| Applications |
This nullipotent stem cell line does not differentiate either spontaneously or in response to retinoic acid. They secrete osteonectin, entactin, and fibronectin which bind to protein A and the 400000 and 200000 soluble laminins, but they do not form a visible extracellular matrix. |
| Comments |
This nullipotent stem cell line does not differentiate either spontaneously or in response to retinoic acid. They express SSEA-1 antigen and alkaline phosphatase on the cell surface. The cells have a cytoskeleton rich in vimentin. They secrete osteonectin, entactin, and fibronectin which bind to protein A and the 400000 and 200000 soluble laminins, but they do not form a visible extracellular matrix. |
| 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 Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin- 0.53 mM 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: 1:5 to 1:8
Medium Renewal: Every 2 to 3 days
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. |
| References |
Damjanov A, et al. Basement membrane components secreted by mouse yolk sac carcinoma cell lines. Differentiation 45: 84-95, 1990. PubMed: 2098280
Gatalica Z, Damjanov I. Effects of alcohols on mouse embryonal carcinoma-substrate adhesion. Histochemistry 95: 189-193, 1990. PubMed: 2081693
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.
|
