Background and Features
S-Clone SF-O3 is a completely serum-free medium for use in research of mouse hematopoietic stem cells. The product was developed based upon the results of research by the Nishikawa Laboratory, Department of Molecular Genetics at the Graduate School of Medicine, Kyoto University. It is used for molecular studies on proliferation and maintenance mechanisms of mouse hematopoietic stem cells and has been reported effective in the same.
The medium is serum-free, which eliminates effects of any unknown factors in serum, and it contains extremely limited protein components, allowing the action of cytokines, etc. to be clearly identified. Further, being composed of known ingredients, the product affords constancy in experimental conditions. (See Reference Literatures: (1), (2), (3), (4), (5))
1. Basal medium (powder) - Constituted from a composite of slightly modified RPMI1640, Dulbecco's MEM, and Ham's F-12.
2. Sodium hydrogen carbonate (powder)
3. Supplement (liquid) - HEPES buffer containing hormone growth factors (insulin, transferrin, sodium selenite and ethanolamine). Protein components other than insulin and transferrin have not been added.
Directions for Use
Preparing the Medium
1. Add 1 bottle of the basal medium (10.7 g) to approximately 950 mL of purified water and mix well to dissolve.
2. To the solution above, add 1 vial (2.2 g) of sodium hydrogen carbonate and dissolve, and further dilute to a total of 1000 mL.
3. Sterilize by filtering through a low adsorption membrane filter of pore size 0.2-0.22 μm (Toyo Roshi Kaisha Ltd., Cat. No. C020A047A or Millipore Corporation, Cat. No. GVWP04700, etc.).
4. Aseptically add 1 vial of the supplement (10 mL) to the filtered 1000 mL of medium.
5. Seal and store refrigerated (2-10°C).
Examples of Usage
1. Method for cultivation of B precursor cells on a mouse stromal cell line ST2 layer using SF-O3 (Add IL-7: 10 U/mL, 2-mercaptoethanol: 100 μmol/L).
2. Seed the ST2 cells at a density of 1.5 x 10⁵ cells/well to make the feeder layer.
3. Seed the mouse bone marrow cells at a density of 2 x 10⁵ cells/well on the ST2 feeder layer and continue to incubate, replacing half of the medium every 4-5 days. Well used: Falcon Primaria Cultureware 6-Well Plate (3846)
4. Method for cultivation of hematopoietic precursor cells in a mouse stromal cell line PA6 layer using SF-O3 (Add bFGF or EGF: 5-10 ng/mL, 2-mercaptoethanol: 100 μmol/L).
5. Seed the PA6 cells at a density of 2 x 10⁵ cells/well to make the feeder layer.
6. Seed the mouse bone marrow cells at a density of 2 x 10⁵ cells/well on the PA6 feeder layer and continue to incubate, replacing half of the medium every 4-5 days. Well used: Falcon PrimariaCultureware 6-Well Plate (3846)
Precautions for Use and Handling
1. The entire bottle of basal medium should be dissolved in a single use. Avoid dissolving the powder in small portion.
2. When dissolving the basal medium, inject carbon dioxide gas (high-quality carbon dioxide or dry ice is suitable) to shift the pH of the medium to acidic. An acidic pH enhances solubility of the medium as well as speeds equilibration (pH 7.2-7.4) of the medium in the carbon dioxide gas incubator.
3. As the supplement is a concentrated solution, it may show precipitation when stored refrigerated. It should be mixed well before adding to the basal medium.
4. Avoid filtering the medium after the supplement has been added, as it may cause lowered growth in some types of cells.
5. The prepared medium is confirmed to be stable for 3 months when stored at 2-10°C, however, it should be used as soon as possible.
6. This product does not contain 2-mercaptoethanol.
7. This product is a reagent for in vitro research use only and should not be used for diagnostic purposes, nor should the grown cells be used for treatment.
8. Treatment of wastes generated by this method should be done according to guidelines in accordance with the local laws and regulations.
