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Eosinophils pp 53-70 | Cite as

Cell Signalling During Human Eosinophil Differentiation

  • Miranda BuitenhuisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1178)

Abstract

Eosinophil differentiation is a complex series of events regulated by cytokines at multiple levels, including proliferation, survival, and maturation. The development of an ex vivo eosinophil differentiation model, using the current knowledge on factors involved in this process, has facilitated efforts to understand the molecular mechanisms underlying human eosinophil development. Differentiation of human hematopoietic progenitor cells, isolated by density centrifugation and immunomagnetic cell separation, towards mature eosinophils, involves a 17-day culture period in the presence of a mixture of cytokines. At early stages of differentiation, these cells can be retrovirally transduced resulting in modulation of the expression of genes of interest to examine their role in eosinophil development. Eosinophil maturation can be analyzed by combining three different methods: histochemical analysis, flow cytometric analysis, and Luxol Fast Blue staining. In addition to this ex vivo differentiation model, human hematopoietic progenitors can be transplanted into immune-deficient mice resulting in the development of all human hematopoietic lineages in the mouse bone marrow, including eosinophils. Although the ex vivo differentiation model can be used separately, combining it with the transplantation model will give insight into not only regulation of human eosinophil development but also hematopoiesis in general.

Key words

Human eosinophil differentiation Hematopoietic progenitors CD34 Retroviral transduction Flow cytometric analysis β2-Microglobulin (−/−) NOD/SCID mice Histochemical analysis 

Notes

Acknowledgements

The author thanks the current and past members of the departments of Respiratory Medicine and Immunology at the University Medical Center Utrecht (the Netherlands), the Department of Hematology at the Erasmus MC (the Netherlands), and the Hematopoietic Stem Cell Laboratory at the Lund Strategic Research Center for Stem Cell Biology and Cell Therapy in Sweden for helping develop these protocols.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  1. 1.Department of HematologyErasmus MCRotterdamThe Netherlands

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