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.

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.

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.

Human Methylcellulose Enriched Media

Name: Human Methylcellulose Enriched Media
Brand: R&D Systems
SKU: HSC005
Rating: 5 (1 reviews)

R&D Systems, part of Bio-Techne | Catalog # HSC005

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Species

Human

Product Summary for Human Methylcellulose Enriched Media

Kit 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.

Kit Contents

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.

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

*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 Human Methylcellulose Enriched Media

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.

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.

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