| Derivation |
The DHFR-G8 cell line was produced by M.C. Hung, et al., in 1986 from the NIH/3T3 mouse fibroblast cell line which was cotransfected with the cNEU-p clone and the pSV2-DHFR plasmid. This cell line was developed from one methotrexate resistant colony by selection in 0.6 uM methotrexate and 10% dialyzed calf serum. These cells contain 50-100 copies of the cNeu-P (normal rat cosmid DNA) and produce approximately 4 x 10(5) molecules of encoded p185 protein per cell. |
| Comments |
The DHFR-G8 cell line was produced by M.C. Hung, et al., in 1986 from the NIH/3T3 mouse fibroblast cell line which was cotransfected with the cNEU-p clone and the pSV2-DHFR plasmid. This cell line was developed from one methotrexate resistant colony by selection in 0.6 uM methotrexate and 10% dialyzed calf serum. These cells contain 50-100 copies of the cNeu-P (normal rat cosmid DNA) and produce approximately 4 x 10(5) molecules of encoded p185 protein per cell. |
| 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 1:8
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 4th edition, published by Wiley-Liss, N.Y., 2000. |
| References |
Hung MC, et al. Molecular cloning of the neu gene: Absence of gross structural alteration in oncogenic alleles. Proc. Natl. Acad. Sci. USA 83: 261-264, 1986. PubMed: 3001730
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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