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MethoCult™SF H4636

含重组细胞因子的无血清甲基纤维素培养基，用于人胚胎干细胞和iPS细胞来源的细胞
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产品号 #04636_C

含重组细胞因子的无血清甲基纤维素培养基，用于人胚胎干细胞和iPS细胞来源的细胞

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概述

MethoCult™SF H4636被推荐用于在无血清条件下培养人胚胎干细胞（ES）和诱导多能干细胞（iPS）衍生的造血祖细胞。MethoCult™SF H4636用于支持红细胞祖细胞（CFU-E和BFU-E）、粒细胞-巨噬细胞祖细胞（CFU-GM、CFU-G、CFU-M）和多能祖细胞(CFU-GEMM；粒细胞、红细胞、巨噬细胞、巨核细胞)。

MethoCult™SF H4636也被推荐用于单个核细胞、CD34+富集细胞和通过其他纯化方法从人骨髓（BM）、动员外周血（MPB）、外周血（PB）和脐带血（CB）样本中分离的细胞的CFU检测。

浏览我们的常见问题（FAQs）在执行CFU化验，并探讨其效用的一部分细胞治疗工作流程.

Contains
• Methylcellulose in Iscove's IMDM
• Bovine serum albumin
• 2-Mercaptoethanol
• Recombinant human (rh) insulin
• Human transferrin (iron-saturated)
• Supplements
• rh stem cell factor (SCF)
• rh interleukin 3 (IL-3)
• rh granulocyte colony-stimulating factor (G-CSF)
• rh granulocyte-macrophage colony-stimulating factor (GM-CSF)
 
Subtype
Semi-Solid Media, Specialized Media
 
Cell Type
Hematopoietic Stem and Progenitor Cells
 
Species
Human
 
Application
Cell Culture, Colony Assay, Functional Assay
 
Brand
MethoCult
 
Area of Interest
Stem Cell Biology
 
Formulation Category
Serum-Free
 

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Data Figures

Procedure Summary for Hematopoietic CFU Assays

Figure 1. Procedure Summary for Hematopoietic CFU Assays

Figure 2. Total Colony Numbers of CD34+ Cells Grown in MethoCult™ SF H4436 and MethoCult™ SF H4636

CD34+ and mononuclear cells derived from CB, mPB and BM were isolated and plated in MethoCult™ media and counted after 14 days. Shown are the number of total colonies grown in serum-free MethoCult™ SF H4436 (Catalog #04436) and MethoCult™ SF H4636 (Catalog #04636) normalized relative to total colonies grown in serum-containing MethoCult™ H4435 (Catalog #04435). MethoCult™ SF H4636 provides improved conditions for colony growth in serum-free conditions as compared to MethoCult™ SF H4436. Shown are means with standard deviation (n = 6).

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
Product Name
MethoCult™ SF H4636
Catalog #
04636
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
MethoCult™ SF H4636
Catalog #
04636
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 (10)

MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays
Brochure
MethoCult™ Media for Performing Hematopoietic Colony-Forming Unit (CFU) Assays
Atlas of Hematopoietic Colonies From Cord Blood
Brochure
Atlas of Hematopoietic Colonies From Cord Blood
Atlas of Human Hematopoietic Colonies
Brochure
Atlas of Human Hematopoietic Colonies
STEMdiff™ Hematopoietic Kit
Brochure
STEMdiff™ Hematopoietic Kit
Hematopoietic Stem and Progenitor Cells - Products for Your Research
Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
How to Prepare and Plate Semi-Solid Medium
1:52
Video
How to Prepare and Plate Semi-Solid Medium
Generating Gene-Edited CD34⁺ Cells From Urine‑Derived iPSCs
45:13
Webinar
Generating Gene-Edited CD34⁺ Cells From Urine‑Derived iPSCs
Lost in Translation - Moving Your Research to Clinical Trials
59:01
Webinar
Lost in Translation - Moving Your Research to Clinical Trials
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays
Mini Review
Hematopoietic Stem and Progenitor Cells (HSPCs): Isolation, Culture, and Assays
The STEMdiff™ Hematopoietic Kit Reproducibly Generates Functional Hematopoietic Progenitor Cells from Human Pluripotent Stem Cells
Scientific Poster
The STEMdiff™ Hematopoietic Kit Reproducibly Generates Functional Hematopoietic Progenitor Cells f...

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 (1)

Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs with Peripheral Blood Derivatives. X. Cao et al. Stem cell reports 2019 jun

Abstract

A renewable source of human monocytes and macrophages would be a valuable alternative to primary cells from peripheral blood (PB) in biomedical research. We developed an efficient protocol to derive monocytes and macrophages from human induced pluripotent stem cells (hiPSCs) and performed a functional comparison with PB-derived cells. hiPSC-derived monocytes were functional after cryopreservation and exhibited gene expression profiles comparable with PB-derived monocytes. Notably, hiPSC-derived monocytes were more activated with greater adhesion to endothelial cells under physiological flow. hiPSC-derived monocytes were successfully polarized to M1 and M2 macrophage subtypes, which showed similar pan- and subtype-specific gene and surface protein expression and cytokine secretion to PB-derived macrophages. hiPSC-derived macrophages exhibited higher endocytosis and efferocytosis and similar bacterial and tumor cell phagocytosis to PB-derived macrophages. In summary, we developed a robust protocol to generate hiPSC monocytes and macrophages from independent hiPSC lines that showed aspects of functional maturity comparable with those from PB.
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Species Human
Contains • Methylcellulose in Iscove's IMDM • Bovine serum albumin • 2-Mercaptoethanol • Recombinant human (rh) insulin • Human transferrin (iron-saturated) • Supplements • rh stem cell factor (SCF) • rh interleukin 3 (IL-3) • rh granulocyte colony-stimulating fac
Formulation Category Serum-Free
PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED. FOR ADDITIONAL INFORMATION ON QUALITY AT STEMCELL, REFER TO WWW.STEMCELL.COM/COMPLIANCE.
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