Complete Growth Medium
The base medium for this cell line is Medium 199 containing 1.5 g/L sodium bicarbonate.
To make the complete growth medium, add the following components to the base medium:
10% fetal bovine serum (final conc.)
3.3 nM epidermal growth factor (EGF) (final conc.) (do not filter).
400 nM hydrocortisone (final conc.)
870 nM zinc-free bovine insulin (final conc.)
20 mM HEPES (final conc.)
The trace elements at the following final concentrations:
See Methods in Cell Biology, Vol. 21B, pg. 200, 1980
- H2SeO3 0.3869 mg/L (Selenious acid)
- MnCl2×4H20 0.0198 mg/L (Manganese chloride)
- Na2SiO3×9H20 14.2100 mg/L (Sodium silicate)
- (NH4)6Mo7O24×4H20 0.1236 mg/L (Ammonium molybdate)
- NH4VO3 0.0585 mg/L (Ammonium vanadate)
- NiSO4×6H20 0.0131 mg/L (Nickle sulfate)
- SnCl2×2H20 0.0113 mg/L (Tin Chloride)
This medium is formulated for use with a 5% CO2 in air atmosphere.
ATCC tested fetal bovine serum is available as ATCC Catalog No. 30-2020 (500ml) and ATCC Catalog No. 30-2021 (100ml).
Volumes are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.
- 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 37°C to facilitate dispersal.
Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes.
Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: Every 2 to 3 days
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase