MethoCult™ GF R3774

Methylcellulose-based medium with recombinant cytokines (without erythropoietin [EPO]) for rat cells

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MethoCult™ GF R3774

Methylcellulose-based medium with recombinant cytokines (without EPO) for rat cells

100 mL
Catalog #03774
602 USD

Overview

MethoCult™ GF R3774 is optimized for growth and enumeration of granulocyte-macrophage progenitor cells (CFU-GM, CFU-G and CFU-M) in colony-forming unit (CFU) assays of rat bone marrow cells.
Contains:
• Methylcellulose in Iscove's MDM
• Fetal bovine serum
• Bovine serum albumin
• 2-Mercaptoethanol
• Stem cell factor (SCF)
• Interleukin 3 (IL-3)
• Granulocyte-macrophage colony-stimulating factor (GM-CSF)
• Supplements
Subtype:
Semi-Solid Media; Specialized Media
Cell Type:
Hematopoietic Stem and Progenitor Cells
Species:
Rat
Application:
Cell Culture; Colony Assay; Functional Assay
Brand:
MethoCult
Area of Interest:
Drug Discovery and Toxicity Testing; Stem Cell Biology

Scientific Resources

Product Documentation

Educational Materials

(3)

Frequently Asked Questions

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)
The Journal of burn care & rehabilitation

Changes in bone marrow-derived myeloid cells from thermally injured rats reflect changes in the progenitor cell population.

Noel JG et al.

Abstract

Bone marrow progenitor cells develop into mature tissue myeloid cells under the influence of colony-stimulating factors. Cytokines that are elevated post-thermal injury have been shown to influence this process. We hypothesize that thermal injury alters myelopoiesis at the level of the progenitor cell. These differences should be visible after in vitro cultures that include colony-stimulating factors. Prior to culture, bone marrow at postburn day 1 (PBD1) was assessed for cell surface markers and the levels of myeloid progenitors. After culture in granulocyte/macrophage-stimulating colony-stimulating factor, the cell surface markers of the cultured cells were determined. PBD1 marrow from thermally injured rats had more progenitor cells responsive to granulocyte/macrophage-stimulating colony-stimulating factor than did sham. Cultured PBD1 marrow produced more CD90(br) MY(br) CD45(dim) CD4(-) MHCII(-) CD11b(dim) eosinophils than did sham. Cultured bone marrow from thermally injured animals produces myeloid cells with an altered phenotype. Similar changes in myelopoiesis may take place in vivo.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.