How many freeze-thaws cycles can Methylcellulose stock solution, Catalog # HSC001, undergo?

We recommend keeping freeze-thaw cycles to a minimum. In our experience, two freeze-thaw cycles of the material received is acceptable, but after that, the viscosity of the product may be affected.

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.

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.

Is lot-specific information on viscosity available for Methyl Cellulose Stock Solution (Catalog # HSC001)?

Methyl cellulose Stock Solution is prepared using Methyl cellulose with an estimated viscosity of 1500 CPS. Because of our stringent quality control assays, we don’t expect large variations in viscosity from lot to lot.

What is the difference between Methylcellulose Stock (Catalog # HSC001) and Base Media (Catalog # HSC002)?

The Methylcellulose Stock (Catalog # HSC001) contains methylcellulose in Iscove's Modified Dulbecco's Medium. The Base Media (Catalog # HSC002) contains methylcellulose in Iscove's Modified Dulbecco's Medium along with serum, albumin, L-glutamine, and 2-mercaptoethanol. The additional components of HSC002 are needed to sustain the viability and growth of HSCs. The advantage to using HSC002 is that R&D Systems has already evaluated the serum, albumin, L-glutamine, and 2-mercaptoethanol in-house to ensure lot-to-lot consistency, eliminating the need for the researcher to evaluate these components individually.

What is the molecular weight of Methylcellulose used in Catalog # HSC001?

The Methylcellulose in Catalog # HSC001 has a molecular weight range of 208272 - 219639 g/mol.

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.

Why is there color difference observed sometimes between two bottles of Methylcellulose Stock Solution (Catalog # HSC001)?

The difference in color can be because of dry ice added to the shipment. Dry ice can cause minor pH changes and slight changes in color, but this will not affect performance.

Methylcellulose Stock Solution Best Seller

Name: Methylcellulose Stock Solution
Brand: R&D Systems
SKU: HSC001
Rating: 4 (3 reviews)

R&D Systems, part of Bio-Techne | Catalog # HSC001

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Product Summary for Methylcellulose Stock Solution

Kit Summary

For the growth and differentiation of human, mouse, and rat hematopoietic stem cells using Iscove’s Modified Dulbecco’s Media.

Key Benefits

Can be supplemented with user-defined cytokines and growth factors
Excellent optical clarity facilitates colony identification
High lot-to-lot consistency decreases variation

Why is it Important to Verify Hematopoietic Progenitor Cell Identity using Established Markers?

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 high quality Methylcellulose Stock Solution with superior optical clarity to support optimal colony growth, enumeration, and identification.

R&D Systems Methylcellulose Stock Solution:

Optical clarity facilitates colony identification.
High lot-to-lot consistency decreases variation.
Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
Can be supplemented with user-defined cytokines and growth factors.
Increased cloning efficiency and improved colony growth compared to agar.

Kit Contents

100 mL of 3% Methylcellulose in Iscove's Modified Dulbecco's Medium.

Contents	Concentration
Methylcellulose (1500 cps) in Iscove’s Modified Dulbecco's Medium	3.0%

Stability and Storage

Methylcellulose Stock 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 label.
Human hematopoietic progenitors derived from different individuals may cause results to vary.

Guide to Choosing Media for the Colony Forming Cell (CFC) Assay

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

Mouse 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
HSC006	Mouse Methylcellulose	90 mL	N/A*	Yes	None
HSC011	StemXVivo^® Methylcellulose Concentrate	50 mL	N/A*	No	None

Complete Mouse Methylcellulose Media

Catalog #	Product Description	Volume	Colonies Selected for	Contains Serum	Cytokines Included
HSC007	Mouse Methylcellulose Complete Media	100 mL	BFU-E CFU-E CFU-G CFU-GEMM CFU-GM CFU-M	Yes	Epo 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.

Scientific Data Images for Methylcellulose Stock Solution

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.

Mouse Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay.

A. 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.

Tips to Identify CFU-GEMM Colonies in the Human Colony Forming Unit Assay.

CFU-GEMMs contain a mixture of granulocytes, erythrocytes, megakaryocytes, and macrophages. Human CFU-GEMMs have the obvious red color of the BFU-E as well as the complex morphological features of both the small, compact granulocytes and the large, round macrophages. Viewing the cluster in multiple focal planes is again important to ensure that the colony contains all of the GEMM cell types. CFU-GEMM appearance is similar for human and mouse cells. For more tips please visit watch our CFC Assay video tutorial.

Formulation, Preparation, and Storage

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.

Background: Hematopoietic Stem Cells

The definitive hematopoietic system is made up of all adult blood cell types including megakaryocytes, erythrocytes, and cells of the myeloid and lymphoid lineages. All of these cells are derived from multipotent hematopoietic stem cells (HSCs) through a succession of precursors with progressively limited potential. Hematopoietic stem cells are tissue-specific stem cells that exhibit remarkable self-renewal capacity and are responsible for the life-long mainteÂnance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continuÂously taking place. They can also be found in cord blood, fetal liver, adult spleen, and peripheral blood. R&D Systems offers several products for studying hematopoietic lineage cells including serum-free media, lineage depleÂtion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

Additional Hematopoietic Stem Cells Products

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Product Specific Notices for Methylcellulose Stock Solution

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