Changes

#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 to the 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. [[Category: Cell Culture]][[Category: Storage]]