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The cells are positive for keratin by immunoperoxidase staining.

The cells are negative for bovine viral diarrhea virus (BVDV).

Electron microscopic studies show many bulb gap junctions (BGJ).

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 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. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to10 minutes.
5. Discard supernatant and resuspend cells in fresh growth medium Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:8 is recommended
Medium Renewal: Twice 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.

Hull RN, et al. Development and characteristics of the rabbit kidney cell line strain, LLC-RK. Proc. Soc. Exp. Biol. Med. 118: 1054-1059, 1965. PubMed: 14277663

Bolin SR, et al. Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus. J. Virol. Methods 48: 211-221, 1994. PubMed: 7989438

Hull RN, Butorac G. The utility of rabbit kidney cell strain, LLC-RK to rubella virus studies. Am. J. Epidemiol. 83: 509-517, 1966. PubMed: 4956596

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.