Generating DMSO Stocks for Cell Culture

From Bridges Lab Protocols
Jump to: navigation, search

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.
  4. Centrifuge 5 min at 1500RPM to pellet cells.
  5. Aspirate media.
  6. Add media (1.8 mL per original plate).
  7. Add sterile DMSO to a final concentration of 10% (0.2 mL per original plate).
  8. Gently resuspend cells and aliquot 1 mL of suspension into cryopreservation vials.
  9. Label vials with name, date, cell type and passage (if known).
  10. Ensure isopropanol is added to the container to above the indicated line.
  11. Place container with vials at -80 for 1-3 days.
  12. Remove cells from container and place in liquid nitrogen storage.