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.

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.

StemXVivo Methylcellulose Concentrate

Name: StemXVivo Methylcellulose Concentrate
Brand: R&D Systems
SKU: HSC011

R&D Systems, part of Bio-Techne | Catalog # HSC011

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Product Summary for StemXVivo Methylcellulose Concentrate

Kit Summary

For the growth and differentiation of human and mouse hematopoietic stem cells using Iscove’s Modified Dulbecco’s Media or any other basal media.

Key Benefits

Is not restricted to use with Iscove's Modified Dulbecco's Media (IMDM)
Supports reproducible in vitro growth of hematopoietic stem and progenitor cells
Can be supplemented with user-defined cytokines and growth factors
Excellent optical clarity facilitates colony identification
High lot-to-lot consistency decreases variation

Why Make Methylcellulose Media from a Concentrated Stock Solution Rather than a Powder?

Although Methylcellulose is soluble at low temperatures, adding powdered methylcellulose to cold water results in clumping of the methylcellulose rather than its dissolution.

Using hot water to dissolve methylcellulose is also not an option since methylcellulose is insoluble in hot water. Therefore, methylcellulose stock solutions are made by adding methylcellulose to hot water (~80 °C) and then decreasing the temperature of the solution until the methylcellulose is fully dissolved. To avoid this time consuming and potentially inconsistent process, R&D Systems offers high quality StemXVivo^® Methylcellulose Concentrate with superior optical clarity to support optimal colony growth, enumeration, and identification.

R&D Systems StemXVivo^® Methylcellulose Concentrate:

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

50 mL of 2.8% Methylcellulose (1500 cps) in water.

Contents	Concentration
Methylcellulose (1500 cps) in water	2.8%

Stability and Storage

The Methylcellulose Concentrate should be stored at ≤-20 °C upon receipt. Storage at 2 °C to 8 °C is not recommended.

Precaution

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.
Results may vary due to variations between hematopoietic progenitors derived from different individuals.

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 StemXVivo Methylcellulose Concentrate

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.

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.

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.

Product Documents for StemXVivo Methylcellulose Concentrate

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Product Specific Notices for StemXVivo Methylcellulose Concentrate

For research use only

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