Protocol
- Pick a low passage number of cells and grow 2-5 10cm dishes.
- At near confluence wash cells twice with PBS -/- and trypsinize normally.
- Collect all the cells in a 15 mL falcon tube.
- Centrifuge 5 min at 1500RPM to pellet cells.
- Aspirate media.
- Add media (1.8 mL per original plate).
- Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate).
- Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials.
- Label vials with name, date, cell type and passage (if known).
- Ensure isopropanol is added to the container to above the indicated line.
- Place container with vials at -80 for 1-3 days.
- Remove cells from container and place in liquid nitrogen storage.
