Volumes are given for a 75 cm² flask. Proportionally increase or reduce the amount of dissociation medium for culture vessels of other sizes.

1. Remove culture medium to a centrifuge tube.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0-3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5-15 minutes). Cells that are difficult to detach can be placed at 37°C to facilitate dispersal.
4. Add 6.0-8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Transfer the cell suspension to the centrifuge tube with the medium and cells from step 1, and centrifuge at 125 x g for 5-10 minutes. Discard the supernatant.
6. Resuspend the cell pellet in the fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
7. Incubate cultures at 37°C.