MethoCult® H4001

Procedures and instruction manuals:

•  MANUAL: Human Colony-Forming Cell (CFC) Assays Using MethoCult™
•  PRODUCT INFORMATION SHEET - 04001, Lot# All:29527-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

MSDS:

•  04001-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. Giovanni F Crosta et al. Scoring CFU-GM colonies in vitro by data fusion: a first account.Exp Hematol 35 (1) 1-12 (January 2007)
2. A Pessina et al. Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.Toxicol Sci 75 (2) 355-367 (October 2003)
3. D A Volpe et al. Myeloid clonogenic assays for comparison of the in vitro toxicity of alkylating agents.Toxicol In Vitro 17 (3) 271-277 (June 2003)

Background References:

1. A Pessina et al. Application of the CFU-GM assay to predict acute drug-induced neutropenia: an international blind trial to validate a prediction model for the maximum tolerated dose (MTD) of myelosuppressive xenobiotics.Toxicol Sci 75 (2) 355-367 (October 2003)
2. A Pessina et al. Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.Toxicol In Vitro 15 (6) 729-740 (December 2001)

Procedure summary for hematopoietic CFC Assays