Human Methylcellulose Serum-Free Enriched Media
Human Methylcellulose Serum-Free Enriched Media Summary
Complete, defined media for the differentiation and enumeration of human hematopoietic stem cells.
Key Benefits
- Defined media components reduce experimental variation
- Excellent optical clarity for easy identification of colonies
- High lot-to-lot consistency decreases variation
Why use R&D Systems Human Methylcellulose Serum-Free Enriched 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 Human Methylcellulose Enriched Media, which contains all growth factors and cytokines needed to support optimal colony growth and enumeration. The Human Methylcellulose Enriched Media is specially formulated for use with CD34+ cells and has been optimized for CFC assays using a purified population of burst-forming and colony-forming erythroid (BFU-E, CFU-E), myeloid (CFU-GM, CFU-G, CFU-M), and mixed lineage (CFU-GEMM) progenitors of human origin. This product is recommended for use in the CFC assay at the end of the long-term culture-initiating cell (LTC-IC) assay.
R&D Systems Human Methylcellulose Serum-free Enriched Media
- Defined media reduces experimental variation.
- Supplemented with premium quality recombinant proteins.
- Optical clarity facilitates colony identification.
- High lot-to-lot consistency decreases variation.
- Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
- Does not require addition of serum or cytokines.
- Increased cloning efficiency and improved colony growth compared to agar.
- 100 mL of Human Methylcellulose Serum-Free Enriched Media.
| 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 |
| Recombinant Human Epo | 3 IU/mL |
| Cholesterol | Trace |
Stability and Storage
Human Methylcellulose Serum-Free Enriched 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, 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.
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 |
| 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 |
*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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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 is used in the Colony Forming Cell Assay using the following procedure:
- Prepare human mononuclear cells
- Add cells to Human Methylcellulose Serum-Free Enriched Media
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Human Methylcellulose Serum-Free Enriched Media (Catalog # HSC005SF):
- 100 mL of Human Methylcellulose Serum-Free Enriched Media
| 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 |
| Recombinant Human Epo | 3 IU/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 Methylcellose Complete Media at room temperature.
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 Cell Resuspension Solution 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. 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 14-16 days.
Use an inverted microscope and a scoring grid to identify and count individual colonies.
FAQs
-
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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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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