Rat Methylcellulose Complete Media Without Epo
Rat Methylcellulose Complete Media Without Epo Summary
Complete media for the differentiation and enumeration of rat colony-forming myeloid progenitors (CFU-GM) and for the identification of Epo-independent erythropoiesis.
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 Rat 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 Rat Methylcellulose Complete Media Without Epo, which contains premium quality recombinant rat cytokines to support the optimal growth and enumeration of colony-forming myeloid progenitors (CFU-GM) of rat origin. The absence of Epo makes this media useful for the identification of Epo-independent stimulators of erythropoiesis.
R&D Systems Rat Methylcellulose Complete Media Without Epo:
- Can be used to identify Epo-independent stimulators of erythropoiesis.
- Includes premium quality rat recombinant cytokines to support growth of CFU-GM.
- Optical clarity facilitates colony identification.
- High lot-to-lot consistency decreases variation.
- Does not require addition of serum or cytokines.
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100 mL of Rat 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 | 25% |
| Bovine Serum Albumin | 2% |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Rat SCF | 50 ng/mL |
| Recombinant Rat GM-CSF | 10 ng/mL |
| Recombinant Rat IL-3 | 10 ng/mL |
Stability and Storage
Rat 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.
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 rat hematopoietic progenitors and is ineffective with human hematopoietic progenitors.
- Derivation of human hematopoietic progenitors from different individuals may cause results to vary.
 View Larger Image |
Rat Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A single cell suspension of rat bone marrow cells was resuspended in Rat Methylcellulose Complete Media Without Epo (Catalog # HSC012) and used in the Colony Forming Cell (CFC) assay. Colonies were viewed under light microscope after an 8-12 day incubation. A. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. B and C. Colony forming unit- macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. |
Rat 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 |
| HSC011 | StemXVivo® Methylcellulose Concentrate |
50 mL | N/A* | No | None |
Complete Rat Methylcellulose Media
| Catalog # | Product Description | Volume | Colonies Selected for | Contains Serum | Cytokines Included |
| HSC012 | Rat Methylcellulose Complete Media Without Epo |
100 mL | CFU-G CFU-GM CFU-M |
Yes | GM-CSF IL-3 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.
Specifications
Product Datasheets
Scientific Data
View Larger
Rat Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A single cell suspension of rat bone marrow cells was resuspended in Rat Methylcellulose Complete Media Without Epo (Catalog # HSC012) and used in the Colony Forming Cell (CFC) assay. Colonies were viewed under light microscope after an 8-12 day incubation.A. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes.B and C. Colony forming unit- macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Rat Methylcellulose Complete Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:
- Prepare rat bone marrow cells
- Add cells and desired supplements to Rat Methylcellulose Complete Media Without Epo
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Rat Methylcellulose Complete Media Without Epo (Catalog # HSC012):
- 100 mL of Rat 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 | 25% |
| Bovine Serum Albumin | 2% |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Rat SCF | 50 ng/mL |
| Recombinant Rat GM-CSF | 10 ng/mL |
| Recombinant Rat IL-3 | 10 ng/mL |
Reagents
- Cells derived from rat bone marrow, spleen, peripheral blood, or fetal liver.
- 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
Prepare a suspension of rat 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 Rat 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 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 Rat Methylcellulose Complete Media Without Epo. The final Methylcellulose concentration should be 1.3%.
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.
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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