Difference between revisions of "Generating DMSO Stocks for Cell Culture"
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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 | ||
==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. Add media up to 15 ml. (To avoid potentially over-trypsinizing, you can add media to each plate immediately after the cells detach, then add media up to the 15 ml mark on the 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, so if you started with 5 plates, the added media will be 5*1.8). |
| − | #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, so if you started with 5 plates, the added DMSO will be 0.2*5). |
| − | #Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials. | + | #Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials. (Resuspend with a pipette tip gently.) |
#Label vials with name, date, cell type and passage (if known). | #Label vials with name, date, cell type and passage (if known). | ||
| − | |||
#Place container with vials at -80 for 1-3 days. | #Place container with vials at -80 for 1-3 days. | ||
#Remove cells from container and place in liquid nitrogen storage. | #Remove cells from container and place in liquid nitrogen storage. | ||
Latest revision as of 18:34, 1 June 2018
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. Add media up to 15 ml. (To avoid potentially over-trypsinizing, you can add media to each plate immediately after the cells detach, then add media up to the 15 ml mark on the falcon tube.)
- Centrifuge 5 min at 1500RPM to pellet cells.
- Aspirate media.
- Add media (1.8 mL per original plate, so if you started with 5 plates, the added media will be 5*1.8).
- Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate, so if you started with 5 plates, the added DMSO will be 0.2*5).
- Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials. (Resuspend with a pipette tip gently.)
- Label vials with name, date, cell type and passage (if known).
- Place container with vials at -80 for 1-3 days.
- Remove cells from container and place in liquid nitrogen storage.
