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ATCC complete growth medium: Dulbeccos modified Eagles medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 90%; fetal bovine serum, 10% Temperature: 37.0C Atmosphere: air, 95%; carbon dioxide (CO2), 5% Growth Conditions: The cells can also be propagated in a serum free medium consisting of 3 parts Dulbeccos modified Eagles medium, 1 part Waymouths MAB 87/3 medium and 30 nM selenium.
Subculturing:
Protocol:
Remove and discard culture medium.
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.
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 37C to facilitate dispersal.
Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
Add appropriate aliquots of the cell suspension to new culture vessels.
Incubate cultures at 37C.
Subcultivation ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
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