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The JB6 Cl 41-5a cell line was established from primary cultures of neonatal BALB/c epidermal cells that had been treated with dimethylsulfoxide (DMSO, 0.01 to 0.1 %).

The line is sensitive to promotion of transformation (P+) by phorbol esters and other tumor promoters.

The cells must be carried at low density to avoid spontaneous transformation.

The cells should be subcultured for at least two passages prior to testing in agar for tumor promotor induced transformation.

For the Anchorage Independent Transformation Assay, suspend 10000 cells in 0.33% agar medium containing 8% fetal bovine serum, with or without added tumor promoter (e.g., 10 ng/mL TPA). Incubate at 37°C, and score colonies greater than 8 cells at 14 days.

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.05% (w/v) Trypsin-0.53 mM 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 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: An inoculation density of 2 x 10⁴ viable cells per 25 cm² flask is recommended
Medium Renewal: 2 to 3 times 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.

Colburn NH, et al. A cell culture assay for tumor-promoter-dependent progression toward neoplastic phenotype: detection of tumor promoters and promotion inhibitors. Teratog. Carcinog. Mutagen. 1: 87-96, 1980. PubMed: 6119803

Sun Y, et al. Dosage-dependent dominance over wild-type p53 of a mutant p53 isolated from nasopharyngeal carcinoma. FASEB J. 7: 944-950, 1993. PubMed: 8344492

Colburn NH, et al. Correlation of anchorage-independent growth with tumorigenicity of chemically transformed mouse epidermal cells. Cancer Res. 38: 624-634, 1978. PubMed: 626967

Dong Z, et al. Blocking of tumor promoter-induced AP-1 activity inhibits induced transformation in JB6 mouse epidermal cells. Proc. Natl. Acad. Sci. USA 91: 609-613, 1984. PubMed: 8290571

Colburn NH, et al. Tumour promoter induces anchorage independence irreversibly. Nature 281: 589-591, 1979. PubMed: 492322

Bernstein LR, Colburn NH. AP1/jun function is differentially induced in promotion-sensitive and resistant JB6 cells. Science 244: 566-569, 1989. PubMed: 2541502

Colburn NH, et al. Dissociation of mitogenesis and late-stage promotion of tumor cell phenotype by phorbol esters: mitogen-resistant variants are sensitive to promotion. Proc. Natl. Acad. Sci. USA 78: 6912-6916, 1981. PubMed: 6947266

Sun Y, et al. Progression toward tumor cell phenotype is enhanced by overexpression of a mutant p53 tumor-suppressor gene isolated from nasopharyngeal carcinoma. Proc. Natl. Acad. Sci. USA 90: 2827-2831, 1993. PubMed: 8464896

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.