MethoCult™ H4100

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Base Methylcellulose Medium for Human Cells

  • MethoCult™ H4100 40 mL
  • Label for MethoCult™ H4100 40 mL
MethoCult™ H4100 40 mL
MethoCult™ H4100 is a base methylcellulose medium for the culture of human cells in semisolid media. The medium is supplied as a 2.6% solution of methylcellulose in Iscove's MDM, allowing the addition of serum, growth factors and other supplements for colony-forming cell (CFC) assays of human cells. The medium can also be used as a base for cloning cell lines in semisolid media.
Product Name Description Catalog # Size Price Quantity
MethoCult™ H4100 Base methylcellulose medium for human cells 04100 40 mL 75.00 USD      
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Application: Colony assays & quantification
Product Type: Specialized cell culture media
Recommended For:
• Assays of human clonogenic hematopoietic progenitor cells
• Testing sera, erythropoietin and cytokines
• Cloning of cell lines
Accessory Products:
• Iscove's Modified Dulbecco's Medium (IMDM) with 2% Fetal Bovine Serum (Catalog #07700)
• Blunt-end Needles (Catalog #28110/28120)
• 3 cc Syringe (Catalog #28230/28240)
• 35 mm Culture Dishes (Catalog #27100/27150)
• 60 mm Gridded Scoring Dishes (Catalog #27500)
Contains:
2.6% Methylcellulose in Iscove's MDM
Area of Interest: Cancer, Hematologic malignancies, Hematopoietic stem cell research, Stem cell biology
Cell Type: Hematopoietic stem & progenitor cells
Medium Type: Methylcellulose-based
Popular Product Line: MethoCult™
Species: Human
Intended Use Statement: FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
ISO Statement: STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485 MEDICAL DEVICE STANDARDS

Procedures and instruction manuals:

Educational resources:

MSDS:

FAQS:


    • Q. WHY USE SEMI-SOLID MEDIA?
      A. Semi-solid media (such as methylcellulose-based or collagen-based) allow the clonal progeny of a single progenitor cell to stay together so you can recognize distinct colonies.
    • Q. WHY USE METHYLCELLULOSE-BASED MEDIA?
      A. Methylcellulose is now widely used as a gelling agent because it permits better growth of erythroid colonies than other types of semi-solid support systems (e.g. agar) while allowing optimal granulocyte/macrophage colony formation. Committed progenitors for both erythroid and granulocyte/macrophage lineages, as well as multi-potential progenitors, can therefore be assayed simultaneously in the same culture dish.
    • Q. IS IT NECESSARY TO ADD ANTIBIOTICS TO THE MEDIA?
      A. No, we do not use antibiotics in our lab for colony assays. Aseptic technique should suffice. However, Penicillin/Streptomycin and an anti-fungal drug like amphotericin B can be added to the methylcellulose. The media should be mixed well prior to the addition of cells. Presence of antibiotics does not inhibit progenitor growth.
    • Q. IS THERE ANYTHING I CAN DO IF MY PLATES ARE CONTAMINATED?
      A. No, once contamination is visible, it is not possible to rescue the cultures by the addition of antibiotics. The bacteria/yeast inhibit colony formation by depleting nutrients or releasing toxic substances.
    • Q. WHY CAN'T I USE A PIPETTE TO DISPENSE METHYLCELLULOSE-BASED MEDIA?
      A. Because methylcellulose is a viscous solution and cannot be accurately dispensed using pipettes due to adherence of the medium to the walls of the pipette. 16-gauge (blunt-end) needles are recommended for accurate dispensing.
    • Q. CAN I 'PLUCK' THE COLONIES?
      A. Yes, colonies can be 'plucked' using a pipettor and 200 µL sterile pipette tips or glass Pasteur pipette with an elongated tip. Colonies should be placed in a volume of 25 - 50 µL and diluted into suitable culture medium.
    • Q. IS THERE A METHOCULT™ FORMULATION SUITABLE FOR HPP-CFC (HIGH PROLIFERATIVE POTENTIAL COLONY FORMING CELL)?
      A. Yes, MethoCult™ H4535 can be used as HPP-CFC does not require EPO. The culture period is usually 28 days. Feeding the media is not required as the growth factors are present in excess in the media. As these colonies can be quite large, overplating can be a problem. It is recommended to plate two different concentrations. The standard concentration, as well as a lower concentration.
    • Q. WHY ARE LOW ADHERENCE DISHES SO IMPORTANT?
      A. Because adherent cells such as fibroblasts can cause inhibition of colony growth and obscure visualization of colonies.
    • Q. CAN METHOCULT™ PRODUCTS BE USED FOR LYMPHOID PROGENITOR CFC ASSAYS?
      A. Human lymphoid progenitors (B,NK, and T) seem to require stromal support for growth therefore cannot be grown in MethoCult™. Mouse pre-B clonogenic progenitors can be grown in MethoCult™ M3630.
    • Q. IS IT POSSIBLE TO SET UP CFC ASSAYS IN A 24-WELL PLATE?
      A. Yes, as long as the same ratio of cells is plated. Optimal plating concentration and the number of wells required to get an accurate estimation of CFC numbers may require optimization:
      • The surface area of a 35 mm dish is ~9.5 cm2 and 1.9 cm2 for the 24 well plate.
    • Q. CAN I STAIN THE COLONIES IN THE METHOCULT™?
      A. Colonies can be stained but only if they are plucked from the dish. However, mouse erythroid colonies can be stained in the dish (Benzidine Staining Protocol). CollagenCult™ products are recommended for staining applications. The 3D matrix can be dehydrated and the colonies can then be fixed onto glass slides for immunohistochemical or enzymatic staining.
    • Q. ARE THERE DIFFERENCES IN COLONY MORPHOLOGY WITH SERUM-FREE MEDIA?
      A. The serum containing media generally give better overall growth (especially CFU-GM colonies, as they will contain more cells) but not more colonies. There is no difference in total CFC’s between serum-free and conditioned or recombinant media.
    • Q. CAN METHOCULT™ BE MADE WITH AN ALTERNATE BASE MEDIA?
      A. Yes, this can be done as a 'Custom' media order. Please contact techsupport@stemcell.com for more information.

