Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Shake flask. 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 which contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually with 5 to 10 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.  Cells that are difficult to detach may be placed at 37¡ãC to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
5. To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube with the medium and cells from step #1 and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium.  Add appropriate aliquots of cell suspension to new culture vessels.
7. Place culture vessels in incubators at 37¡ãC.