Mouse Methylcellulose Complete Media Without Epo
Mouse Methylcellulose Complete Media Without Epo Summary
For the differentiation and enumeration of murine colony-forming myeloid progenitors (CFU-GM) and for the detection of Epo-independent erythropoiesis, optimized with premium quality cytokines.
Key Benefits
- Suitable for identification of Epo-independent erythropoiesis
- Excellent optical clarity facilitates colony identification
- High lot-to-lot consistency decreases variation
- Optimized for enumeration of colony-forming myeloid progenitors (CFU-GM)
Why use R&D Systems Mouse Methylcellulose Complete Media Without Epo for Colony Forming Cell Assays?
Colony forming cell (CFC) assays, which are used to enumerate and quantify multi-potent and single lineage hematopoietic progenitors, can be time consuming and laborious.
Successful growth and enumeration of cell colonies is dependent on factors such as accurate cell counts, the presence of growth factors and/or cytokines, adequate humidity, and the use of high quality media. R&D Systems offers Mouse Methylcellulose Complete Media Without Epo, which contains all growth factors and cytokines needed to support optimal growth and enumeration of colony-forming myeloid progenitors (CFU-GM) of mouse origin. The absence of Epo makes this media useful for the identification of Epo-independent stimulators of erythropoiesis.
R&D Systems Mouse Methylcellulose Complete Media Without Epo:
- Can be used to identify Epo-independent stimulators of erythropoiesis.
- Optical clarity facilitates colony identification.
- High lot-to-lot consistency decreases variation.
- Supports reproducible in vitro growth of CFU-GM.
- Does not require addition of serum or cytokines.
- Increased cloning efficiency and improved colony growth compared to agar.
- 100 mL of Mouse Methylcellulose Complete Media Without Epo.
| Contents | Concentration (when diluted to a final volume of 100 mL) |
| Methylcellulose (1500 cps) in Iscove’s Modified Dulbecco's Medium |
1.4% |
| Fetal Bovine Serum | 15% |
| Bovine Serum Albumin | 2% |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Human Insulin | 10 μg/mL |
| Recombinant Transferrin | 200 μg/mL |
| Recombinant Mouse SCF | 50 ng/mL |
| Recombinant Mouse IL-3 | 10 ng/mL |
| Recombinant Mouse IL-6 | 10 ng/mL |
Stability and Storage
Mouse Methylcellulose Complete Media Without Epo should be stored at ≤-20 °C upon receipt. Storage at 2 °C to 8 °C is not recommended.
Precautions
- The acute and chronic effects of overexposure to this media are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling this media.
- The human Transferrin used in this product was derived from human plasma, which has been tested and found negative for HIV-1/2 antibodies, Hepatitis B surface antigen, Hepatitis C antibody, Syphilis, ALT Test and NAT-PCR (HAV, HIV, HBC, HCV, and Parovirus B19) by FDA approved methods. Handle as if capable of transmitting infection, and dispose of according to applicable regulations.
Limitations
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- The reagent should not be used beyond the expiration date indicated on the vial labels.
- The media is optimized to assay mouse hematopoietic progenitors and is ineffective with human hematopoietic progenitors.
- Derivation of mouse hematopoietic progenitors from different individual animals may cause results to vary.
Mouse Methylcellulose Stock and Base Media
| Catalog # | Product Description | Volume | Colonies Selected for | Contains Serum | Cytokines Included |
| HSC001 | Methylcellulose Stock Solution | 100 mL | N/A* | No | None |
| HSC006 | Mouse Methylcellulose | 90 mL | N/A* | Yes | None |
| HSC011 | StemXVivo® Methylcellulose Concentrate |
50 mL | N/A* | No | None |
Complete Mouse Methylcellulose Media
| Catalog # | Product Description | Volume | Colonies Selected for | Contains Serum | Cytokines Included |
| HSC007 | Mouse Methylcellulose Complete Media | 100 mL | BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M |
Yes | Epo IL-3 IL-6 SCF |
| HSC008 | Mouse Methylcellulose Complete Media Without Epo |
100 mL | CFU-G CFU-GM CFU-M |
Yes | IL-3 IL-6 SCF |
| HSC009 | Mouse Methylcellulose Complete Media for Pre-B Cells |
100 mL | CFU-Pre-B | Yes | IL-7 |
*Base media and stock solutions do not contain cytokines and will not support colony growth unless conditioned media, cytokines, or other culture supplements are added.
