MethoCult® H4434 Classic

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Methylcellulose-Based Medium with Recombinant Cytokines for Human Cells

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  • MethoCult® H4434 Classic 100 mL
  • Label for MethoCult® H4434 Classic 100 mL
  • Tube label for MethoCult® H4434 Classic 3 mL
MethoCult® H4434 Classic 100 mL
MethoCult® H4434 Classic (MethoCult® GF H4434) is optimized for the detection and quantification of human hematopoietic progenitors in bone marrow (BM), mobilized peripheral blood (MPB), peripheral blood (PB) and cord blood (CB) samples using colony-forming cell (CFC) assays. It is suitable for CD34+-enriched cells, mononuclear cells and cells isolated by other purification methods.
 
MethoCult® H4434 Classic is formulated to support optimal growth of erythroid progenitors (CFU-E and BFU-E), granulocyte-macrophage progenitors (CFU-GM, CFU-G and CFU-M) and multi-potential granulocyte, erythroid, macrophage and megakaryocyte progenitors (CFU-GEMM).
Product Name Description Catalog # Size Price Quantity
MethoCult® H4434 Classic Pre-aliquoted methylcellulose medium with recombinant cytokines and EPO for human cells 04444 24 x 3 mL 500.00 USD      
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MethoCult® H4434 Classic Methylcellulose medium with recombinant cytokines and EPO for human cells 04434 100 mL 406.00 USD      
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Call us: +1.604.877.0713  
Toll Free: 1.800.667.0322  
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Fax us: +1.604.877.0704
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Recommended for:
Assays of human clonogenic hematopoietic progenitor cells from bone marrow, peripheral blood, cord blood, leukapheresis products and purified CD34+ cells
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)
Intended Use Statement: For Research Use Only. Not for Therapeutic or Diagnostic Use.
Contains:
• Methylcellulose in Iscove's MDM
• Fetal Bovine Serum
• Bovine Serum Albumin
• 2-Mercaptoethanol
• rh Stem Cell Factor
• rh GM-CSF
• rh IL-3
• rh Erythropoietin
• Supplements
Product Type: Specialized cell culture media
Application: Colony assays & quantification
Area of Interest: Cancer, Cord blood banking, Hematologic malignancies, Hematopoietic stem cell research, Stem cell biology, Transplantation
Cell Type: Hematopoietic stem & progenitor cells
Medium Type: Methylcellulose-based
Popular Product Line: MethoCult
Species: Human

