MethoCult® Express

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Methylcellulose Medium for 7-Day Colony-Forming Cell Assays of Human Hematopoietic Cells

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  • MethoCult® Express 100 mL
  • Label for MethoCult® Express 100 mL
  • Tube label for MethoCult® Express 3 mL
MethoCult® Express 100 mL
MethoCult® Express is recommended for the detection and quantification of human hematopoietic progenitors in cord blood (CB) samples using CFC assays after a minimum culture period of seven days. It is recommended for red blood cell (RBC) depleted CB samples, whole CB samples that have been cryopreserved and thawed, and CB mononuclear cells.
 
MethoCult® Express is optimized for the detection and counting of human hematopoietic progenitor cells after much shorter periods than the 14 - 16 days of standard CFC assays. In MethoCult® Express, colonies containing at least 20 cells can be counted as early as after seven days of culture. At this time, most colonies are immature and have not yet differentiated into morphologically distinguishable colony types. Therefore the colonies counted after seven days of culture give information about the total frequency of hematopoietic progenitor cells present in the sample without distinction between different progenitor types.
If MethoCult® Express cultures are maintained for 14 - 16 days, colonies derived from erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (CFU-GM, CFU-M, CFU-G), and multi-potential granulocyte, erythroid, macrophage and megakaryocyte progenitors (CFU-GEMM) can be counted.
Product Name Description Catalog # Size Price Quantity
MethoCult™ Express Pre-aliquoted methylcellulose medium for 7-day colony-forming cell assays of human hematopoietic cells 04447 24 x 3 mL 809.00 USD      
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MethoCult™ Express Methylcellulose medium for 7-day colony-forming cell assays of human hematopoietic cells 04437 100 mL 610.00 USD      
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Recommended for:
Detection of human clonogenic hematopoietic progenitor cells from cord blood using CFC assays after a minimum culture period of seven days
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
• Cytokines, including erythropoietin (EPO)
• Supplements
Product Type: Specialized cell culture media
Application: Colony assays & quantification
Area of Interest: Cord blood banking, 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.

Background References:

  1. Vinod Prasad et al. Unrelated donor umbilical cord blood transplantation for inherited metabolic disorders in 159 pediatric patients from a single center: influence of cellular composition of the graft on transplantation outcomes.Blood 112 (7) 2979-2989 (June 27, 2008)
  2. K H Yoo et al. The impact of post-thaw colony-forming units-granulocyte/macrophage on engraftment following unrelated cord blood transplantation in pediatric recipients.Bone Marrow Transplant 39 (9) 515-521 (May 2007)
  3. H Yang et al. Association of post-thaw viable CD34+ cells and CFU-GM with time to hematopoietic engraftment.Bone Marrow Transplant 35 (9) 881-887 (May 2005)
  4. Luciano Rodriguez et al. Predictive utility of the attached segment in the quality control of a cord blood graft.Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation 11 (4) 247-251 (April 2005)
  5. A P Iori et al. Pre-transplant prognostic factors for patients with high-risk leukemia undergoing an unrelated cord blood transplantation.Bone Marrow Transplant 33 (11) 1097-1105 (June 2004)
  6. A R Migliaccio et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity.Blood 96 (8) 2717-2722 (October 15, 2000)
  7. D E Hogge et al. Quantitation of primitive and lineage-committed progenitors in mobilized peripheral blood for prediction of platelet recovery post autologous transplant.Bone Marrow Transplant 25 (6) 589-598 (March 2000)
  8. 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)
  9. 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)

Product Name

Description

Catalog #
HetaSep™ For Isolating Human Nucleated Cells from Peripheral Blood 07806
Ficoll-Paque™ PLUS Density Gradient Medium for the Isolation of Mononuclear Cells 07907
Ficoll-Paque™ PREMIUM Density Gradient Medium for the Isolation of Mononuclear Cells 07908
Ammonium Chloride Solution Reagent for the Lysis of Red Blood Cells 07800

Colonies in MethoCult® Express


Colonies in MethoCult® Express
Colonies derived from cord blood progenitors in MethoCult® Express at (A) day 7 and (B) day 14 with a 2X objective lens; at (C) day 7 and (D) day 14 with a 4X objective lens


Correlation between colony numbers after Day 7 with MethoCult® Express and Day 14 with MethoCult® H4034 Optimum (Catalog #04034/04044) using cryopreserved whole cord blood


Correlation between colony numbers after Day 7 with MethoCult® Express and Day 14 with MethoCult® H4034 Optimum (Catalog #04034/04044) using cryopreserved whole cord blood
Cryopreserved whole cord blood samples (n = 5) were thawed and set up in MethoCult® Express and MethoCult® GF H4034 without further processing. Colonies in MethoCult® Express were counted after 7 days and colonies in MethoCult® GF H4034 were counted after 14 days. Correlation coefficient: 0.973; p = 0.005 (Pearson Product Moment Correlation).


Correlation between colony numbers after Day 7 with MethoCult® Express and Day 14 with MethoCult® H4034 Optimum (Catalog #04034/04044).


Correlation between colony numbers after Day 7 with MethoCult® Express and Day 14 with MethoCult® H4034 Optimum (Catalog #04034/04044).
Samples included cryopreserved whole cord blood (5), cryopreserved cord blood with red blood cells depleted by HetaSep™ (3), fresh cord blood with red blood cells depleted by HetaSep™ (3) and cryopreserved cord blood mononuclear cells isolated by density sedimentation over Ficoll-Paque™ PLUS (3). Samples (n = 14) were set up in MethoCult® Express and MethoCult® GF H4034 and colonies were counted after 7 and 14 days, respectively. Correlation coefficient: 0.997; p <0.0001 (pearson="" product="" moment="">


Correlation between colony numbers after Day 7 and Day 14 with MethoCult® Express


Correlation between colony numbers after Day 7 and Day 14 with MethoCult® Express
Samples included cryopreserved whole cord blood samples (5), cryopreserved cord blood with red blood cells depleted by HetaSep™ (3), fresh cord blood with red blood cells depleted by HetaSep™ (3), cryopreserved cord blood mononuclear cells isolated by density sedimentation over Ficoll-Paque™ PLUS (11) and fresh cord blood mononuclear cells isolated by density sedimentation over Ficoll-Paque™ PLUS (2). Samples (n = 24) were set up in MethoCult® Express and colonies were counted after 7 and 14 days. Correlation coefficient: 0.991; p <0.0001 (pearson="" product="" moment="">


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