| Applications |
293/CHE-Fc was established by cotransfecting human embryonic 293 cell (ATCC CRL-1573) with the plasmid pCHE-Fc and a plasmid encoding Genetic (G418) resistance. The cell line is stably transfected to secrete the chimeric protein (CHE-Fc), a recombinant soluble chirmeric protein useful as a probe to detect 9-O-Acetylated sialic acids. CHE-Fc is a bifunctional molecule that can be used either to cleave acetyl groups at the ninth position of sialic acids or as a binding probe for 9-O-sialic acids. |
| Comments |
293/CHE-Fc was established by cotransfecting human embryonic 293 cell (ATCC CRL-1573) with the plasmid pCHE-Fc and a plasmid encoding Genetic (G418) resistance. The cell line is stably transfected to secrete the chimeric protein (CHE-Fc), a recombinant soluble chirmeric protein useful as a probe to detect 9-O-Acetylated sialic acids. CHE-Fc is the fusion protein of hemagglutinin-esterase from Influenza C with human IgG1 Fc. CHE-Fc is a bifunctional molecule that can be used either to cleave acetyl groups at the ninth position of sialic acids or as a binding probe for 9-O-sialic acids. 293/CHE-Fc cells are G418 resistant. |
| 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-0.03% (w/v) 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: 1:6 to 1:10
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 |
Klein A, et al. 9-O-Acetylated sialic acids have widespread but selective expression: Analysis using a chimeric dual-function probe derived from influenza C hemaggIutinin-estrase. Proc. Natl. Acad. Sci. USA 91: 7782-7786, 1994. PubMed: 8052660
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.
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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