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1. Remove and discard culture medium.
2. Briefly rinse the cell layer with or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution or Dulbecco?s Phosphate Buffered Saline (D-PBS) to remove all traces of serum that contains trypsin inhibitor.
3. Add 1.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until the 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°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
6. Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels. An inoculum of 5 x 10³ to 7 x 10³ viable cells/cm² is recommended.
7. Place culture vessels in incubators at 37°C. Maintain cultures at a cell concentration between 3 x 10⁴ and 5 x 10⁴ cells/cm².

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