| Comments |
xrs5 is a X-ray sensitive Chinese Hamster Ovary mutant cell line which was derived from CHO-K1 cells by treating the cells with ethyl methanesulphonate and subsequent growth in agar. These cells belong to X-ray complementation group 5 and are mutant in the p86 subunit of the Ku autoantigen. Ku has been shown to be involved in DNA double-strand break repair and V(D)J recombination. The cells will revert to wild type on treatment with azacytidine. |
| 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).
- 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: 1:6 to 1:10
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 |
Jeggo PA, Kemp LM. X-ray-sensitive mutants of Chinese hamster ovary cell line. Isolation and cross-sensitivity to other DNA-damaging agents. Mutat. Res. 112: 313-327, 1983. PubMed: 6197643
Rathmell WK, Chu G. Involvement of the Ku autoantigen in the cellular response to DNA double-strand breaks. Proc. Natl. Acad. Sci. USA 91: 7623-7627, 1994. PubMed: 8052631
Rathmell WK, Chu G. A DNA end-binding factor involved in double-strand break repair and V(D)J recombination. Mol. Cell. Biol. 14: 4741-4748, 1994. PubMed: 7516471
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.
|
