| Comments |
Defects in the hematopoietic microenvironment, associated with the Steel (Sl) mutation in
mice, have been shown to be due to abnormalities in the production or presentation of the protein product of the Steel gene.
This product is termed stem cell factor (SCF) or mast cell growth factor (MGF). It exists as a locally secreted or membrane-bound protein.
Stable stromal cell transfectants can differentially process the two forms of human SCF protein product, membrane-bound and secreted SCF.
Membrane-bound SCF is available in the transfected Sl/Sl4 cell line, Sl/Sl4 hSCF220 (ATCC CRL-2453).
Secreted SCF is available in the transfected Sl/Sl4 cell line, Sl/Sl4 hSCF248 (ATCC CRL-2454). |
| 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. Subculture when flasks reach 80% to 90% confluency.
- 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 coated with 0.1% gelatin.
- Incubate cultures at 37°C.
Subcultivation Ratio: 1:10 to 1:30
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 |
Toksoz D, et al. Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor. Proc. Natl. Acad. Sci. USA 89: 7350-7354, 1992. PubMed: 1380155
Majumdar MK, et al. Identification and mutation of primary and secondary proteolytic cleavage sites in murine stem cell factor cDNA yields biologically active, cell-associated protein. J. Biol. Chem. 269: 1237-1242, 1994. PubMed: 7507105
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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