Difference between revisions of "Generating DMSO Stocks for Cell Culture"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (Created page with '==Materials== *Cells in 10cm dishes, at 90-95% confluence. *Cryopreservation Container (Nalgene 5100-0001) *Sterile DMSO ==Protocol== #Pick a low passage number of cells and gro...') |
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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 #430487 | ||
*Sterile DMSO | *Sterile DMSO | ||
==Protocol== | ==Protocol== | ||
#Pick a low passage number of cells and grow 2-5 10cm dishes. | #Pick a low passage number of cells and grow 2-5 10cm dishes. | ||
| − | #At near confluence wash cells twice with PBS -/- and trypsinize normally | + | #At near confluence wash cells twice with PBS -/- and trypsinize normally. |
| − | #Collect all the cells in a 15 mL falcon tube | + | #Collect all the cells in a 15 mL falcon tube. |
| − | #Centrifuge 5 min at 1500RPM to pellet cells | + | #Centrifuge 5 min at 1500RPM to pellet cells. |
| − | #Aspirate media | + | #Aspirate media. |
| − | #Add media (1.8 mL per original plate) | + | #Add media (1.8 mL per original plate). |
| − | #Add sterile DMSO to a final concentration of 10% (0.2 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 | + | #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. | ||
Revision as of 15:41, 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.
