| Applications |
Passage 3 cells were transformed with a retrovirus vector (LXSN16E6E7 produced by ATCC CRL-2203) in the presence of polybrene. The transformed cells are resistant to G418 and sensitive to Hygromycin B. E6E7 sequences were detected by PCR in cells at passage 15. Major Histocompatibility Complex class I or II molecules were not expressed on these cells, but PCR analyses revealed presence of the genes for directing synthesis of HLA antigens. CCD 1105 KIDTr is sensitive to Coxsackieviruses A9 and B5, measles virus, poliovirus 1 and rhinovirus. |
| Comments |
Fetal kidney was digested with collagenase: trypsin mixture. The digestion products were plated in REGM (Renal Epithelial Cell Growth Medium which include 0.5% FBS) from Clonetics on collagen l-fibronectin-BSA coated flasks. Passage 3 cells were transformed with a retrovirus vector (LXSN16E6E7 produced by ATCC CRL-2203) in the presence of polybrene. The transformed cells are resistant to G418 and sensitive to Hygromycin B. This line stains positively with antibody for cytokeratin and is acid phosphatase positive. After 50 population doublings, the cells continue dividing and retain cuboidal morphology. E6E7 sequences were detected by PCR in cells at passage 15. Major Histocompatibility Complex class I or II molecules were not expressed on these cells, but PCR analyses revealed presence of the genes for directing synthesis of HLA antigens. CCD 1105 KIDTr is sensitive to Coxsackieviruses A9 and B5, measles virus, poliovirus 1 and rhinovirus. |
| 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: 1:3 to 1:5
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 |
Zabrenetzky V, et al. The isolation, immortalization and characterization of human fetal keratinocytes. In Vitro Cell. Dev. Biol. 33: Part II, p. 34A, 1997.
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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