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?

Name: Human Methylcellulose Complete Media Without Epo
Brand: R&D Systems
SKU: HSC004
Rating: 5 (1 reviews)

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.

Human Methylcellulose Complete Media Without Epo

Catalog #: HSC004
5 Citations 1 Review Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
HSC004

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

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Product Details

Procedure

Citations (5)

FAQs

Reviews (1)

Summary Product Datasheets Data Images Background

Human Methylcellulose Complete Media Without Epo Summary

Kit Summary

For the differentiation and enumeration of granulocyte-macrophage progenitor cells and for detection of Epo-independent erythropoiesis, optimized with premium quality cytokines.

Key Benefits

Suitable for detection of Epo-independent erythropoiesis
Excellent optical clarity facilitates colony identification
High lot-to-lot consistency decreases variation
Optimized for enumeration of myeloid progenitor cells

Why use R&D Systems Human Methylcellulose Complete Media Without Epo 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 Complete Media Without Epo, which contains all of the growth factors and cytokines needed to support optimal growth and enumeration of colony-forming myeloid progenitors. The absence of Epo makes this media useful for the identification of Epo-independent stimulators of erythropoiesis. Human Methylcellulose Complete Media Without Epo is specially formulated and has been optimized for CFC assays using colony-forming myeloid progenitors (CFU-GM, CFU-G, CFU-M) of human origin. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.

R&D Systems Human Methylcellulose Complete Media without EPO:

Can be used to identify Epo-independent stimulators of erythropoiesis.
Optical clarity facilitates colony identification.
High lot-to-lot consistency decreases variation.
Supports reproducible in vitro growth of myeloid progenitor cells.
Does not require addition of growth factors or cytokines.
Increased cloning efficiency and improved colony growth compared to agar.

Kit Contents

100 mL of Human Methylcellulose Complete Media Without Epo and 15 mL 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	10 ng/mL
Recombinant Human IL-3	10 ng/mL

Cell Resuspension Solution (15 mL)

Contents	Concentration
Fetal Bovine Serum in Iscove’s Modified Dulbecco’s Medium	50%

Stability and Storage

Human Methylcellulose Complete Media Without Epo 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
HSC002SF	Human Methylcellulose Seum-Free Base Media	90 mL	N/A*	No	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
HSC010SF	Human Methylcellulose Serum-Free Enriched Media without Epo	100 mL	CFU-G CFU-GM CFU-M	No	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

Shipping Conditions

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.

Species

Human

Product Datasheets

Product Datasheet

COA

SDS

Scientific Data

Cell Morphology

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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.

Related Research Areas

Hematopoietic Stem Cell Markers

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Human Methylcellulose Complete Media Without Epo is used in the Colony Forming Cell Assay using the following procedure:

Prepare human mononuclear cells
Add cells to Human Methylcellulose Complete Media Without Epo
Plate and incubate cells
Identify and count colonies

View Human Procedure

Reagents Provided

Reagents supplied in the Human Methylcellulose Complete Media (Catalog # HSC004):

100 mL of Human Methylcellulose Complete 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	10 ng/mL
Recombinant Human IL-3	10 ng/mL

Cell Resuspension Solution (15 mL)

Contents	Concentration
Fetal Bovine Serum in Iscove’s Modified Dulbecco’s Medium	50%

Other Supplies Required

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

Procedure Overview

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.

Prepare mononuclear cells by Ficoll-Paque gradient centrifugation

Thaw aliquots of Human Methylcellose Complete Media Without Epo at room temperature.

Thaw aliquots of Methylcellulose Stock Solution 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 Complete Media Without Epo. 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.

Place two 35 mm plates into a 10 cm plate

Use an inverted microscope and a scoring grid to identify and count individual colonies.

Citations for Human Methylcellulose Complete Media Without Epo

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.

5 Citations: Showing 1 - 5
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Identification of curaxin as a potential new therapeutic for JAK2 V617F mutant patients
Authors: Pearson, S;Blance, R;Yan, F;Hsieh, YC;Geary, B;Amaral, FMR;Somervaille, TCP;Kirschner, K;Whetton, AD;Pierce, A;
PloS one 2023-05-30
miR-23b/SP1/c-myc forms a feed-forward loop supporting multiple myeloma cell growth.
Authors: Fulciniti M, Amodio N, Bandi R, Cagnetta A, Samur M, Acharya C, Prabhala R, D'Aquila P, Bellizzi D, Passarino G, Adamia S, Neri A, Hunter Z, Treon S, Anderson K, Tassone P, Munshi N
Blood Cancer J, 2016-01-15;6(0):e380. 2016-01-15
Combination drug scheduling defines a "window of opportunity" for chemopotentiation of gemcitabine by an orally bioavailable, selective ChK1 inhibitor, GNE-900.
Authors: Blackwood E, Epler J, Yen I, Flagella M, O'Brien T, Evangelista M, Schmidt S, Xiao Y, Choi J, Kowanetz K, Ramiscal J, Wong K, Jakubiak D, Yee S, Cain G, Gazzard L, Williams K, Halladay J, Jackson P, Malek S
Mol Cancer Ther, 2013-07-19;12(10):1968-80. 2013-07-19
Acellular bone marrow extracts significantly enhance engraftment levels of human hematopoietic stem cells in mouse xeno-transplantation models.
Authors: Zibara K, Hamdan R, Dib L
PLoS ONE, 2012-07-02;7(7):e40140. 2012-07-02
Autocrine insulin-like growth factor-I signaling promotes growth and survival of human acute myeloid leukemia cells via the phosphoinositide 3-kinase/Akt pathway.
Authors: Doepfner KT, Spertini O, Arcaro A
Leukemia, 2007-06-21;21(9):1921-30. 2007-06-21

FAQs

What are the differences between in Methylcellulose Complete Media with EPO (Catalog # HSC003) and without EPO (Catalog # HSC004)?
- EPO is a potent cytokine used to stimulate differentiation of HSCs. EPO is needed for erythrocyte development. Media without EPO offers researchers the ability to expand and differentiate their HSCs and then to test supplemental drugs or compounds of interest.
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.
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.
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.