Mouse Methylcellulose Base Media
R&D Systems, part of Bio-Techne | Catalog # HSC006
Key Product Details
Summary for Mouse Methylcellulose Base Media
For the support of mouse hematopoietic stem cell growth and differentiation.
Key Benefits
- Can be supplemented with user-defined growth factors
- High lot-to-lot consistency decreases variation
- Excellent optical clarity for easy identification of colonies
Why use R&D Systems Mouse Methylcellulose Base Media 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 Base Media with superior optical clarity to support optimal colony growth, enumeration, and identification. The Mouse Methylcellulose Base Media contains components that have been optimized for CFC assays. Individual researchers can customize the media by adding cells and other culture supplements tailored to their specific research. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.
R&D Systems Mouse Methylcellulose Base Media:
- Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
- Can be supplemented with user-defined cytokines and growth factors.
- Increased cloning efficiency and improved colony growth compared to agar.
- Optical clarity facilitates colony identification.
- High lot-to-lot consistency decreases variation.
- 90 mL of Mouse Methylcellulose Base Media.
| 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 |
Stability and Storage
Mouse Methylcellulose Base Media 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 label.
- Derivation of mouse hematopoietic progenitors from different individual animals may cause results to vary.
- The media is optimized to assay mouse hematopoietic progenitors and is ineffective with human hematopoietic progenitors.
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 |
*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.
| HSC Differentiation &Lineage Marker Schematic View Interactive Pathway |
The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long maintenance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continuously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage depletion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.
|
Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. Burst forming unit-erythroid (BFU-E) colonies are defined as clusters with a minimal of 30 cells that can be seen from day 7 onward. Each individual cluster consisted of tiny, irregular shaped cells that may appear fused together. Each cluster normally contains 5-8 cells, and the size of the cluster is similar to that of a single macrophage. The cluster may vary in sizes and color. A large BFU-E is usually bright red and is differentiable even without the use of a microscope. Smaller BFU-E may not appear red in color but is distinguishable based on the morphology.B.Colony forming unit-macrophage (CFU-M; left) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. Colony forming unit-granulocyte (CFU-G; right) are clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils, or neutrophils.C. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. The morphology is similar to the CFU-M and CFU-G descriptions.D.Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to the lineage of erythroid, granulocytes, macrophages, and megakaryocytes as the name indicates. It can be identified as reddish colored cells (erythroid) mixed with colorless cells (granulocytes, macrophages, and megakaryocytes) in a single colony. This progenitor is typically the largest colony on the culture dish; occasionally CFU-GM may attain a size comparable or larger than that of CFU-GEMM. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Mouse Methylcellulose Base Media is used in the Colony Forming Cell Assay using the following procedure:
- Prepare mouse mononuclear cells
- Add cells and desired supplements to Mouse Methylcellulose Base Media
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Mouse Methylcellulose Base Media (Catalog # HSC006):
- 90 mL of Mouse Methylcellulose Base Media
| 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 |
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 Base Media 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 10 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 Methylcellulose Enriched Media. 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 by gently rotating the plate.
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.
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