| 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: 1:8 to 1:20
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.
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| References |
Beavers LS, et al. Recombinant and chimeric KS1/4 antibodies directed against a human adenocarcinoma antigen. US Patent 4,975,369 dated Dec 4 1990
Berg DT, et al. E1a-responsive mammalian host/vector system for the stable high-level expression of secreted proteins. Nucleic Acids Res. 20: 5485-5486, 1992. PubMed: 1437573
Grinnell BW, et al. Gamma-carboxylated isoforms of recombinant human protein S with different biologic properties. Blood 76: 2546-2554, 1990. PubMed: 2148275
Burck PJ, et al. Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain. J. Biol. Chem. 265: 5170-5177, 1990. PubMed: 2108167
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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