This product has been used in:

  1. Nina Fenouille et al. Persistent activation of the Fyn/ERK kinase signaling axis mediates imatinib resistance in chronic myelogenous leukemia cells through upregulation of intracellular SPARC.Cancer Res 70 (23) 9659-9670 (December 1, 2010)
  2. Beno??t Laurent et al. High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription.Blood 115 (3) 687-695 (January 21, 2010)
  3. Melanie Fakler et al. Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2-mediated resistance.Blood 113 (8) 1710-1722 (February 19, 2009)
  4. Tinke L Vormer et al. Anchorage-independent growth of pocket protein-deficient murine fibroblasts requires bypass of G2 arrest and can be accomplished by expression of TBX2.Mol Cell Biol 28 (24) 7263-7273 (December 2008)
  5. Dale A Moulding et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia.J Exp Med 204 (9) 2213-2224 (September 3, 2007)
  6. Nobuyuki Onai et al. Activation of the Flt3 signal transduction cascade rescues and enhances type I interferon-producing and dendritic cell development.J Exp Med 203 (1) 227-238 (January 23, 2006)
  7. Eleni Thanopoulou et al. Engraftment of NOD/SCID-beta2 microglobulin null mice with multilineage neoplastic cells from patients with myelodysplastic syndrome.Blood 103 (11) 4285-4293 (June 1, 2004)
  8. I Dybedal et al. Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression.Blood 102 (1) 118-126 (July 1, 2003)
  9. Junko Iwasaki-Arai et al. Enforced granulocyte/macrophage colony-stimulating factor signals do not support lymphopoiesis, but instruct lymphoid to myelomonocytic lineage conversion.J Exp Med 197 (10) 1311-1322 (May 19, 2003)
  10. Encarnacion Montecino-Rodriguez et al. Bipotential B-macrophage progenitors are present in adult bone marrow.Nature Immunology 2 (1) 83-88 (January 2001)

