Complete Growth Medium
The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: 0.2 Units/ml bovine insulin; fetal bovine serum to a final concentration of 10%.
Phenol Red is a structural mimic for estrogen. Therefore the estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture (RefBerthois Y, et al. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc. Natl. Acad. Sci. USA 83: 2496–2500, 1986. PubMed: 3458212). The Phenol Red-free version of RPMI 1640, is ATCC catalog No. 30-2602.
Volumes used in this protocol are for 75cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: Subculture cells before or upon reaching confluence. Do not allow cells to become super-confluent.
- Remove and discard culture medium.
- Briefly rinse the cell layer with either Hanks' Balanced Salt Solution (HBSS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
- Add 1.0 to 2.0 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 10 to 15 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 x 104 to 4 x 104 viable cells/cm2 is recommended.
- Incubate cultures at 37C. We recommend that you maintain cultures at a cell concentration between 3 x 104 and 2 x 105 cells/cm2.
Subcultivation Ratio: 1:2 to 1:3 is recommended
Medium Renewal: every 2 to 3 days
Freeze medium: culture medium, 95%; DMSO, 5%
Storage temperature: liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Duration: The depositor states that supplementing the growth medium with insulin is optional.