Volumes used in this protocol are for 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.03% (w/v) 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 supension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per 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 culture medium described above supplemented with 5% (v/v) DMSO.xa0xa0Cell culture tested DMSO is available as ATCC Catalog No. 4-X.

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Evans CH, DiPaolo JA. Neoplastic transformation of guinea pig fetal cells in culture induced by chemical carcinogens. Cancer Res. 35: 1035-1044, 1975. PubMed: 1116142

Evans CH, et al. Colony inhibition mediated by nonimmune leukocytes in vitro and skin reactivity in vivo as indices of tumorigenicity of guinea pig cultures transformed by chemical carcinogens. Cancer Res. 35: 1045-1052, 1975. PubMed: 1116143

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.