Background References:

  1. R E Donahue et al. High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.Blood 95 (2) 445-452 (January 15, 2000)
  2. L Gribaldo et al. Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow.Exp Hematol 27 (11) 1593-1598 (November 1999)
  3. D E Hogge et al. Enhanced detection, maintenance, and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human steel factor, interleukin-3, and granulocyte colony-stimulating factor.Blood 88 (10) 3765-3773 (November 15, 1996)
  4. A L Petzer et al. Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.Proc Natl Acad Sci U S A 93 (4) 1470-1474 (February 20, 1996)
  5. E Conneally et al. Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer.Blood 87 (2) 456-464 (January 15, 1996)
  6. A M Farese et al. Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia.Blood 87 (2) 581-591 (January 15, 1996)
  7. H Mayani et al. Cytokine-induced selective expansion and maturation of erythroid versus myeloid progenitors from purified cord blood precursor cells.Blood 81 (12) 3252-3258 (June 15, 1993)

Product Name

Description

Catalog #

Iscove's MDM with 2% FBS Medium for Preparing and Washing Samples for Colony-Forming Cell (CFC) Assays 07700
35 mm Culture Dishes 35 mm Culture Dishes 27100
60 mm Gridded Scoring Dishes Dishes for Reproducible and Accurate Scoring of Colonies 27500
Blunt-End Needles, 16 Gauge 16 Gauge Blunt-end Needles 28110
3 cc Syringes 3 cc Syringes 28230
SCF, Human, Recombinant Stem Cell Factor 02630
IL-3, Human, Recombinant Interleukin-3 02503
GM-CSF, Human, Recombinant Granulocyte-Macrophage colony-Stimulating factor (GM-CSF) 02532
G-CSF, Human, Recombinant Granulocyte Colony-Stimulating Factor (G-CSF) 02615
IL-6, Human, Recombinant Interleukin-6 02506
10% Bovine Serum Albumin in Iscove's MDM
10% Bovine Serum Albumin (BSA) in Iscove's MDM
09300
L-Glutamine L-Glutamine (200 mM) 07100
Fetal Bovine Serum for Human Myeloid Colony-Forming Cells For Preparation of Methylcellulose-Based Medium for Human Myeloid Colony-forming Cells 06100
EasySep™ Human CD34 Positive Selection Kit Immunomagnetic Positive Selection Kit 18056
EasySep™ Human Whole Blood CD34 Positive Selection Kit Immunomagnetic Positive Selection Kit 18086
EasySep™ Human Cord Blood CD34 Positive Selection Kit Immunomagnetic Positive Selection Kit 18096
EasySep™ Human Progenitor Cell Enrichment Kit Immunomagnetic Negative Selection Kit 19056
RosetteSep™ Human Bone Marrow Progenitor Cell Pre-Enrichment Cocktail Immunodensity Negative Selection Cocktail 15027
Lymphoprep™ Density Gradient Medium for the Isolation of Mononuclear Cells 07801
Ammonium Chloride Solution Reagent for the Lysis of Red Blood Cells 07800
RosetteSep™ Human CD45 Depletion Cocktail Immunodensity Depletion Cocktail 15122
RosetteSep™ Human Cord Blood Debulking Cocktail Immunodensity Depletion Cocktail 15126
RosetteSep™ Human Cord Blood Progenitor Cell Enrichment Cocktail Immunodensity Negative Selection Cocktail 15026
StemSep™ Anti-Human CD41 TAC Immunomagnetic Selection Reagent 14050
StemSep™ Human CD34 Positive Selection Cocktail Immunomagnetic Column-Based Positive Selection Kit 14756
Anti-Mouse CD49f Antibody, Clone GoH3 Rat Monoclonal Antibody Against Human, Mouse, Rhesus CD49f (Integrin α6) 60037

Procedure summary for hematopoietic CFC Assays


Procedure summary for hematopoietic CFC Assays



Examples of colonies derived from human hematopoietic progenitors


Examples of colonies derived from human hematopoietic progenitors



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