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入门套件为MethoCult™H4434经典

完整的造血CFU检测试剂盒
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产品号 #（选择产品）

产品号 #04464_C

完整的造血CFU检测试剂盒

产品组分包括

  • 入门套件MethoCult™H4434经典（目录#04464）
    • MethoCult™H4434经典，24 x 3 mL管（目录#04444）
    • Iscove改良Dulbecco培养基（IMDM），含2%胎牛血清，100 mL（目录#07700）
    • 氯化铵溶液，100 mL（目录#07800）
    • 35毫米培养皿，10个/包（目录#27100）
    • 60毫米网格计分盘，20个/包（目录#100-0085）
    • 钝头针，30支/袋（目录#28130）
    • 3cc注射器，30个/袋（目录#28230）
    • 殖民地地图集（目录#28700）
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产品说明书及文档
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概述

MethoCult™H4434 Classic入门试剂盒（MethoCult™GF H4434）推荐用于建立使用集落形成单位（CFU）测定评估人类造血祖细胞的程序的初始阶段的实验室。这些产品支持来自人骨髓、外周血、脐带血、白细胞分离产品和纯化的富集祖细胞的克隆造血祖细胞的生长。H4434是一种完全基于甲基纤维素的培养基，支持红细胞祖细胞（BFU-E和CFU-E）、粒细胞-巨噬细胞祖细胞（CFU-GM、CFU-G和CFU-M）和多潜能粒细胞、红细胞、巨噬细胞、巨核细胞祖细胞（CFU-GEMM）的生长。每个试剂盒包含教学材料，除了所有试剂和材料需要进行24次重复分析。

浏览我们的常见问题（FAQs）进行CFU化验和探索其作为细胞治疗工作流程一部分的效用.

Subtype
Semi-Solid Media, Specialized Media
 
Cell Type
Hematopoietic Stem and Progenitor Cells
 
Species
Human, Non-Human Primate
 
Application
Cell Culture, Colony Assay, Functional Assay
 
Brand
MethoCult
 
Area of Interest
Stem Cell Biology
 

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Data Figures

Procedure Summary for Hematopoietic CFU Assays

Figure 1. Procedure Summary for Hematopoietic CFU Assays

Examples of Colonies Derived from Human Hematopoietic Progenitors

Figure 2. Examples of Colonies Derived from Human Hematopoietic Progenitors

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type
Product Name
Catalog #
Lot #
Language
Document Type
Product Information Sheet 1
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Product Information Sheet 2
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Product Information Sheet 3
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
19A98311 or higher
Language
English
Document Type
Product Information Sheet 4
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Product Information Sheet 5
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Product Information Sheet 6
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Product Information Sheet 7
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Technical Manual
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
Starter Kit for MethoCult™ H4434 Classic
Catalog #
04464
Lot #
All
Language
English

Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Research Area
Workflow Stages

Resources and Publications

Educational Materials (4)

Hematopoietic Stem and Progenitor Cells - Products for Your Research
Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays
Brochure
MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays
Identification of Colonies Derived from Human Hematopoietic Progenitors
Wallchart
Identification of Colonies Derived from Human Hematopoietic Progenitors
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays
Mini Review
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays

Frequently Asked Questions

Why use semi-solid media?

Semi-solid media (methylcellulose-based MethoCult™ and collagen-based MegaCult™-C) allow the clonal progeny of a single progenitor cell to remain spatially isolated from other colonies within a culture, so they may be separately identified and counted.

Why use methylcellulose-based media?

Methylcellulose permits better growth of erythroid colonies than other types of semi-solid support systems (eg. agar) while allowing optimal myeloid colony formation. When appropriate cytokines are present, committed progenitor cells of both erythroid and granulocyte/macrophage lineages (CFU-GM, CFU-G, CFU-M) as well as multi-potential progenitor cells (CFU-GEMM), can be assayed simultaneously in the same culture dish.

Is it necessary to add antibiotics to the media?

No, aseptic technique should be sufficient to maintain sterile cultures. However, antibiotics (eg. Penicillin/Streptomycin) or anti-fungals (eg. Amphotericin B) may be added to the methylcellulose medium if desired.

Is there anything I can do if my cultures appear contaminated?

No, once contamination is visible, it is not possible to rescue the cultures by the addition of antibiotics. Bacteria and yeast inhibit colony formation by depleting nutrients or by releasing toxic substances.

Why can't I use a pipette to dispense methylcellulose-based media?

Methylcellulose is a viscous solution that cannot be accurately dispensed using a pipette due to adherence of the medium to the walls of the pipette tip. Blunt-End, 16 Gauge needles (Catalog #28110), in combination with 3 cc Syringes (Catalog #28230) are recommended for accurate dispensing of MethoCult™.

Can I 'pluck' the colonies for individual analysis?

Yes, colonies can be 'plucked' using a pipette with 200 µL sterile pipette tips or using a glass Pasteur pipette with an elongated tip. Individual colonies should be placed in a volume of 25 - 50 µL of medium, and diluted into suitable culture medium for further culture or analysis.

Why are low adherence dishes so important?

Adherent cells such as fibroblasts can cause inhibition of colony growth and obscure visualization of colonies.

Can MethoCult™ products be used for lymphoid progenitor CFU assays?

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 (Catalog #03630).

Is it possible to set up CFU assays in a 24-well plate?

Yes, as long as a plating concentration optimized for the smaller surface area of a well in a 24-well plate (1.9 cm2 as compared to ~9.5 cm2 for a 35 mm dish) is used for these assays. The number of replicate wells required to get an accurate estimation of CFU numbers may also need to be increased.

Can I stain colonies in MethoCult™ medium?

The cells in individual colonies in MethoCult™ can be stained, eg., for analysis of morphology or phenotype, after they are plucked from the dish and washed free of methylcellulose. Colonies grown in collagen-based MegaCult™-C medium can be used for immunohistochemical or enzymatic staining in situ after dehydration and fixation onto glass slides.

Are there differences in colony morphology with serum-free media?

Serum-containing media generally give better overall growth (colonies may appear larger) but there are no large differences in total colony numbers when CFU assays using serum-free media and serum-containing media are compared, provided that identical cytokines are present.

Can MethoCult™ be made with alternate base media?

Yes, this can be done as a 'custom' media order. Please contact techsupport@stemcell.com for more information.

Is there a MethoCult™ formulation suitable for HPP-CFC (high proliferative potential colony forming cell)?

Yes, MethoCult™ H4535 (Catalog #04535) can be used for the HPP-CFC assay as it does not contain EPO. The culture period is usually 28 days. It is not necessary to feed these cultures as growth factors in the medium are present in excess. As HPP-CFCs can be quite large, overplating can be a problem. It is recommended to plate cells at two or more different concentrations.

Publications (7)

Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6 Guye P et al. Nature Communications 2015 JAN

Abstract

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.
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High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells. Donahue RE et al. Blood 2000 JAN

Abstract

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)
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Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow. Gribaldo L et al. Experimental hematology 1999 NOV

Abstract

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs.
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Species Human, Non-Human Primate
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