StemXVivo Methylcellulose Concentrate

Catalog # Availability Size / Price Qty
HSC011
Human Hematopoietic Colony Formation Using the Methylcellulose-based Colony Forming Cell Assay. 
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Product Details
Procedure
Citations (3)
FAQs
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StemXVivo Methylcellulose Concentrate Summary

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
HSC002SF Human Methylcellulose
Serum-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

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
HSC008 Mouse Methylcellulose Complete Media Without Epo 100 mL CFU-G
CFU-GM
CFU-M
Yes IL-3
IL-6
SCF
HSC009 Mouse Methylcellulose Complete Media for Pre-B Cells 100 mL CFU-Pre-B Yes IL-7

*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.

Product Datasheets

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Scientific Data

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

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.

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

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.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, Methylcellulose Stock Solution is used in the Colony Forming Cell Assay using the following procedure:

  • Prepare human mononuclear cells or mouse/rat bone marrow cells
  • Add cells and desired supplements to Methylcellulose Stock Solution
  • Plate and incubate cells
  • Identify and count colonies
 

 

Reagents Provided

Reagent supplied in the StemXVivo® Methylcellulose Concentrate (Catalog # HSC011):

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

 

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

 

Other Supplies Required

Reagents

  • Cells derived from mouse bone marrow, spleen, peripheral blood, or fetal liver. Mice are routinely used between 6 - 12 weeks.
  • Iscove's Modified Dulbecco’s Media (IMDM)
  • Fetal Bovine Serum
  • IMDM/2% Fetal Bovine Serum
  • (Optional) Flow Cytometry Mouse Lyse Buffer (Catalog # FC003)

Materials

  • 100 mm culture plates
  • 35 mm culture plates
  • 15 mL centrifuge tubes
  • 10 mL syringes
  • 3 mL syringes
  • 5 mL vials
  • 16 gauge 1½ inch needle
  • 14 gauge laboratory pipetting needle
  • Serological pipettes
  • Pipettes and pipette tips

Equipment

  • 37 °C and CO2 humidified incubator
  • Centrifuge
  • Vortex mixer
  • Hemocytometer
  • Inverted Microscope

 

Procedure Overview

Procedure for the Human Colony Forming Cell Assay

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 StemXVivo® Methylcellulose Concentrate at room temperature.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

Resuspend mononuclear cells in 10 mL of IMDM.

Resuspend mononuclear cells in IMDM

Perform a cell count.

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.

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube

Remove the supernatant.

Resuspend the cells in IMDM to the desired stock cell number to generate a 10X stock concentration.

Remove the supernatant

Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and StemXVivo® Methylcellulose Stock Solution. The final Methylcellulose concentration should be 1.27%.

Combine the appropriate volume of 10X cell stock

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.

Vortex the samples vigorously

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.

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

 

Procedure for the Mouse Colony Forming Cell Assay

Pass a suspension of mouse bone marrow cells through a 70 μm nylon strainer to remove clumps and debris.

Remove red blood cells if necessary.

Wash the cells with IMDM/2% FBS by centrifugation at 300 x g for 8 minutes and pool the cells.

Pass a suspension of mouse bone marrow cells through a nylon strainer

Remove the supernatant.

Resuspend the cells in 10 mL of IMDM/2% FBS.

Remove the supernatant

Thaw aliquots of StemXVivo® Methylcellulose Stock Solution at room temperature for approximately 30 minutes.

Thaw aliquots of Methylcellulose Stock Solution at room temperature

Perform a cell count.

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.

Transfer the appropriate volume of cells plus a slight excess into a new 15 mL centrifuge tube

Remove the supernatant.

Resuspend the cells in IMDM to the desired stock cell number to generate a 10X stock concentration.

Remove the supernatant

Combine the appropriate volume of 10X cell stock with the desired cell culture supplements/cytokines, and StemXVivo® Methylcellulose Stock Solution. The final Methylcellulose concentration should be 1.27%.

Combine the appropriate volume of 10X cell stock

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.

Vortex the samples vigorously

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 8-12 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.

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

Citations for StemXVivo Methylcellulose Concentrate

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.

3 Citations: Showing 1 - 3
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  1. Loss of EZH2 Reprograms BCAA Metabolism to Drive Leukemic Transformation
    Authors: Z Gu, Y Liu, F Cai, M Patrick, J Zmajkovic, H Cao, Y Zhang, A Tasdogan, M Chen, L Qi, X Liu, K Li, J Lyu, KE Dickerson, W Chen, M Ni, ME Merritt, SJ Morrison, RC Skoda, RJ DeBerardin, J Xu
    Cancer Discov, 2019-06-12;0(0):.  2019-06-12
  2. Proteoglycan-targeting applied to hypoxia-activated prodrug therapy in chondrosarcoma: first proof-of-concept
    Authors: A Voissiere, V Weber, Y Gerard, F Rédini, F Raes, JM Chezal, F Degoul, C Peyrode, E Miot-Noira
    Oncotarget, 2017-09-27;8(56):95824-95840.  2017-09-27
  3. Enrichment of the Cancer Stem Phenotype in Sphere Cultures of Prostate Cancer Cell Lines Occurs through Activation of Developmental Pathways Mediated by the Transcriptional Regulator DeltaNp63alpha.
    Authors: Portillo-Lara R, Alvarez M
    PLoS ONE, 2015-06-25;10(6):e0130118.  2015-06-25

FAQs

  1. 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.

  2. 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.

View all Stem Cell Product FAQs

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