Human Methylcellulose Base Media

Catalog # Availability Size / Price Qty
HSC002
Product Details
Procedure
Citations (8)
FAQs
Reviews

Human Methylcellulose Base Media Summary

Kit Summary

For the growth and differentiation of human hematopoietic stem cells.

Key Benefits

  • Can be supplemented with user-defined cytokines and growth factors
  • Excellent optical clarity facilitates colony identification
  • High lot-to-lot consistency decreases variation
 

 

Why is it Important to Verify Hematopoietic Progenitor Cell Identity using Established Markers?

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 Base Media with superior optical clarity to support optimal colony growth, enumeration, and identification. The Human Methylcellulose Base Media contains components that have been optimized for CFC assays. Individual researchers can customize the media by adding cells and other culture supplements tailored to their specific research. This product can also be used in the long-term culture-initiating cell (LTC-IC) assay.

R&D Systems Human Methylcellulose Base Media:

  • 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
  • 90 mL of Human Methylcellulose Base 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

Cell Resuspension Solution (15 mL)

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

Stability and Storage

Human Methylcellulose Base 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 label.
  • 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
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

*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 Base Media is used in the Colony Forming Cell Assay using the following procedure:

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

 

Reagents Provided

Reagents supplied in the Human Methylcellulose Base Media (Catalog # HSC002):

  • 90 mL of Human Methylcellulose Base 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

 

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

  • 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 Methylcellulose Stock Solution 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 5 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 Base 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 Base 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.

8 Citations: Showing 1 - 8
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  1. Single-cell epigenomic variability reveals functional cancer heterogeneity
    Authors: UM Litzenburg, JD Buenrostro, B Wu, Y Shen, NC Sheffield, A Kathiria, WJ Greenleaf, HY Chang
    Genome Biol, 2017;18(1):15.  2017
  2. Mobilization studies in mice deficient in sphingosine kinase 2 support a crucial role of the plasma level of sphingosine-1-phosphate in the egress of hematopoietic stem progenitor cells
    Authors: M Adamiak, L Chelvaraja, KR Lynch, WL Santos, A Abdel-Lati, MZ Ratajczak
    Oncotarget, 2017;8(39):65588-65600.  2017
  3. Bone marrow stem/progenitor cell mobilization in C57BL/6J and BALB/c mice.
    Authors: Lee, Hakmo, Che, Jeong-Hw, Oh, Ju Eun, Chung, Sung Soo, Jung, Hye Seun, Park, Kyong So
    Lab Anim Res, 2014;30(1):14-20.  2014
  4. 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
  5. STAT3 mediates oncogenic addiction to TEL-AML1 in t(12;21) acute lymphoblastic leukemia.
    Authors: Mangolini M, de Boer J, Walf-Vorderwulbecke V, Pieters R, den Boer M, Williams O
    Blood, 2013;122(4):542-9.  2013
  6. The AAA+ ATPase RUVBL2 is a critical mediator of MLL-AF9 oncogenesis.
    Authors: Osaki H, Walf-Vorderwulbecke V, Mangolini M, Zhao L, Horton S, Morrone G, Schuringa J, de Boer J, Williams O
    Leukemia, 2013;27(7):1461-8.  2013
  7. 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;7(7):e40140.  2012
  8. Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.
    Authors: Ratajczak MZ, Lee H, Wysoczynski M, Wan W, Marlicz W, Laughlin MJ, Kucia M, Janowska-Wieczorek A, Ratajczak J
    Leukemia, 2010;24(5):976-85.  2010

FAQs

  1. What is the difference between Methylcellulose Stock (Catalog # HSC001) and Base Media (Catalog # HSC002)?

    • The Methylcellulose Stock (Catalog # HSC001) contains methylcellulose in Iscove's Modified Dulbecco's Medium. The Base Media (Catalog # HSC002) contains methylcellulose in Iscove's Modified Dulbecco's Medium along with serum, albumin, L-glutamine, and 2-mercaptoethanol. The additional components of HSC002 are needed to sustain the viability and growth of HSCs. The advantage to using HSC002 is that R&D Systems has already evaluated the serum, albumin, L-glutamine, and 2-mercaptoethanol in-house to ensure lot-to-lot consistency, eliminating the need for the researcher to evaluate these components individually.
  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.

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