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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 into new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: 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.

Klebe RJ, et al. Mapping of a human genetic regulator element by somatic cell genetic analysis. Proc. Natl. Acad. Sci. USA 66: 1220-1227, 1970. PubMed: 4920091

Littlefield JW. Selection of Hybrids from Matings of Fibroblasts in vitro and Their Presumed Recombinants. Science 145: 709, 1964. PubMed: 14168277

Klebe RJ, et al. Controlled production of proliferating somatic cell hybrids. J. Cell Biol. 45: 74-82, 1970. PubMed: 4318843

Felluga B, et al. Electron microscope observations on virus particles associated with a transplantable renal adenocarcinoma in BALB-cf-Cd mice. J. Natl. Cancer Inst. 43: 319-333, 1969. PubMed: 5797837

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.