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 - 0.53 mM EDTA solution to remove all traces of serum that 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.
6. Incubate cultures at 37°C.

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.

Giaccone G, et al. Neuromedin B is present in lung cancer cell lines. Cancer Res. 52: 2732s-2736s, 1992. PubMed: 1563005

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.