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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
1. 0.005 mg/ml Insulin
2. 0.01 mg/ml Transferrin
3. 30nM Sodium selenite (final conc.)
4. 10 nM Hydrocortisone (final conc.)
5. 10 nM beta-estradiol (final conc.)
6. extra 2mM L-glutamine (for final conc. of 4.5 mM)
7. 5% fetal bovine serum (final conc.)

1. Remove and discard culture medium.
2. 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 to remove all traces of serum which contains trypsin inhibitor.
3. 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 1.5 x 10⁴ to 2 0 x 10⁴ viable cells/cm² is recommended.

Subcultivation Ratio: 1:3 to 1:8


6. Incubate cultures at 37°C. Subculture when cell concentration is between 9 x 10⁴ and 1.7 x 10⁵ cells/cm².

NCI-Navy Medical Oncology Branch Cell Line Supplement. J. Cell. Biochem. suppl. 24: 1996.