Note: Please refer to the "S-Clone SF-O3" Material Safety Data Sheet (MSDS) before use.
MSDS for SF-O3 Cat#1303 [PDF]
Storage and Shelf Life
Store at 2-10°C. Avoid freezing.
The shelf life is 1 year following manufacture (expiration date is indicated on the outer box).
* Basal medium: 1 L-equivalent x 1 bottle
* Sodium hydrogen carbonate: 2.2 g x 1 vial
* Supplement: 10 mL x 1 vial
Nishikawa, S. et al. Stromal cell-dependent bone marrow culture with a nearly protein-free defined medium. Immunology Letters 40, 163–169 (1994) doi: 10.1016/0165-2478(94)90188-0.
Yasunaga, M., Adachi, S., Itoh, N. & Nishikawa, S.-I. Making the in-vitro model closer to actual B lymphopoiesis in the bone marrow. Seminars in Immunology 7, 185–196 (1995) doi: 10.1016/1044-5323(95)90046-2.
Yasunaga, M., Wang, F., Kunisada, T., Nishikawa, S. & Nishikawa, S. Cell cycle control of c-kit+IL-7R+ B precursor cells by two distinct signals derived from IL-7 receptor and c-kit in a fully defined medium. Journal of Experimental Medicine 182, 315–323 (1995) doi: 10.1084/jem.182.2.315.
Sase, H., Watabe, T., Kawasaki, K., Miyazono, K. & Miyazawa, K. VEGFR2-PLCγ1 axis is essential for endothelial specification of VEGFR2+ vascular progenitor cells. Journal of Cell Science 122, 3303–3311 (2009) doi: 10.1242/jcs.049908.
Yasunaga, M. et al. Involvement of Fyn tyrosine kinase in progression of cytokinesis of B lymphocyte progenitor. J Cell Biol 132, 91–99 (1996) doi: 10.1083/jcb.132.1.91.
Yasunaga, M. et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nature Biotechnology 23, 1542–1550 (2005) doi: 10.1038/nbt1167.
Sarrazin, S. et al. MafB Restricts M-CSF-Dependent Myeloid Commitment Divisions of Hematopoietic Stem Cells. Cell 138, 300–313 (2009) doi: 10.1016/j.cell.2009.04.057.
Nakajima, H. et al. TIMP-3 recruits quiescent hematopoietic stem cells into active cell cycle and expands multipotent progenitor pool. Blood 116, 4474–4482 (2010) doi: 10.1182/blood-2010-01-266528.
Jakt, L. M., Moriwaki, S. & Nishikawa, S. A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization. Development 140, 216–225 (2013) doi: 10.1242/dev.086975.
Feng, J. et al. Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells. Immunity 55, 405-422.e11 (2022) doi: 10.1016/j.immuni.2022.01.016.
Omatsu, Y. et al. Runx1 and Runx2 inhibit fibrotic conversion of cellular niches for hematopoietic stem cells. Nature Communications 13, 2654 (2022) doi: 10.1038/s41467-022-30266-y.
Jin, X. et al. Epigenetic downregulation of Socs2 contributes to mutant N-Ras-mediated hematopoietic dysregulation. Disease Models & Mechanisms 15, dmm049088 (2022) doi: 10.1242/dmm.049088.
Sorimachi, Y. et al. p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts. Journal of Biological Chemistry 296, (2021) doi: 10.1016/j.jbc.2021.100563.
Li, K. et al. Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing. Nature Communications 11, 485 (2020) doi: 10.1038/s41467-020-14362-5.
Product Insert SF-O3 [PDF]
MSDS for SF-O3 [PDF]
FOR RESEARCH USE ONLY, SHOULD NOT BE USED FOR DIAGNOSTIC PURPOSE, NOR SHOULD THE GROWN CELLS BE USED FOR TREATMENT.
A serum-free medium for use in research of hematopoietic stem cells
Manufactured by : Sekisui Medical Co., LTD
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