MegaCult™-C Complete Kit Without Cytokines

Complete kit for human CFU-Mk assays
MegaCult™-C Complete Kit Without Cytokines

Complete kit including collagen, media, staining kit and chamber slide kit for human CFU-Mk assays

1 Kit
Catalog # 04970
5,451 USD
MegaCult™-C Medium Without Cytokines

Medium without cytokines

24 x 1.7 mL
Catalog # 04900
1,152 USD
Collagen Solution

For preparation of collagen gels and for coating cell culture surfaces

35 mL
Catalog # 04902
519 USD
MegaCult™-C Staining Kit for CFU-Mk

Staining kit for human CFU-Mk assays

1 Kit
Catalog # 04962
2,520 USD
Double Chamber Slide Kit

For the culture, fixation and staining of collagen-based cultures

1 Kit
Catalog # 04963
1,517 USD

Overview

MegaCult™-C Complete Kit Without Cytokines includes all reagents (except cytokines) required for colony-forming unit (CFU) assays of megakaryocyte progenitor cells (CFU-Mk) in a collagen-based culture medium, including the detection of human CFU-Mk colonies by immunocytochemical staining. The addition of appropriate cytokines, as chosen by the user, is required.
Components
  • MegaCult™-C Complete Kit Without Cytokines (Catalog #04970)
    • Collagen Solution, 35 mL (Catalog #04902)
    • MegaCult™-C Medium Without Cytokines, 24 x 1.7 mL (Catalog #04900)
    • Double Chamber Slide Kit (Catalog #04963)
    • MegaCult™-C Staining Kit for CFU-Mk (Catalog #04962)
Subtype
Semi-Solid Media, Specialized Media
Cell Type
Hematopoietic Stem and Progenitor Cells
Species
Human
Application
Cell Culture, Colony Assay, Functional Assay
Brand
MegaCult
Area of Interest
Stem Cell Biology
Formulation
Serum-Free

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
MegaCult™-C Medium Without Cytokines
Catalog #
04900
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
Collagen Solution
Catalog #
04902
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
MegaCult™-C Staining Kit for CFU-Mk
Catalog #
04962
Lot #
All
Language
English
Document Type
Product Information Sheet
Product Name
Double Chamber Slide Kit
Catalog #
04963
Lot #
All
Language
English
Document Type
Manual
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 6
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 7
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 8
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 9
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet 10
Product Name
MegaCult™-C Complete Kit Without Cytokines
Catalog #
04970
Lot #
All
Language
English
Document Type
Safety Data Sheet
Product Name
Collagen Solution
Catalog #
04902
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
MegaCult™-C Staining Kit for CFU-Mk
Catalog #
04962
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
MegaCult™-C Staining Kit for CFU-Mk
Catalog #
04962
Lot #
All
Language
English

Educational Materials (2)

Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
Scientific Poster
Detection of Human Bipotential Erythroid-Megakaryocytic Progenitors in Serum-Free Collagen Gels

Frequently Asked Question

Why is the MegaCult™-C formulation serum free?

MegaCult™-C is formulated without FBS to avoid inhibition of CFU-Mk growth by TGF beta and Platelet Factor-4, which are often present in the serum.

Why use semi-solid media?

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.
Read More

Product 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.

Data and Publications

Publications (27)

Blood 2009 MAR Role for MKL1 in megakaryocytic maturation. Cheng E-C et al.

Abstract

Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34(+) cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.
Toxicology in vitro : an international journal published in association with BIBRA 2009 FEB Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs. Pessina A et al.

Abstract

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.
Blood 2009 FEB Incomplete restoration of Mpl expression in the mpl-/- mouse produces partial correction of the stem cell-repopulating defect and paradoxical thrombocytosis. Lannutti BJ et al.

Abstract

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors, where it initiates critical cell survival and proliferation signals after stimulation by its ligand, thrombopoietin (TPO). As a result, a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore, they show very low-level expression of Mpl on platelets and megakaryocytes, and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However, impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels, which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis.
Blood 2009 APR Mef2C is a lineage-restricted target of Scl/Tal1 and regulates megakaryopoiesis and B-cell homeostasis. Gekas C et al.

Abstract

The basic helix-loop-helix transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Scl(fl/fl) fetal liver progenitor cell lines. Analysis of Mef2C(-/-) embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre(+)Mef2C(fl/fl) mice exhibited platelet defects similar to those observed in Scl-deficient mice. The platelet counts were reduced, whereas platelet size was increased and the platelet shape and granularity were altered. Furthermore, megakaryopoiesis was severely impaired in vitro. Chromatin immunoprecipitation microarray hybridization analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl-independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B-cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages.
Blood 2008 MAY Transgenic expression of JAK2V617F causes myeloproliferative disorders in mice. Xing S et al.

Abstract

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice, which expressed a lower level of JAK2(V617F), showed moderate elevations of blood cell counts, whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid, megakaryocytic, and granulocytic hyperplasia in the bone marrow and spleen, displayed splenomegaly, and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood, spleen, and bone marrow, and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs.
Journal of immunological methods 2008 MAR Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system. Boyer L et al.

Abstract

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end, we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells, and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo, while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence, we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase, the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings, two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly, these cocktails were either deprived of thrombopoietin or of stem cell factor, two cytokines known to favor megakaryopoiesis and HPC expansion, respectively. Based on these results, a short resource-efficient two-phase culture protocol for the production of Mks near purity (textgreater95%) from human CD34+ CB cells has been established.
View All Publications

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