| 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. Subcultivation Ratio: 1:3 to 1:5
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:5 is recommended
Medium Renewal: 2 to 3 times per week |
| References |
Fogh J. Human tumor cells in vitro. New York: Plenum Press; 1975.
Fogh J, et al. Absence of HeLa cell contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst. 58: 209-214, 1977. PubMed: 833871
Goodfellow M, et al. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59: 221-226, 1977. PubMed: 77210034
Guldberg P, et al. Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Cancer Res. 57: 3660-3663, 1997. PubMed: 9288767
Hay RJ, Caputo JL, Macy, ML, Eds. (1992) ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo JL. 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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