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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. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:2 to 1:6
Medium Renewal: Two times a week

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.

Complete growth medium described above supplemented with 5% (v/v) DMSO.  Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

Cohen LA. Isolation and characterization of a serially cultivated, neoplastic, epithelial cell line from the N-nitrosomethylurea induced rat mammary adenocarcinoma. In Vitro 18: 565-575, 1982. PubMed: 7118137

Cohen LA, Karmali RA. Endogenous prostaglandin production by established cultures of neoplastic rat mammary epithelial cells. In Vitro 20: 119-126, 1984. PubMed: 6423517

Vignon F, et al. Hormonal regulation in two rat mammary cancer cell lines: glucocorticoid and androgen receptors. Mol. Cell. Endocrinol. 13: 191-202, 1979. PubMed: 109329

Okazaki IJ, et al. Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. J. Biol. Chem. 271: 22052-22057, 1996. PubMed: 8703012

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.