| 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-0.5% (w/v) polyvinylpyrrolidone solution to remove all traces of serum ,which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA- polyvinylpyrrolidone solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
- 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 28°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:4 is recommended
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 |
Paw BH, Zon LI. Primary fibroblast cell culture. Methods Cell Biol. 59: 39-43, 1999. PubMed: 9891354
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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