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. Michele S Redell et al. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor.Blood 117 (21) 5701-5709 (May 26, 2011)
  2. Olena Dotsenko et al. Bone marrow resident and circulating progenitor cells in patients undergoing cardiac surgery.Ann Thorac Surg 90 (6) 1944-1951 (December 2010)
  3. Naoya Takayama et al. Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells.J Exp Med 207 (13) 2817-2830 (December 20, 2010)
  4. Sang-Wook Park et al. Efficient differentiation of human pluripotent stem cells into functional CD34+ progenitor cells by combined modulation of MEK/ERK and BMP4 signaling pathways.Blood (September 30, 2010)
  5. Jie Jiang et al. Crucial roles for protein kinase C isoforms in tumor-specific killing by apoptin.Cancer Res 70 (18) 7242-7252 (September 15, 2010)
  6. Vijay P S Rawat et al. The vent-like homeobox gene VENTX promotes human myeloid differentiation and is highly expressed in acute myeloid leukemia.Proc Natl Acad Sci U S A (September 10, 2010)
  7. Clinton Campbell et al. The human stem cell hierarchy is defined by a functional dependence on Mcl-1 for self-renewal capacity.Blood 116 (9) 1433-1442 (June 4, 2010)
  8. Judith E Karp et al. Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide.Blood 113 (20) 4841-4852 (May 14, 2009)
  9. Min Liang et al. Targeted transduction of CD34+ hematopoietic progenitor cells in nonpurified human mobilized peripheral blood mononuclear cells.J Gene Med 11 (3) 185-196 (March 2009)
  10. J Dierov et al. BCR/ABL induces chromosomal instability after genotoxic stress and alters the cell death threshold.Leukemia 23 (2) 279-286 (February 2009)
  11. Rachid Safi et al. Pharmacological manipulation of the RAR/RXR signaling pathway maintains the repopulating capacity of hematopoietic stem cells in culture.Mol Endocrinol 23 (2) 188-201 (February 2009)
  12. E Ortiz-Sanchez et al. Enhanced cytotoxicity of an anti-transferrin receptor IgG3-avidin fusion protein in combination with gambogic acid against human malignant hematopoietic cells: functional relevance of iron, the receptor, and reactive oxygen species.Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 23 (1) 59-70 (January 2009)
  13. Ellen Weisberg et al. Antileukemic effects of the novel, mutant FLT3 inhibitor NVP-AST487: effects on PKC412-sensitive and -resistant FLT3-expressing cells.Blood 112 (13) 5161-5170 (December 15, 2008)
  14. Anna van Rhenen et al. The novel AML stem cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells.Blood 110 (7) 2659-2666 (October 1, 2007)
  15. M Penicka et al. One-day kinetics of myocardial engraftment after intracoronary injection of bone marrow mononuclear cells in patients with acute and chronic myocardial infarction.Heart 93 (7) 837-841 (July 2007)
  16. Andrew C Boquest et al. CpG methylation profiles of endothelial cell-specific gene promoter regions in adipose tissue stem cells suggest limited differentiation potential toward the endothelial cell lineage.Stem Cells 25 (4) 852-861 (April 2007)
  17. Sandra Gottschling et al. Human mesenchymal stromal cells regulate initial self-renewing divisions of hematopoietic progenitor cells by a beta1-integrin-dependent mechanism.Stem Cells 25 (3) 798-806 (March 2007)
  18. Ellen Weisberg et al. Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias.Blood 109 (5) 2112-2120 (March 1, 2007)
  19. Rentian Feng et al. SDX-308, a nonsteroidal anti-inflammatory agent, inhibits NF-kappaB activity, resulting in strong inhibition of osteoclast formation/activity and multiple myeloma cell growth.Blood 109 (5) 2130-2138 (March 1, 2007)
  20. James Edwin Thompson et al. Enhanced growth of myelodysplastic colonies in hypoxic conditions.Exp Hematol 35 (1) 21-31 (January 2007)
  21. Yasuo Miura et al. Mesenchymal stem cell-organized bone marrow elements: an alternative hematopoietic progenitor resource.Stem Cells 24 (11) 2428-2436 (November 2006)
  22. Takahiro Suzuki et al. Highly efficient ex vivo expansion of human hematopoietic stem cells using Delta1-Fc chimeric protein.Stem Cells 24 (11) 2456-2465 (November 2006)
  23. Paola Secchiero et al. Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL).Blood 107 (10) 4122-4129 (May 15, 2006)
  24. Taina Jaatinen et al. Global gene expression profile of human cord blood-derived CD133+ cells.Stem Cells 24 (3) 631-641 (March 1, 2006)
  25. Patrick B Senatus et al. Restoration of p53 function for selective Fas-mediated apoptosis in human and rat glioma cells in vitro and in vivo by a p53 COOH-terminal peptide.Mol Cancer Ther 5 (1) 20-28 (January 2006)
  26. Ki-Ryang Koh et al. Immunomodulatory derivative of thalidomide (IMiD CC-4047) induces a shift in lineage commitment by suppressing erythropoiesis and promoting myelopoiesis.Blood 105 (10) 3833-3840 (May 15, 2005)
  27. Shanbao Cai et al. Mitochondrial targeting of human O6-methylguanine DNA methyltransferase protects against cell killing by chemotherapeutic alkylating agents.Cancer Res 65 (8) 3319-3327 (April 15, 2005)
  28. Karene Mahtouk et al. An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti-IL-6 antibody-induced apoptosis.Blood 103 (5) 1829-1837 (March 1, 2004)
  29. Dylan T Jones et al. Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs.Blood 103 (5) 1855-1861 (March 1, 2004)
  30. Hermann Eichler et al. Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration.Stem Cells 21 (2) 208-216 (2003)
  31. Eun-Sun Yoo et al. Adherent cells generated during long-term culture of human umbilical cord blood CD34+ cells have characteristics of endothelial cells and beneficial effect on cord blood ex vivo expansion.Stem Cells 21 (2) 228-235 (2003)
  32. Ilham Saleh Abuljadayel et al. Induction of stem cell-like plasticity in mononuclear cells derived from unmobilised adult human peripheral blood.Curr Med Res Opin 19 (5) 355-375 (2003)
  33. Steven P Zielske et al. In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.J Clin Invest 112 (10) 1561-1570 (November 2003)
  34. Chandini M Thirukkumaran et al. Reovirus oncolysis as a novel purging strategy for autologous stem cell transplantation.Blood 102 (1) 377-387 (July 1, 2003)
  35. Bernhard Schiedlmeier et al. High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation.Blood 101 (5) 1759-1768 (March 1, 2003)

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 #
3 cc Syringes 3 cc Syringes 28230
MethoCult® H4035 Optimum without EPO Methylcellulose-Based Medium with Recombinant Cytokines (without EPO) for Human Cells 04035
MethoCult® H4435 Enriched Methylcellulose Medium with Recombinant Cytokines and EPO for Human Cells 04435
MethoCult® H4034 Optimum Methylcellulose-Based Medium with Recombinant Cytokines and EPO for Human Cells 04034
MethoCult® H4535 Enriched without EPO Methylcellulose-Based Medium with Recombinant Cytokines (without EPO) for Human Cells 04535
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
MethoCult® H4534 Classic without EPO
Methylcellulose-Based Medium with Recombinant Cytokines (without EPO) for Human Cells
 04534
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
Ficoll-Paque™ PLUS Density Gradient Medium for the Isolation of Mononuclear Cells 07907
Ficoll-Paque™ PLUS 07917
Ficoll-Paque™ PREMIUM Density Gradient Medium for the Isolation of Mononuclear Cells 07908
Ficoll-Paque™ PREMIUM 07918
Ammonium Chloride Solution Reagent for the Lysis of Red Blood Cells 07800
Ammonium Chloride Solution 07850
Human Bone Marrow Proficiency Testing Program To Standardize and Reduce Variation in Colony Assays 00602
Frozen Cord Blood Proficiency Testing Program To Reduce Variation in Colony Assays 00608
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

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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