| Applications |
C6/lacZ7 is a subclone of the C6/lacZ cell line (ATCC CRL-2199) that was established in 1989 from the N-nitrosomethylurea-induced glial rat, outbred strain, C6 cell line. The cells were cultured in G418 for 14 days, cloned, and evaluated for beta-gal production. The cells constitutively express the lacZ reporter gene product, E. coli derived beta-gal, as revealed on tissue sections by histochemical stain, and single tumor cells can be identified. Lymphocytes and other responding cells can be identified by double labeling with antibodies on the same slide. The contrast between stained cells and background facilitates image analysis. The depositor has confirmed that the C6/lacZ7 subclone grows in rat brain, and that the lacZ marker can be detected by histochemistry for b-gal. This is one of few models that permit quantitative analysis of microscopic tumor in the brain. The beta-gal expression is less stable than for 9L/lacZ. Cells should be used soon after thawing or re-cloned. Methods for the use of C6/lacZ may be found in Cancer Res. |
| Derivation |
C6/lacZ7 is a subclone of the C6/lacZ cell line (ATCC CRL-2199) that was established in 1989 from the N-nitrosomethylurea-induced glial rat, outbred strain, C6 cell line. The cells constitutively express the lacZ reporter gene product, E. coli derived beta-gal, as revealed on tissue sections by histochemical stain, and single tumor cells can be identified. |
| Comments |
C6/lacZ7 is a subclone of the C6/lacZ cell line (ATCC CRL-2199) that was established in 1989 from the N-nitrosomethylurea-induced glial rat, outbred strain, C6 cell line. C6 cells were infected with the BAG replication deficient retroviral vector carrying the E. coli lacZ gene encoding beta-gal and the Tn5 neomycin gene, which confers resistance to G418. The cells were cultured in G418 for 14 days, cloned, and evaluated for beta-gal production. To obtain the subclone, C6/lacZ was cloned in 1996 at limiting dilution. The cells constitutively express the lacZ reporter gene product, E. coli derived beta-gal, as revealed on tissue sections by histochemical stain, and single tumor cells can be identified. Lymphocytes and other responding cells can be identified by double labeling with antibodies on the same slide. The contrast between stained cells and background facilitates image analysis. The subclone was chosen because it has a more stable expression of the lacZ marker. The depositor has confirmed that the C6/lacZ7 subclone grows in rat brain, and that the lacZ marker can be detected by histochemistry for b-gal. This is one of few models that permit quantitative analysis of microscopic tumor in the brain. The tumor mimics important features of human brain tumor growth and spread. The beta-gal expression is less stable than for 9L/lacZ. Cells should be used soon after thawing or re-cloned. The cloned cells have been shown to retain the marker for up to eleven weeks in culture and up to three weeks in the brain. Methods for the use of C6/lacZ may be found in Cancer Res. 52: 1018-1025, 1992; ibid., 53: 176-182, 1993. |
| 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.
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:6 to 1:12
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 |
Lampson LA, et al. Exploiting the lacZ reporter gene for quantitative analysis of disseminated tumor growth within the brain: use of the lacZ gene product as a tumor antigen, for evaluation of antigenic modulation, and to facilitate image analysis of tumor growth in situ. Cancer Res. 53: 176-182, 1993. PubMed: 8416743
Lampson LA, et al. Disseminating tumor cells and their interactions with leukocytes visualized in the brain. Cancer Res. 52: 1018-1025, 1992. PubMed: 1737331
Lampson LA, et al. A new model permits study of the immune response to individual tumor cells within the brain. Neurology 40: 396-397, 1990.
Dutta T, et al. Robust ability of IFN-gamma to upregulate class II MHC antigen expression in tumor bearing rat brains. J. Neuro-Oncol. 64: 31-44, 2003. PubMed: 12952284
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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