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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: 1:8 to 1:10
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.

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

Ehmann UK, et al. To grow mouse mammary epithelial cells in culture. J. Cell Biol. 98: 1026-1032, 1984. PubMed: 6699079

Dulbecco R, et al. Differentiation of a rat mammary cell line in vitro. Proc. Natl. Acad. Sci. USA 76: 1256-1260, 1979. PubMed: 375233

Ehmann UK, et al. Long-term proliferation of mouse thymic epithelial cells in culture. In Vitro Cell. Dev. Biol. 22: 738-748, 1986. PubMed: 3782011

Ehmann UK, et al. Cultured proliferating rat mammary epithelial cells. In Vitro Cell. Dev. Biol. 27A: 749-754, 1991. PubMed: 1717432

Ehmann UK, et al. Gap-junctional communication between feeder cells and recipient normal epithelial cells correlates with growth stimulation. In Vitro Cell. Dev. Biol. Anim. 37: 100-110, 2001. PubMed: 11332735

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.