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Propagation:           T
he base medium for this cell line is  Dulbecco's Modified Eagle's Medium. To make the complete growth medium, add thefetal bovine serum to a final concentration of 10%.
Temperature: 39.0¡ãC; (Max. 40.0 ¡ãC, Min. 38.0¡ãC)
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 that 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 39¡ãC 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 39¡ãC.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:10 is recommended
Medium Renewal: Twice per week
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