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edits
Changes
m
#Ensure isopropanol is added to the container to above the indicated line.
clarified some calculations, removed the isopropanol step
==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 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, 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.
