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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 0.25% (w/v) Trypsin-0.53mM 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 with 5% FBS and aspirate cells by gently pipetting.
5. Transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
6. Discard supernatant and resuspend cells in fresh culture medium. Add appropriate aliquots of cell suspension to new culture vessels.
7. Place culture vessels in incubators at 37°C.

Subcultivation Ratio: 1:3 to 1:5
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.

Atmosphere: Air, 95%; Carbon dioxide (CO₂), 5%

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

Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.

Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.

Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.

Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.