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1. 0.02 mg/ml insulin
2. 0.01 mg/ml transferrin
3. 25 nM sodium selenite
4. 50 nM Hydrocortisone
5. 1 ng/ml Epidermal Growth Factor (do not filter)
6. 0.01 mM ethanolamine
7. 0.01 mM phosphorylethanolamine
8. 100 pM triiodothyronine
9. 0.5% (w/v) bovine serum albumin
10. 10 mM HEPES
11. 0.5 mM sodium pyruvate
12. 2mM L-glutamine(for final conc. of 4.5 mM)
13. 10% FBS

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 and aspirate cells by gently pipetting.
5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 10 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 1:8
Medium Renewal: Every 2 to 3 days

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

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.