Generating DMSO Stocks for Cell Culture

Materials

• Cells in 10cm dishes, at 90-95% confluence.
• Cryopreservation Container (Nalgene 5100-0001)
• Cryopreservation Vials (Corning 430487)
• Sterile DMSO

Protocol

1. Pick a low passage number of cells and grow 2-5 10cm dishes.
2. At near confluence wash cells twice with PBS -/- and trypsinize normally.
3. 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.)
4. Centrifuge 5 min at 1500RPM to pellet cells.
5. Aspirate media.
6. Add media (1.8 mL per original plate, so if you started with 5 plates, the added media will be 5*1.8).
7. 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).
8. Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials. (Resuspend with a pipette tip gently.)
9. Label vials with name, date, cell type and passage (if known).
10. Place container with vials at -80 for 1-3 days.
11. Remove cells from container and place in liquid nitrogen storage.