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.

Human Methylcellulose Serum-Free Enriched Media

Name: Human Methylcellulose Serum-Free Enriched Media
Brand: R&D Systems
SKU: HSC005SF

R&D Systems, part of Bio-Techne | Catalog # HSC005SF

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Product Datasheet / COA / SDS

Key Product Details

Summary for Human Methylcellulose Serum-Free Enriched Media

Kit Summary

Complete, defined media for the differentiation and enumeration of human hematopoietic stem cells.

Key Benefits

Defined media components reduce experimental variation
Excellent optical clarity for easy identification of colonies
High lot-to-lot consistency decreases variation

Why use R&D Systems Human Methylcellulose Serum-Free 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 Serum-free Enriched Media

Defined media reduces experimental variation.
Supplemented with premium quality recombinant proteins.
Optical clarity facilitates colony identification.
High lot-to-lot consistency decreases variation.
Supports reproducible in vitro growth of hematopoietic stem and progenitor cells.
Does not require addition of serum or cytokines.
Increased cloning efficiency and improved colony growth compared to agar.

Kit Contents

100 mL of Human Methylcellulose Serum-Free Enriched Media.

Contents	Concentration (when diluted to a final volume of 100 mL)
Methylcellulose (1500 cps) in Iscove’s Modified Dulbecco's Medium	1.4%
Human Transferrin	300 μg/mL
Bovine Serum Albumin	3.6%
Recombinant Human Insulin	16 μg/mL
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
Cholesterol	Trace

Stability and Storage

Human Methylcellulose Serum-Free Enriched Media 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.
The human Transferrin used in this product was derived from human plasma, which has been tested and found negative for HIV-1/2 antibodies, Hepatitis B surface antigen, Hepatitis C antibody, Syphilis, and p24 antigen by FDA approved methods. Handle as if capable of transmitting infection, and dispose of according to applicable regulations.

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.

HSC Differentiation &Lineage Marker Schematic View Interactive Pathway

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 maintenance of the hematopoietic system. HSCs are rare cells that reside in adult bone marrow where hematopoiesis is continuously 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 depletion antibodies and kits, and reagents for performing colony forming cell (CFC) assays.

Product Specifications for Human Methylcellulose Serum-Free Enriched Media

Species	Human
Source	N/A

Scientific Data Examples for Human Methylcellulose Serum-Free 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.

Preparation & Storage

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.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Human Methylcellulose Serum-Free Enriched Media is used in the Colony Forming Cell Assay using the following procedure:

Prepare human mononuclear cells
Add cells to Human Methylcellulose Serum-Free Enriched Media
Plate and incubate cells
Identify and count colonies

View Human Procedure

Reagents Provided

Reagent supplied in the Human Methylcellulose Serum-Free Enriched Media (Catalog # HSC005SF):

100 mL of Human Methylcellulose Serum-Free Enriched Media

Contents	Concentration (when diluted to a final volume of 100 mL)
Methylcellulose (1500 cps) in Iscove’s Modified Dulbecco's Medium	1.4%
Human Transferrin	300 μg/mL
Bovine Serum Albumin	3.6%
Recombinant Human Insulin	16 μg/mL
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
Cholesterol	Trace

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 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 Serum-Free 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.

Place two 35 mm plates into a 10 cm plate

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

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