2X Freezing media = 80% FBS + 20% tissue-culture grade DMSO
- Change media 2-4 hours prior to freezing. Cells must be 80% confluent and healthy.
- Prepare 2X freezing media and label cryovials with date/name/number of cells. Pre-chill on ice.
- When cells are ready to be frozen, aspirate off old media and wash with 10ml pre-warmed PBS. Add PBS gently to the side of the plate to avoid disrupting the cells.
- After aspirating off PBS, add 2ml of pre-warmed trypsin. Be sure to cover the entire plate by carefully swirling it.
- Incubate in a 37C incubator for 5 minutes and check under the microscope. If you see big clumps of cells, incubate additional 2-5 minutes. Do not over-trypsinize.
- While cells are incubating, add 5ml of warmed medium into 15ml Falcon tube.
- Once majority of the cells are uniformly dispersed into small clumps or single cells, inactivate trypsin by adding 5ml of medium. Gently pipette up and down using 10ml pipette 10-15 times without making bubbles.
- Transfer the cells into the 15ml Falcon tube and take a small amount to count the number of cells.
- Spin down the cells by centrifuging for 5 minutes at 1000rpm, and meanwhile count the cells using hemocytometer. Aspirate off the supernatant and resuspend the pellet in pre-chilled medium.
- Add equal volume of freezing media slowly while swirling. The final concentration of DMSO must be 10%.
- Aliquot 0.5-1ml into each cryovial. 2-4 million cells/vial can be revived to a 10cm plate.