Splitting Cells

Protocol:
1- Warm Media, PBS and Trypsin to 37 degrees
2- Dilute Trypsin: 1ml of Trypsin + 9ml of PBS to make a 1X Tryspin solution
3- Pour out the old media in your flask
4- Wash the cells with 5ml of PBS
5- Add 1ml of Trypsin to strip/detach the cells
6- Incubate the flasks for 5 minutes
7- Check under the microscope to confirm that your cells have indeed detached.
8- After incubating add 5ml of media to neutralize or quench the Trypsin
9- Take up all the cells in the flask using a serological pipette and place into a 15ml centrifuge tube
10- Centrifuge at 400rcf for 3 minutes
11- Pour out the supernatant, whilst being careful not to disturb or pour out your cell pellet at the bottom of the tube
12- Add 10ml of new media to the same tube and pipette up and down to mix the cells with the media
13- Next pipette 5ml of this into one T75 and add another 5ml of media to the T75 flask to make the total volume 10ml.
14- Pipette the other 5ml into another T75 and add 5ml of media
15- Label and Incubate