Organ: skin Strain: BALB/c Tissue: epidermis Disease: normal
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Tumorigenic:
No
Age:
newborn
Comments:
This is one of several lines that are useful in studies of the molecular events in tumor promotion and for development of promotion assays (see also ATCC CRL-2002 - RT101, ATCC CRL-2010 - JB6 Cl 41-5a and ATCC CRL-2012 - T36274).
The JB6 Cl 30-7b cell line was derived from primary cultures of neonatal BALB/c epidermal cells that had been treated with dimethylsulfoxide (DMSO, 0.01 to 0.1 %).
The line is resistant to promotion of transformation (P-) by phorbol esters.
These cells can be used in comparisons with P+ cells for studying differential responses to tumor promoters.
Never allow the cells to become confluent.
The cells must be carried at low density to avoid spontaneous transformation.
Propagation:
ATCC complete growth medium: Minimum essential medium (Eagle) with 2 mM L-glutamine, 95%; fetal bovine serum, 5% Temperature: 37.0°C
Subculturing:
Protocol: The cells must be carried at low density to avoid spontaneous transformation.Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. 1. Remove and discard culture medium.2. 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.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. An inoculation density of 2 x 104 viable cells per 25 sq. cm. flask is recommended or a spilt ratio of 1:6 to 1:8.6. Incubate cultures at 37°C. Subcultivation Ratio: An inoculation density of 2 X 10 exp4 viable cells per 25 sq. cm. flask is recommended Medium Renewal: 1 to 2 times per week
Rap the flask smartly against a hard surface to get a single cell suspension.
Add fresh medium, aspirate and dispense into new flasks. Culture at 36C in a humidified atmosphere of 5% CO2 - 95% air.
References:
22309: 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 22427: 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 22908: Colburn NH, et al. Tumour promoter induces anchorage independence irreversibly. Nature 281: 589-591, 1979. PubMed: 492322 22959: Bernstein LR, Colburn NH. AP1/jun function is differentially induced in promotion-sensitive and resistant JB6 cells. Science 244: 566-569, 1989. PubMed: 2541502 23279: 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
