MethoCult™ GF H84535

Methylcellulose-based medium with recombinant cytokines (without EPO), IVD
Catalog #
84535_C
Methylcellulose-based medium with recombinant cytokines (without EPO), IVD
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Overview

MethoCult™ 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).

This product carries the CE mark and is available as a Class I in vitro diagnostic device in select regions.

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, STEMCELL has conducted a complete review of our MethoCult™ CE-IVD products. We have determined that our current CE-marked IVD MethoCult™ products will no longer carry the CE mark as “For In-Vitro Diagnostic Use” and will be labelled “For Research Use Only” labelling. These changes will take effect in December 2021. For more information please contact the Quality Assurance and Regulatory Department at qaschangenote@stemcell.com.

Browse our Frequently Asked Questions (FAQs) on performing the CFU assay and explore its utility as part of the cell therapy workflow.
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

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet 1
Product Name
MethoCult™ GF H84535
Catalog #
84535
Lot #
All
Language
English
Document Type
Product Information Sheet 2
Product Name
MethoCult™ GF H84535
Catalog #
84535
Lot #
All
Language
Multi
Document Type
Product Information Sheet 1
Product Name
MethoCult™ GF H84545
Catalog #
84545
Lot #
All
Language
Multi
Document Type
Product Information Sheet 2
Product Name
MethoCult™ GF H84545
Catalog #
84545
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
MethoCult™ GF H84535
Catalog #
84535
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
MethoCult™ GF H84545
Catalog #
84545
Lot #
All
Language
English

Educational Materials (5)

Brochure
2018-2019 Hematopoietic Training Catalog
Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
Brochure
MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays
Wallchart
Identification of Colonies Derived from Human Hematopoietic Progenitors
Mini Review
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays

Frequently Asked Question

Why use semi-solid media?

Semi-solid media (methylcellulose-based MethoCult™ and collagen-based MegaCult™-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 MethoCult™.

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 MethoCult™. Mouse pre-B clonogenic progenitors can be grown in MethoCult™ 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 MethoCult™ medium?

The cells in individual colonies in MethoCult™ 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 MegaCult™-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, MethoCult™ 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.
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Product Applications

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.

Data and Publications

Publications (1)

Circulation research 2003 SEP Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues. Pesce M et al.

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.
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