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MethoCultâ„¢ GF H84535 is a complete methylcellulose-based medium for the growth and enumeration of hematopoietic progenitor cells in colony-forming unit (CFU) assays of human bone marrow, mobilized peripheral blood, peripheral blood and cord blood samples. It is suitable for use with mononuclear cells, CD34+-enriched cells and other purified cell populations. H84535 has been formulated to support the optimal growth of granulocyte and macrophage progenitors (CFU-GM, CFU-G and CFU-M).
Please Note: In light of the new EU Regulation 2017/746 (IVDR) of the European Parliament and of the Council on in vitro diagnostic medical devices, which is expected to become effective in 2022, º£½ÇÆƽâ°æ recently deregistered and removed the CE IVD claims associated with these products, and they are now labeled “For Research Use Onlyâ€. Please note there have been no changes to the form, function and quality assurances associated with these products. For more information please contact the Quality Assurance and Regulatory Department at qaschangenote@stemcell.com.
Contains
• Methylcellulose in Iscove's MDM
• Fetal bovine serum
• Bovine serum albumin
• 2-Mercaptoethanol
• Recombinant human stem cell factor (SCF)
• Recombinant human interleukin 3 (IL-3)
• Recombinant human interleukin 6 (IL-6)
• Recombinant human granulocyte colony-stimulating factor (G-CSF)
• Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF)
• Supplements
Subtype
Semi-Solid Media, Specialized Media
Cell Type
Hematopoietic Stem and Progenitor Cells
Species
Human
Application
Cell Culture, Colony Assay, Functional Assay, In Vitro Diagnostic
Brand
MethoCult
Area of Interest
Cord Blood Banking, Stem Cell Biology, Transplantation Research
This product is designed for use in the following research area(s) as part
of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we
offer to support each research area.
Semi-solid media (methylcellulose-based MethoCultâ„¢ and collagen-based MegaCultâ„¢-C) allow the clonal progeny of a single progenitor cell to remain spatially isolated from other colonies within a culture, so they may be separately identified and counted.
Why use methylcellulose-based media?
Methylcellulose permits better growth of erythroid colonies than other types of semi-solid support systems (eg. agar) while allowing optimal myeloid colony formation. When appropriate cytokines are present, committed progenitor cells of both erythroid and granulocyte/macrophage lineages (CFU-GM, CFU-G, CFU-M) as well as multi-potential progenitor cells (CFU-GEMM), can be assayed simultaneously in the same culture dish.
Is it necessary to add antibiotics to the media?
No, aseptic technique should be sufficient to maintain sterile cultures. However, antibiotics (eg. Penicillin/Streptomycin) or anti-fungals (eg. Amphotericin B) may be added to the methylcellulose medium if desired.
Is there anything I can do if my cultures appear contaminated?
No, once contamination is visible, it is not possible to rescue the cultures by the addition of antibiotics. Bacteria and yeast inhibit colony formation by depleting nutrients or by releasing toxic substances.
Why can't I use a pipette to dispense methylcellulose-based media?
Methylcellulose is a viscous solution that cannot be accurately dispensed using a pipette due to adherence of the medium to the walls of the pipette tip. Blunt-End, 16 Gauge needles (Catalog #28110), in combination with 3 cc Syringes (Catalog #28230) are recommended for accurate dispensing of MethoCultâ„¢.
Can I 'pluck' the colonies for individual analysis?
Yes, colonies can be 'plucked' using a pipette with 200 µL sterile pipette tips or using a glass Pasteur pipette with an elongated tip. Individual colonies should be placed in a volume of 25 - 50 µL of medium, and diluted into suitable culture medium for further culture or analysis.
Why are low adherence dishes so important?
Adherent cells such as fibroblasts can cause inhibition of colony growth and obscure visualization of colonies.
Can MethoCult™ products be used for lymphoid progenitor CFU assays?
Human lymphoid progenitors (B, NK and T) seem to require stromal support for growth therefore cannot be grown in MethoCultâ„¢. Mouse pre-B clonogenic progenitors can be grown in MethoCultâ„¢ M3630 (Catalog #03630).
Is it possible to set up CFU assays in a 24-well plate?
Yes, as long as a plating concentration optimized for the smaller surface area of a well in a 24-well plate (1.9 cm2 as compared to ~9.5 cm2 for a 35 mm dish) is used for these assays. The number of replicate wells required to get an accurate estimation of CFU numbers may also need to be increased.
Can I stain colonies in MethoCultâ„¢ medium?
The cells in individual colonies in MethoCultâ„¢ can be stained, eg., for analysis of morphology or phenotype, after they are plucked from the dish and washed free of methylcellulose. Colonies grown in collagen-based MegaCultâ„¢-C medium can be used for immunohistochemical or enzymatic staining in situ after dehydration and fixation onto glass slides.
Are there differences in colony morphology with serum-free media?
Serum-containing media generally give better overall growth (colonies may appear larger) but there are no large differences in total colony numbers when CFU assays using serum-free media and serum-containing media are compared, provided that identical cytokines are present.
Can MethoCult™ be made with alternate base media?
Yes, this can be done as a 'custom' media order. Please contact techsupport@stemcell.com for more information.
Is there a MethoCult™ formulation suitable for HPP-CFC (high proliferative potential colony forming cell)?
Yes, MethoCultâ„¢ H4535 (Catalog #04535) can be used for the HPP-CFC assay as it does not contain EPO. The culture period is usually 28 days. It is not necessary to feed these cultures as growth factors in the medium are present in excess. As HPP-CFCs can be quite large, overplating can be a problem. It is recommended to plate cells at two or more different concentrations.
Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues.
Pesce M et al.
Circulation research 2003 SEP
Abstract
Human umbilical cord blood (UCB) contains high numbers of endothelial progenitors cells (EPCs) characterized by coexpression of CD34 and CD133 markers. Prior studies have shown that CD34+/CD133+ EPCs from the cord or peripheral blood (PB) can give rise to endothelial cells and induce angiogenesis in ischemic tissues. In the present study, it is shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and, unexpectedly, to skeletal muscle cells in mice. In fact, the treated limbs exhibited enhanced arteriole length density and regenerating muscle fiber density. Under similar experimental conditions, CD34- cells did not enhance the formation of new arterioles and regenerating muscle fibers. In nonischemic limbs CD34+ cells increased arteriole length density but did not promote formation of new muscle fibers. Endothelial and myogenic differentiation ability was maintained in CD34+ cells after ex vivo expansion. Myogenic conversion of human cord blood CD34+ cells was also observed in vitro by coculture onto mouse myoblasts. These results show that human cord blood CD34+ cells differentiate into endothelial and skeletal muscle cells, thus providing an indication of human EPCs plasticity. The full text of this article is available online at http://www.circresaha.org.
Medium for preparing and washing samples for colony-forming unit (CFU) assays
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MethoCultâ„¢ GF H84535
New look, same high quality and support! You may notice that your instrument or reagent packaging looks slightly different from images displayed on the website, or from previous orders. We are updating our look but rest assured, the products themselves and how you should use them have not changed. Learn more
Quality Statement:
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT º£½ÇÆƽâ°æ, REFER TO WWW.º£½ÇÆƽâ°æ.COM/COMPLIANCE.
