| Applications |
Phenylalanine hydroxylase is expressed consitutively, and production can be increased by treatment with glucorticoids or cAMP derivatives. The cells are resistant to 6-thioguanine and sensitive to HAT medium. They are deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT-, HPRT-). |
| Comments |
This line is a derivative of the H4-II-E-C3 rat hepatoma cell line (see ATCC CRL-1600). Phenylalanine hydroxylase is expressed consitutively, and production can be increased by treatment with glucorticoids or cAMP derivatives. The cells are resistant to 6-thioguanine and sensitive to HAT medium. They are deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT-, HPRT-). |
| 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: 1:2 to 1:6
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 |
Haggerty DF, et al. Phenylalanine hydroxylase in cultured hepatoxytes. I. Hormonal control of enzyme levels. J. Biol. Chem. 248: 223-232, 1973. PubMed: 4348207
Peraino C, et al. Hepatomas in tissue culture compared with adapting liver in vivo. Natl. Cancer Inst. Monogr. 13: 229-245, 1964. PubMed: 14143233
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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