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:
0.2 mg/ml G -418
fetal bovine serum to a final concentration of 10%
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-0.53 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 pipetting gently.
- Add appropriate aliquots of cell suspension to new culture vessels. An inoculum of 4 x 103 to 2 x 104 viable cells/cm2 is recommended. Maintain cultures at a cell concentration between 1 X 104 and 1 X 105 cells/cm2
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended
Medium Renewal: Two to three times weekly
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture of Animal Cells: a Manual of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%