Mouse Methylcellulose Complete Media
R&D Systems, part of Bio-Techne | Catalog # HSC007
Key Product Details
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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 Complete Media is used in the Colony Forming Cell Assay using the following procedure:
- Prepare mouse bone marrow cells
- Add cells to Mouse Methylcellulose Complete Media
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Mouse Methylcellulose Complete Media (Catalog # HSC007):
- 100 mL of Mouse Methylcellulose Complete 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 |
| Recombinant Human SCF | 50 ng/mL |
| Recombinant Human IL-3 | 10 ng/mL |
| Recombinant Human IL-6 | 10 ng/mL |
| Recombinant Human Epo | 5 IU/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 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 Methylcellulose Complete 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.
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.
