| 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.
- 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 10 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.
ALTERNATIVELY, cultures also can be grown as a suspension and maintained by addition or replacement of fresh medium. Start cultures at 2 x 104 cells/mL and maintain between 2 x 104 and 1 x 106 cells/mL.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: 2 to 3 times per week
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
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| References |
Stanley P. Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes. Mol. Cell. Biol. 1: 687-696, 1981. PubMed: 9279382
Deutscher SL, Hirschberg CB. Mechanism of galactosylation in the Golgi apparatus. A Chinese hamster ovary cell mutant deficient in translocation of UDP-galactose across Golgi vesicle membranes. J. Biol. Chem. 261: 96-100, 1986. PubMed: 3510203
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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