Difference between revisions of "Generating DMSO Stocks for Cell Culture"

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m
m (clarified some calculations, removed the isopropanol step)
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==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 tot eh 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).
#Ensure isopropanol is added to the container to above the indicated line.
 
 
#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.

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

  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 tot eh 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.