| 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 culture medium. Keep the floating cells to transfer with attached cells..
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that 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: A subcultivation ratio of 1:3 to 1:4 is recommended
Medium Renewal: Two to three times weekly
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 |
Hsei V, et al. Methods of treating inflammatory diseases with anti-IL-8 antibody fragment-polymer conjugates. US Patent 6,468,532 dated Oct 22 2002
Hsei V, et al. Methods of treating inflammatory disease with anti-IL-8 antibody fragment-polymer conjugates. US Patent 6,458,355 dated Oct 1 2002
Gonzalez TN, et al. Humanized anti-IL-8 monoclonal antibodies. US Patent 6,133,426 dated Oct 17 2000
Gonzalez TN, et al. Humanized anti-IL-8 monoclonal antibodies. US Patent 6,117,980 dated Sep 12 2000
Gonzalez TN, et al. Nucleic acids encoding humanized anti-IL-8 monoclonal antibodies. US Patent 6,025,158 dated Feb 15 2000
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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