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(Note: The L-15 medium formulation was devised for use in a free gas exchange with atmospheric air. A CO2 and air mixture is detrimental to cells when using this medium for cultivation)

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. Remove and discard culture medium.
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 within 5 to 15 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^oC to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension into new culture vessels.
6. Incubate cultures at 37°C.

Medium Renewal: Replace the medium every 2 to 3 days.

Twenty-Fourth Annual Meeting of the Tissue Culture Association, Boston, MA. In Vitro 8: 443, 1973

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