Complete Growth Medium
ACL-4 medium (serum-free)
The base medium for this cell line is ATCC formulated DMEM: F12 Medium Catalog No. 30-2006. To make the complete growth medium, add the following components to the base medium:
- 0.02 mg/ml insulin
- 0.01 mg/ml transferrin
- 25 nM sodium selenite (final conc.)
- 50 nM Hydrocortisone (final conc.)
- 1 ng/ml Epidermal Growth Factor (do not filter)
- 0.01 mM ethanolamine (final conc.)
- 0.01 mM phosphorylethanolamine (final conc.)
- 100 pM triiodothyronine (final conc.)
- 0.5% (w/v) bovine serum albumin (final conc.)
- 10 mM HEPES
- 0.5 mM sodium pyruvate (final conc.)
- extra 2mM L-glutamine (for final conc. of 4.5mM)
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.
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 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 xg 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
Freeze medium: Complete growth medium supplemented with 10% fetal bovine serum and 5% DMSO
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%