Human Methylcellulose Serum-Free Enriched Media Without Epo
Human Methylcellulose Serum-Free Enriched Media Without Epo Summary
Complete, defined media for the differentiation and enumeration of myeloid progenitor cells and for the detection of Epo-independent erythropoiesis.
Key Benefits
- Suitable for detection of Epo-independent erythropoiesis
- Defined media components reduce experimental variation
- Optimized for enumeration of myeloid progenitor cells
- Excellent optical clarity facilitates colony identification
- High lot-to-lot consistency decreases variation
Why use R&D Systems Human Methylcellulose Serum-Free Enriched 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. Additionally, experimental variation can be reduced by using defined medium. R&D Systems offers Human Methylcellulose Serum-Free Enriched Media Without Epo, a define medium which contains all of the growth factors and cytokines needed to support optimal colony growth and enumeration. The Human Methylcellulose Serum-Free Enriched Media Without Epo is specially formulated and has been optimized for CFC assays detecting colony-forming myeloid progenitors (CFU-GM, CFU-G, CFU-M) in human bone marrow, cord blood, mobilized peripheral blood, and peripheral blood samples. It can also be used in CFC assays for purified human hematopoietic progenitor cells. This product is recommended for use in the CFC assays, when a serum-Free defined condition is desired.
R&D Systems Human Methylcellulose Serum-Free Enriched Media Without Epo:
- Optical clarity facilitates colony identification.
- Suitable for detection of Epo-independent erythropoiesis.
- High lot-to-lot consistency decreases variation.
- Supports reproducible in vitro growth of myeloid progenitor cells.
- Defined media reduces experimental variation.
- Increased cloning efficiency and improved colony growth compared to agar.
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100 mL of Human Methylcellulose Serum-Free Enriched 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% |
| Human Transferrin | 300 μg/mL |
| Bovine Serum Albumin | 3.6% |
| Recombinant Human Insulin | 16 μg/mL |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Human SCF | 50 ng/mL |
| Recombinant Human GM-CSF | 20 ng/mL |
| Recombinant Human G-CSF | 20 ng/mL |
| Recombinant Human IL-3 | 20 ng/mL |
| Recombinant Human IL-6 | 20 ng/mL |
| Cholesterol | Trace |
Stability and Storage
Human Methylcellulose Serum-Free Enriched 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, and p24 antigen 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 human hematopoietic progenitors and is ineffective with mouse hematopoietic progenitors.
- Derivation of human hematopoietic progenitors from different individuals may cause results to vary.
 View Larger Image |
Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. A. Colony forming unit-granulocyte (CFU-G) are clonogenic progenitors of granulocytes that give rise to a homogeneous population of eosinophils, basophils, or neutrophils. B. Colony forming unit-granulocyte, macrophage (CFU-GM) are progenitors that give rise to colonies containing a heterogeneous population of macrophages and granulocytes. C. Colony forming unit-macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages. |
Human 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 |
| HSC002 | Human Methylcellulose Base Media |
90 mL | N/A* | Yes | None |
| HSC002SF | Human Methylcellulose Serum-Free Base Media |
90 mL | N/A* | No | None |
| HSC011 | StemXVivo® Methylcellulose Concentrate |
50 mL | N/A* | No | None |
Complete Human Methylcellulose Media
| Catalog # | Product Description | Volume | Colonies Selected for | Contains Serum | Cytokines Included |
| HSC003 | Human Methylcellulose Complete Media | 100 mL | BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M |
Yes | Epo GM-CSF IL-3 SCF |
| HSC004 | Human Methylcellulose Complete Media without Epo | 100 mL | CFU-G CFU-GM CFU-M |
Yes | SCF GM-CSF IL-3 |
| HSC005 | Human Methylcellulose Enriched Media |
100 mL | BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M |
Yes | Epo G-CSF GM-CSF IL-3 IL-6 SCF |
| HSC005SF | Human Methylcellulose Serum-Free Enriched Media |
100 mL | BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M |
No | Epo G-CSF GM-CSF IL-3 IL-6 SCF |
| HSC010SF | Human Methylcellulose Serum-Free Enriched Media without Epo |
100 mL | CFU-G CFU-GM CFU-M |
No | G-CSF GM-CSF 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.
