Complete Growth Medium
The base medium for this cell line is F-12K Medium (ATCC 30-2004). To make the complete growth medium, add the following components to the base medium:
Do not filter complete medium.
- non heat-inactivated fetal bovine serum (FBS; ATCC 30-2020) to a final concentration of 20%
- 25 ng/ml cholera toxin (Sigma cat# C8052)
- 10 ng/ml mouse Epidermal Growth Factor (Corning cat# 354010)
- 0.005 mM phosphoethanolamine (Sigma cat# P0503)
- 100 pg/ml hydrocortisone (Sigma cat# H0135)
- 45 nM sodium selenite (Sigma cat# 9133)
- 0.005 mg/ml human recombinant insulin (Life Technologies cat# 12585-014)
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 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
Subculture Ratio: 1:2 to 1:4
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.
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%