Cryopreservation of Cells

Protocol:
1- Warm Media, FBS, PBS and Trypsin to 37 degrees
2- Dilute Trypsin: 1ml of Trypsin + 9ml of PBS to make a 1X Tryspin solution
3- Add 1ml of Dimethyl Sulfoxide (DMSO) to 9ml of Foetal Bovine Serum (FBS) to make cell storage medium
4- Pour out the old media in your flask
5- Wash the cells with 5ml of PBS
6- Add 1ml of Trypsin to strip/detach the cells
7- Incubate the flasks for 5 minutes
8- Check under the microscope to confirm that your cells have indeed detached.
9- After incubating add 5ml of media to neutralize or quench the Trypsin
10- Take up all the cells in the flask using a serological pipette and place into a 15ml centrifuge tube
11- Centrifuge at 400rcf for 3 minutes
12- Pour out the supernatant, whilst being careful not to disturb or pour out your cell pellet at the bottom of the tube
12- Add 1ml of the cell storage media (1ml DMSO + FBS) to the same tube and pipette up and down to mix the cells with the media
13- Next pipette 1ml of this into a Cryo tube.
14- Label the Cryo tubes with Cell name, passage number, the date and your name
15- Freeze at -80C