Specifications
Product Datasheets
Scientific Data
View Larger
Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. Colony forming unit-erythroid (CFU-E) are clonogenic progenitors that produce only one or two clusters with each cluster containing from 8 to approximately 100 hemoglobinized erythroblasts. It represents the more mature erythroid progenitors that have less proliferative capacity.B. Colony forming unit-granulocyte (CFU-G) 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. Burst forming unit-erythroid (BFU-E) colonies can be described as small (3 to 8 clusters), intermediate (9 to 16 clusters), or large (more than 16 clusters) according to the number of clusters present. These are primitive erythroid progenitors that have high proliferative capacity.E. Colony forming unit-macrophage (CFU-M) are clonogenic progenitors of macrophages that give rise to a homogenous population of macrophages.F. Colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) are multi-lineage progenitors that give rise to erythroid, granulocyte, macrophage and megakaryocyte lineages, as the name indicates.
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, Human Methylcellulose Serum-Free Enriched Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:
- Prepare mouse bone marrow cells
- Add cells to Human Methylcellulose Serum-Free Enriched Media Without Epo
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Human Methylcellulose Serum-Free Enriched Media Without Epo (Catalog # HSC010SF):
- 100 mL of Human Methylcellulose Serum-Free Enriched 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% |
| Human Transferrin | 300 µg/mL |
| Fetal Bovine Serum | 3.6% |
| Bovine Serum Albumin | 3.6% |
| L-Glutamine | 2 mM |
| 2-Mercaptoethanol | 5 x 10-5 M |
| Recombinant Human SCF | 50 ng/mL |
| Human GM-CSF | 20 ng/mL |
| Recombinant Human G-CSF | 20 ng/mL |
| Recombinant Human IL-3 | 10 ng/mL |
| Recombinant Human IL-6 | 10 ng/mL |
| Cholesterol | trace |
Reagents
- Cells derived from bone marrow, blood, or enriched CD34+ cells
- Iscove’s Modified Dulbecco’s Media (IMDM)
- Ca2+/Mg2+-Free Hank's Balanced Salt Solution (HBSS)
- Ficoll-Paque™ PLUS (GE Healthcare) or equivalent
Materials
- 100 mm culture plates
- 35 mm culture plates
- 15 mL centrifuge tubes
- 50 mL centrifuge tubes
- 10 mL syringes
- 3 mL syringes
- 5 mL vials
- 16 gauge 1½ inch needle
- 14 gauge laboratory pipetting needle
- Heparinized syringes or Vacutainers®
- Serological pipettes
- Pipettes and pipette tips
Equipment
- 37 °C and CO2 humidified incubator
- Centrifuge
- Vortex mixer
- Hemocytometer
- Inverted Microscope
Prepare mononuclear cells by Ficoll-Paque gradient centrifugation.
Wash the cells two times with HBSS and pool the cells.
Centrifuge the cells at 400 x g for 10 minutes.
Thaw aliquots of Human Methylcellulose Serum-Free Enriched Media Without Epo at room temperature for approximately 30 minutes.
Resuspend mononuclear cells in 10 mL of IMDM.
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 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 Human Methylcellulose Serum-Free Enriched 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 14-16 days.
Use an inverted microscope and a scoring grid to identify and count individual colonies.
FAQs
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Can the CFU assay using Methycellulose based media be performed using frozen PBMCs instead of fresh PBMCs?
Yes, the CFU assay can be performed using frozen PBMCs. The PBMCs can be frozen in DMEM containing 10% FBS and 10% DMSO.
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