| Subculturing |
Volumes used in this protocol are for a 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 - 0.53 mM 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.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:6 to 1:12 is recommended
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, 4th edition, published by Wiley - Liss, N.Y., 2000. |
| References |
Aden DP, Knowles BB. Cell surface antigens coded by the human chromosome 7. Immunogenetics 3: 209-221, 1976.
Trinchieri G, et al. Cell-mediated cytotoxicity to SV40-specific tumour-associated antigens. Nature 261: 312-314, 1976. PubMed: 179019
Doherty PC, et al. H-2 gene expression in required for T cell-mediated lysis of virus-infected target cells. Nature 266: 361-362, 1977. PubMed: 300845
Doherty PC, et al. Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype. J. Exp. Med. 148: 534-543, 1978. PubMed: 100569
Wiktor TJ, et al. In vitro evidence of cell-mediated immunity after exposure of mice to both live and inactivated rabies virus. Proc. Natl. Acad. Sci. USA 74: 334-338, 1977. PubMed: 299948
This cell line was established from a tumor which developed after treatment with methylcholanthrene.
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.
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