Human Methylcellulose Enriched Media Summary
Complete media with G-CSF and IL-6 for the differentiation and enumeration of CD34+ human hematopoietic stem cells.
Key Benefits
- Optimized for use with CD34+ cells
- High lot-to-lot consistency decreases variation
- Excellent optical clarity for easy identification of colonies
Why use R&D Systems Human Methylcellulose 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 Enriched Media:
- Optimized for use with CD34+ cells.
- Optical clarity facilitates colony identification.
- High lot-to-lot consistency decreases variation.
- Does not require addition of serum or cytokines.
- Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
- Increased cloning efficiency and improved colony growth compared to agar.
- 100 mL of Human Methylcellulose Enriched Media and 15 mL of Cell Resuspension Solution.
| 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 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 |
Cell Resuspension Solution (15 mL)
| Contents | Concentration |
| Fetal Bovine Serum in Iscove’s Modified Dulbecco’s Medium |
50% |
Stability and Storage
Human Methylcellulose Enriched Media and the Cell Resuspension Solution 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 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 Enriched Media is used in the Colony Forming Cell Assay using the following procedure:
- Prepare human mononuclear cells
- Add cells to Human Methylcellulose Enriched Media
- Plate and incubate cells
- Identify and count colonies
Reagent supplied in the Human Methylcellulose Enriched Media (Catalog # HSC005):
- 100 mL of Human Methylcellulose Enriched Media and 15 mL of Cell Resuspension Solution
| 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 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 |
Cell Resuspension Solution (15 mL)
| Contents | Concentration |
| Fetal Bovine Serum in Iscove's Modified Dulbecco's Medium |
50% |
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 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.
Citations for Human Methylcellulose Enriched Media
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 10
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Streptavidin-drug conjugates streamline optimization of antibody-based conditioning for hematopoietic stem cell transplantation
Authors: Yelamali, AR;Chendamarai, E;Ritchey, JK;Rettig, MP;DiPersio, JF;Persaud, SP;
bioRxiv : the preprint server for biology 2024-02-12
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Identification of a c-MYB-directed therapeutic for acute myeloid leukemia
Authors: K Clesham, V Walf-Vorde, L Gasparoli, C Virely, S Cantilena, A Tsakaneli, S Inglott, S Adams, S Samarasing, J Bartram, G Williams, J de Boer, O Williams
Leukemia, 2022-04-02;0(0):. 2022-04-02
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KAT6A and ENL Form an Epigenetic Transcriptional Control Module to Drive Critical Leukemogenic Gene-Expression Programs
Authors: F Yan, J Li, J Milosevic, R Petroni, S Liu, Z Shi, S Yuan, JM Reynaga, Y Qi, J Rico, S Yu, Y Liu, S Rokudai, N Palmisiano, SE Meyer, PJ Sung, L Wan, F Lan, BA Garcia, BZ Stanger, DB Sykes, MA Blanco
Cancer Discovery, 2022-03-01;12(3):792-811. 2022-03-01
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CD7-deleted hematopoietic stem cells can restore immunity after CAR T cell therapy
Authors: MY Kim, ML Cooper, MT Jacobs, JK Ritchey, J Hollaway, TA Fehniger, JF DiPersio
JCI Insight, 2021-08-23;6(16):. 2021-08-23
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Therapeutic delivery of siRNA with polymeric carriers to down-regulate STAT5A expression in high-risk B-cell acute lymphoblastic leukemia (B-ALL)
Authors: M Mohseni, C Kucharski, RB K C, M Nasrullah, X Jiang, H Uluda?, J Brandwein
PLoS ONE, 2021-06-22;16(6):e0251719. 2021-06-22
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The DPY30 subunit in SET1/MLL complexes regulates the proliferation and differentiation of hematopoietic progenitor cells.
Authors: Yang Z, Augustin J, Chang C, Hu J, Shah K, Chang C, Townes T, Jiang H
Blood, 2014-08-18;124(13):2025-33. 2014-08-18
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Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
Authors: Moriarity, Branden, Rahrmann, Eric P, Beckmann, Dominic, Conboy, Caitlin, Watson, Adrienne, Carlson, Daniel F, Olson, Erik R, Hyland, Kendra A, Fahrenkrug, Scott C, McIvor, R Scott, Largaespada, David A
PLoS ONE, 2014-05-05;9(5):e96114. 2014-05-05
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Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa.
Authors: Tolar J, McGrath J, Xia L, Riddle M, Lees C, Eide C, Keene D, Liu L, Osborn M, Lund T, Blazar B, Wagner J
J Invest Dermatol, 2013-12-06;134(5):1246-54. 2013-12-06
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Hematopoietic differentiation of induced pluripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome).
Authors: Tolar J, Park I, Xia L, Lees C, Peacock B, Webber B, McElmurry R, Eide C, Orchard P, Kyba M, Osborn M, Lund T, Wagner J, Daley G, Blazar B
Blood, 2010-10-29;117(3):839-47. 2010-10-29
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Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device.
Authors: Wojciechowski JC, Narasipura SD, Charles N, Mickelsen D, Rana K, Blair ML, King MR
Br. J. Haematol., 2008-01-22;140(6):673-81. 2008-01-22
FAQs
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What is the difference between the Complete (Catalog # HSC003) and Enriched (Catalog # HSC005) Methylcellulose?
The Complete Methylcellulose Media (Catalog # HSC003) contains four growth factors (rhSCF, rhGM-CSF, rhIL-3, and rhEPO) whereas the Enriched Media (Catalog # HSC005) contains six growth factors (rhSCF, rhGM-CSF, rhIL-3, rhEPO, rhG-CSF, and rhIL-6) for hematpoietic stem cell (HSC) differentiation.
HSC003 is typically used with an unpurified heterogeneous population of cells. PBMCs may be mixed with HSCs and the included growth factors will stimulate the PBMCs to express other growth factors to promote differentiation of HSCs.
HSC005 is typically used with purified HSCs and it includes growth factors to differntiate HSCs without assistance from growth factors expressed from PBMCs.
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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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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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