| Comments |
Thymidine kinase-deficient derivative of ts13, originally isolated as a temperature-sensitive mutant of the baby hamster kidney cell line BHK-21. The tk-ts13 cell line is a derivative of BHK-21. The cells are temperature sensitive for growth, and are thymidine kinase deficient (TK-). The line is an efficient host for transfection studies. The cells should be grown at 34C. |
| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- 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.
- 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.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels. Inoculate new cultures at 2 x 104 cells/cm2
- Incubate cultures at 34°C.
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. |
| References |
Talavera A, Basilico C. Temperature sensitive mutants of BHK cells affected in cell cycle progression. J. Cell. Physiol. 92: 425-436, 1977. PubMed: 903382
Black ME, et al. Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy. Proc. Natl. Acad. Sci. USA 93: 3525-3529, 1996. PubMed: 8622970
Shen YM, et al. Superimposition of temperature regulation on yeast promoters. Mol. Cell. Biol. 2: 1145-1154, 1982. PubMed: 6294505
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.
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