Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

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 that 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 21 to 23°C 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 centrifuge tube and spin at approximately 125 xg 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.
7. Incubate cultures at 21 to 23°C without CO2.