| Comments |
SVEC4-10EE2 is an endothelial cell line derived from the same solid tumor as SVEC4-10EHR1 (see CRL-2161) in a nude mouse injected with SVEC4-10 (see ATCC CRL-2181). The cells were cloned in 1989 by limiting dilution, and are distinguished by their inability to differentiate on a synthetic basement-like membrane. They form tube-like structures in the presence of suramin, a drug which blocks growth factor receptors. The cells exhibit a stable cobblestone morphology in contrast to its sibling from the same tumor (SVEC4-10EHR1). The cells express the cell surface major histocompatibility complex class I antigen, H-2 k of the parental cell line, and express Factor VIII related antigen, high affinity receptors for low density lipoprotein and vascular cell adhesion molecule (VCAM). The cells stain positively for SV40 T antigen. |
| 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.
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:4 to 1:6
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 |
O'Connell KA, Edidin M. A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells. J. Immunol. 144: 521-525, 1990. PubMed: 2153170
O'Connell KA, Rudmann AA. Cloned spindle and epithelioid cells from murine Kaposi's sarcoma-like tumors are of endothelial origin. J. Invest. Dermatol. 100: 742-745, 1993. PubMed: 8496612
O'Connell K, et al. Endothelial cells transformed by SV40 T antigen cause Kaposi's sarcomalike tumors in nude mice. Am. J. Pathol. 139: 743-749, 1991. PubMed: 1928299
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.
Biosafety in Microbiological and Biomedical Laboratories, 5th ed. HHS. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention. Washington DC: U.S. Government Printing Office; 2007. The entire text is available online.
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