Procedure:
To insure the highest level of viability, be sure to warm media and Trypsin/ EDTA to 37ºC before using it on the cells.

Cells should be split when they reach confluency. A split ratio of 1:5 to 1:7 is recommended. Volumes used in this protocol are for 225 cm² (T225); proportionally reduce or increase amount of dissociation medium for culture flasks of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 1X PBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
3. Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).
4. Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.
5. Transfer all cells into a centrifuge bottle or tube and centrifuge at 270 xg for 5 minutes.
6. Remove and discard the supernatant
7. Add 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
8. Add more complete growth medium to cell suspension as needed to plate cells at approximately 5x10⁶/T225 flask.
9. Place flasks in incubator @ 37°C with a 5% CO₂ in air atmosphere.