Specifications
Product Datasheets
Scientific Data
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Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogene-ous population of macrophages and granulocytes.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Mouse Methylcellulose Complete Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:
- Prepare mouse bone marrow cells
- Add cells to Mouse Methylcellulose Complete Media Without Epo
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Mouse Methylcellulose Complete Media Without Epo (Catalog # HSC008):
- 100 mL of Mouse Methylcellulose Complete Media Without Epo.
| Contents | Concentration (when diluted to a final volume of 100 mL) |
| Methylcellulose (1500 cps) in Iscove's Modified Dulbecco's Medium |
1.4% |
| Fetal Bovine Serum | 15% |
| Bovine Serum Albumin | 2% |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Human Insulin | 10 µg/mL |
| Human Transferrin | 200 µg/mL |
| Recombinant Human SCF | 50 ng/mL |
| Recombinant Human IL-3 | 10 ng/mL |
| Recombinant Human IL-6 | 10 ng/mL |
Reagents
- Cells derived from mouse bone marrow, spleen, peripheral blood, or fetal liver. Mice are routinely used between 6 - 12 weeks.
- Iscove's Modified Dulbecco's Media (IMDM)
- Fetal Bovine Serum
- IMDM/2% Fetal Bovine Serum
- (Optional) Flow Cytometry Mouse Lyse Buffer (Catalog # FC003)
Materials
- 100 mm culture plates
- 35 mm culture plates
- 15 mL centrifuge tubes
- 10 mL syringes
- 3 mL syringes
- 5 mL vials
- 16 gauge 1½ inch needle
- 14 gauge laboratory pipetting needle
- Serological pipettes
- Pipettes and pipette tips
Equipment
- 37 °C and CO2 humidified incubator
- Centrifuge
- Vortex mixer
- Hemocytometer
- Inverted Microscope
Pass a suspension of mouse bone marrow cells through a 70 µm nylon strainer to remove clumps and debris.
Remove red blood cells if necessary.
Wash the cells with IMDM/2% FBS by centrifugation at 300 x g for 8 minutes and pool the cells.
Remove the supernatant.
Resuspend the cells in 10 mL of IMDM/2% FBS
Thaw aliquots of Mouse Methylcellulose Complete Media Without EPO at room temperature for approximately 30 minutes.
Perform a cell count.
Transfer the appropriate volume of cells (plus a slight excess) into a new 15 mL centrifuge tube.
Centrifuge at 300 x g for 8 minutes.
Remove the supernatant.
Resuspend the cells in IMDM/2% FBS to the desired stock cell number to generate a 10X stock concentration.
Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and Mouse Mouse Methylcellulose Complete Media Without EPO. The final Methylcellulose concentration should be 1.27%.
Vortex the samples vigorously.
Wait approximately 20 minutes to allow air bubbles to escape.
Add 1.1 mL of the cell mixture to a 35 mm culture plate using a 3 mL syringe and a 16 gauge needle.
Spread the media evenly.
Place two 35 mm plates into a 10 cm plate.
Add one uncovered 35 mm plate that contains 3-4 mL of sterile water.
Cover the 10 cm plate and place it in a 37 °C and 5% CO2 incubator.
Incubate the cells for 8-12 days.
Use an inverted microscope and a scoring grid to identify and count individual colonies.
Citation for Mouse Methylcellulose Complete Media Without Epo
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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The Parkinson's disease-linked Leucine-rich repeat kinase 2 (LRRK2) is required for insulin-stimulated translocation of GLUT4
Authors: N Funk, M Munz, T Ott, K Brockmann, A Wenninger-, R Kühn, D Vogt-Weise, F Giesert, W Wurst, T Gasser, S Biskup
Sci Rep, 2019-03-14;9(1):4515. 2019-03-14
FAQs
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Why does the Human, Mouse and Rat colony forming assay protocol (CFC assay protocol) recommed use of non-tissue culture treated petri dishes?
The CFC assay promotes the growth of cells as colonies suspended in methylcellulose. However, if you use tissue culture treated dishes, the cells will also adhere and grow out on the bottom of the plate. Sometimes this appears as a round colony that is sticking and growing out on the edges (like an egg) and sometimes you can see patches of a monolayer. This makes it difficult to see the suspended colonies.
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Burst Forming Unit-Erythroid (BFU-E ) colonies representing erythorid progenitors appear to be low in frequency. Is there a strategy to count these colonies and visualize them?
It is true that BFU-E colonies are low in frequency. To count and see good BFU-E colonies, the CFC assay is set up at two cell densities. For counting BFU-E colonies, a 10X cell concentration of 1.5-3x105 cells/mL is used. For properly visualizing the BFU-E colonies, an assay at half that cell density is used.
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