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 used in this protocol are for 75 cm2
flasks; 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 Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 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 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 37C to facilitate dispersal.
- Add 2.0 to 3.0 ml of complete growth medium and aspirate cells by gently pipetting.
Transfer cell suspension to a centrifuge tube and spin at approximately 125 X g for 5 to 10 minutes. Discard supernatant.
- Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37C.
Medium Renewal: Every 2 to 3 days
Freeze medium: RPMI 1640 Medium, 85%; fetal bovine serum, 10%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase