MethoCult® H4034 Optimum

Procedures and instruction manuals:

•  MANUAL: Human Colony-Forming Cell (CFC) Assays Using MethoCult™
•  PRODUCT INFORMATION SHEET - 04044, Lot# All:29529-PIS_1_2_0.pdf
•  PRODUCT INFORMATION SHEET - 04034, Lot# All:29529-PIS_1_2_0.pdf

Educational resources:

•  BROCHURE: The World Leader in Tools For Hematopoietic Stem Cell Research
•  BROCHURE: Cell Separation and Culture Tools for Quantitative Analysis of JAK2 Mutationals in MPD
•  BROCHURE: MethoCult® for Hematopoietic Colony-Forming Cell Assays
•  WALLCHART: Hematopoietic Colonies Derived from Human Cord Blood Progenitors Wallchart
•  SCIENTIFIC POSTER: Automation of Colony-Forming Cell Assays for Measuring Hematopoietic Progenitors in Cord Blood
•  SCIENTIFIC POSTER: A Rapid Automated Colony-Forming Cell Assay for Determination of Hematopoietic Progenitors in Cord Blood
•  BROCHURE: STEMvision™ Automated and Standardized CFC Enumeration
•  VIDEO: Procedure for Setting Up the CFC Assay
•  BROCHURE: MethoCult™ Procedure
•  TECH BULLETIN: HetaSep™ Protocol
•  TECH BULLETIN: The CFC Assay For Cord Blood

MSDS:

•  04034_04044-MSDS.pdf

FAQS:

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  • 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. Etsuko Sekimoto et al. A single-chain Fv diabody against human leukocyte antigen-A molecules specifically induces myeloma cell death in the bone marrow environment.Cancer Res 67 (3) 1184-1192 (February 1, 2007)
2. Yukari Muguruma et al. Reconstitution of the functional human hematopoietic microenvironment derived from human mesenchymal stem cells in the murine bone marrow compartment.Blood 107 (5) 1878-1887 (March 1, 2006)
3. Masayoshi Kobune et al. Indian hedgehog gene transfer augments hematopoietic support of human stromal cells including NOD/SCID-beta2m-/- repopulating cells.Blood 104 (4) 1002-1009 (August 15, 2004)
4. 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)
5. Yutaka Kawano et al. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells.Blood 101 (2) 532-540 (January 15, 2003)
6. Tetsuro Tamaki et al. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle.J Cell Biol 157 (4) 571-577 (May 13, 2002)
7. Y Shimakura et al. Murine stromal cell line HESS-5 maintains reconstituting ability of Ex vivo-generated hematopoietic stem cells from human bone marrow and cytokine-mobilized peripheral blood.Stem Cells 18 (3) 183-189 (2000)
8. K Matsumoto et al. In vitro proliferation potential of AC133 positive cells in peripheral blood.Stem Cells 18 (3) 196-203 (2000)
9. N Niitsu et al. Antileukemic efficacy of 2-deoxycoformycin in monocytic leukemia cells.Blood 96 (4) 1512-1516 (August 15, 2000)
10. A Limon et al. High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.Blood 90 (9) 3316-3321 (November 1, 1997)

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)

Procedure summary for hematopoietic CFC Assays

Examples of colonies derived from CFU-GM in MethoCult® H4034 Optimum

Examples of colonies derived from BFU-E in MethoCult® H4034 Optimum