Human Methylcellulose Enriched Media

Catalog # Availability Size / Price Qty
HSC005
Product Details
Procedure
Citations (5)
FAQs
Reviews (1)

Human Methylcellulose Enriched Media Summary

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.
 

 

Data Examples

View Larger Image

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

 

 

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.

Product Datasheets

Preparation and Storage

Stability & 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

HSC Differentiation &
Lineage Marker Schematic


Hematopoietic Stem Cells & Lineage-specific Markers

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.

Alternate Names
Hematopoietic Stem Cells

Assay Procedure

Refer to the product datasheet for complete product details.

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

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

 

Reagents Provided

Reagent supplied in the Human Methylcellulose Enriched Media (Catalog # HSC005):

  • 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%

 

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.

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 Cell Resuspension Solution 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 Human Methylcellulose Enriched Media. 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.

Place two 35 mm plates into a 10 cm plate

Citations for Human Methylcellulose Enriched Media

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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  1. The DPY30 subunit in SET1/MLL complexes regulates the proliferation and differentiation of hematopoietic progenitor cells.
    Authors: Yang Z, Augustin J, Chang C, Hu J, Shah K, Chang C, Townes T, Jiang H
    Blood, 2014;124(13):2025-33.  2014
  2. Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa.
    Authors: Tolar J, McGrath J, Xia L, Riddle M, Lees C, Eide C, Keene D, Liu L, Osborn M, Lund T, Blazar B, Wagner J
    J Invest Dermatol, 2014;134(5):1246-54.  2014
  3. Simple and efficient methods for enrichment and isolation of endonuclease modified cells.
    Authors: Moriarity, Branden, Rahrmann, Eric P, Beckmann, Dominic, Conboy, Caitlin, Watson, Adrienne, Carlson, Daniel F, Olson, Erik R, Hyland, Kendra A, Fahrenkrug, Scott C, McIvor, R Scott, Largaespada, David A
    PLoS ONE, 2014;9(5):e96114.  2014
  4. Hematopoietic differentiation of induced pluripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome).
    Authors: Tolar J, Park I, Xia L, Lees C, Peacock B, Webber B, McElmurry R, Eide C, Orchard P, Kyba M, Osborn M, Lund T, Wagner J, Daley G, Blazar B
    Blood, 2011;117(3):839-47.  2011
  5. Capture and enrichment of CD34-positive haematopoietic stem and progenitor cells from blood circulation using P-selectin in an implantable device.
    Authors: Wojciechowski JC, Narasipura SD, Charles N, Mickelsen D, Rana K, Blair ML, King MR
    Br. J. Haematol., 2008;140(6):673-81.  2008

FAQs

  1. 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.

  2. 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.

View all Stem Cell Product FAQs

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Human Methylcellulose Enriched Media
By Anonymous on 04/03/2018