MethoCult® M3630

Procedures and instruction manuals:

•  MANUAL: Mouse Colony-Forming Cell Assays Using MethoCult®
•  PRODUCT INFORMATION SHEET - 03630, Lot# All:29512-PIS_1_1_2.pdf

Educational resources:

•  BROCHURE: The World Leader in Tools For Hematopoietic Stem Cell Research
•  TECH BULLETIN: MethoCult® formulation optimized for mouse BFU-E detection (Catalog #29953)
•  WALLCHART: Mouse Hematopoietic Progenitors Wallchart
•  VIDEO: Procedure for Setting Up the CFC Assay

MSDS:

•  04050-MSDS.pdf
•  03630-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. Kazuko Miyazaki et al. Enhanced expression of p210BCR/ABL and aberrant expression of Zfp423/ZNF423 induce blast crisis of chronic myelogenous leukemia.Blood 113 (19) 4702-4710 (May 7, 2009)
2. Michael G Kharas et al. Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells.J Clin Invest 118 (9) 3038-3050 (September 2008)
3. Samantha L Finstad et al. Diminished potential for B-lymphoid differentiation after murine leukemia virus infection in vivo and in EML hematopoietic progenitor cells.J Virol 81 (13) 7274-7279 (July 2007)
4. Hongjie Li et al. Ewing sarcoma gene EWS is essential for meiosis and B lymphocyte development.J Clin Invest 117 (5) 1314-1323 (May 2007)
5. Annie Bourdeau et al. TC-PTP-deficient bone marrow stromal cells fail to support normal B lymphopoiesis due to abnormal secretion of interferon-{gamma}.Blood 109 (10) 4220-4228 (May 15, 2007)
6. Michael G Kharas et al. KLF4 suppresses transformation of pre-B cells by ABL oncogenes.Blood 109 (2) 747-755 (January 15, 2007)
7. Wei-Chun Chou et al. STAT3 positively regulates an early step in B-cell development.Blood 108 (9) 3005-3011 (November 1, 2006)
8. Michael J Nemeth et al. Hmgb3 regulates the balance between hematopoietic stem cell self-renewal and differentiation.Proc Natl Acad Sci U S A 103 (37) 13783-13788 (September 12, 2006)
9. Weili Chen et al. A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy.Blood 108 (2) 669-677 (July 15, 2006)
10. Mathijs Baens et al. Selective expansion of marginal zone B cells in Emicro-API2-MALT1 mice is linked to enhanced IkappaB kinase gamma polyubiquitination.Cancer Res 66 (10) 5270-5277 (May 15, 2006)
11. Cheng Cheng Zhang et al. Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal.Proc Natl Acad Sci U S A 103 (7) 2184-2189 (February 14, 2006)
12. P Louise Coletta et al. Lymphodepletion in the ApcMin/+ mouse model of intestinal tumorigenesis.Blood 103 (3) 1050-1058 (February 1, 2004)
13. Nicolas Pineault et al. Induction of acute myeloid leukemia in mice by the human leukemia-specific fusion gene NUP98-HOXD13 in concert with Meis1.Blood 101 (11) 4529-4538 (June 1, 2003)
14. Toni Portis et al. Epstein-Barr virus LMP2A interferes with global transcription factor regulation when expressed during B-lymphocyte development.J Virol 77 (1) 105-114 (January 2003)
15. S Teglund et al. Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.Cell 93 (5) 841-850 (May 29, 1998)

Background References:

1. M R Hough et al. Reduction of early B lymphocyte precursors in transgenic mice overexpressing the murine heat-stable antigen.J Immunol 156 (2) 479-488 (January 15, 1996)
2. M E Lemieux et al. Characterization and purification of a primitive hematopoietic cell type in adult mouse marrow capable of lymphomyeloid differentiation in long-term marrow "switch" cultures.Blood 86 (4) 1339-1347 (August 15, 1995)

Procedure summary for hematopoietic CFC Assays