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.

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Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% 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¡ãC 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 to new culture vessels.
6. Incubate cultures at 37¡ãC.

Subcultivation Ratio: 1:4 to 1:8 is recommended

Medium Renewal: Every 2 to3 days

Freeze medium: Culture medium, 95%; DMSO, 5%

Storage temperature: liquid nitrogen vapor phase

Atmosphere: air, 95%; carbon dioxide (CO2), 5%

Temperature: 37¡ãC