Complete Growth Medium
The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: heat-inactivated fetal bovine serum to a final concentration of 15%.
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) trypsin- 0.53mM EDTA-0.5% (w/v) polyvinylpyrrolidone solution to remove all traces of serum ,which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA- polyvinylpyrrolidone solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
- 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 28°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.
culture medium 95%; DMSO, 5%