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

1. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
2.  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 within 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°C to facilitate dispersal.
3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. 
4. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1 X 10⁴ viable cells/cm² is recommended
5. Incubate cultures at 37°C. 

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.   

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