MethoCult™ H4100

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.

Procedures and instruction manuals:

•  MANUAL: Human Colony-Forming Cell (CFC) Assays Using MethoCult™
•  PRODUCT INFORMATION SHEET - 04100, Lot# All:29532-PIS_1_1_0.pdf

Educational resources:

•  BROCHURE: The World Leader in Tools For Hematopoietic Stem Cell Research
•  VIDEO: Procedure for Setting Up the CFC Assay
•  WALLCHART: Choose Your MethoCult™ For Human CFU Assays Wallchart

MSDS:

•  04100-MSDS.pdf

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	60037

Procedure summary for hematopoietic CFC Assays

Examples of colonies derived from human hematopoietic progenitors