Generating DMSO Stocks for Cell Culture: Difference between revisions

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 ==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.
+#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.
 #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).
-#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.