| Applications |
SL/Sl4 hSCF220 is a transfected SL/Sl4 cell line that expresses membrane-bound stem cell factor (SCF).
This cell line was derived from the Sl/Sl4 cell line (ATCC CRL-2452) by transfection with spliced forms of hSCF cDNAs inserted into the pJT-1 (hSCF220) vector and expressed off the simian virus 40 (SV40) early promoter.
Defects in the HM, 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.
Sl/Sl4 is a SV40 large T antigen immortalized stromal cell line derived from the hematopoietic microenvironment (HM) of a fetal murine homozygous (Sl/Sl) SCF-deficient embryo.
It exists as a locally secreted or membrane-bound protein.
SL/Sl4 hSCF220 cells do not secrete detectable amounts of SCF.
Secreted SCF is available in the transfected Sl/Sl4 cell line, Sl/Sl4 hSCF248 (ATCC CRL-2454). |
| Comments |
SL/Sl4 hSCF220 is a transfected SL/Sl4 cell line that expresses membrane-bound stem cell factor (SCF). This cell line was derived from the Sl/Sl4 cell line (ATCC CRL-2452) by transfection with spliced forms of hSCF cDNAs inserted into the pJT-1 (hSCF220) vector and expressed off the simian virus 40 (SV40) early promoter. It was cotransfected with the p48 vector encoding hygromycin B using the Lipofectin method. Defects in the HM, 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. Sl/Sl4 is a SV40 large T antigen immortalized stromal cell line derived from the hematopoietic microenvironment (HM) of a fetal murine homozygous (Sl/Sl) SCF-deficient embryo. This product is termed stem cell factor (SCF) or mast cell growth factor (MGF). It exists as a locally secreted or membrane-bound protein. Both forms show biological activity when assayed on human hematopoietic cells. SL/Sl4 hSCF220 cells that express the membrane-bound form (hSCF220) of SCF supported the maintenance of human progenitor cells in culture for up to four weeks This was 1-2 weeks longer than SL/Sl4 hSCF248. SL/Sl4 hSCF220 cells do not secrete detectable amounts of SCF. Secreted SCF is available in the transfected Sl/Sl4 cell line, Sl/Sl4 hSCF248 (ATCC CRL-2354). |
| 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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