Generating DMSO Stocks for Cell Culture: Difference between revisions
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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.