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Basophil Stimulation and Signaling Pathways

  • Edward F. KnolEmail author
  • Bernhard F. GibbsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1192)

Abstract

Despite growing use of flow cytometry to analyze the functional characteristics of primary basophils the intracellular signaling cascades that control their ability to elaborate various inflammatory mediators and cytokines remain comparatively obscure. Additionally, some studies require the analysis of pro-allergic and inflammatory mediators, such as histamine, LTC4, and various basophil-derived cytokines (e.g., IL-4 and IL-13). Elucidation of intracellular signaling proteins by Western blotting, cytosolic free calcium concentration by spectrofluorophotometry, and detection of mediator releases, as well as analysis of gene expressions by RT-PCR, generally require relatively large numbers of purified basophils. In selected assays, flow cytometry can enable the analysis of relatively low cell numbers and purity for the expression of intracellular signaling proteins or measurement of cytosolic free calcium concentrations by basophil-specific gating strategies. Unfortunately, many aspects of signal transduction relevant to human basophils cannot be readily extrapolated from the use of basophil or mast cell lines. This chapter therefore focuses on how to employ primary human basophils for studying mediator releases and signaling characteristics.

Key words

Basophils Signaling IgE receptors Western blotting Flow cytometry Inhibitors 

Notes

Acknowledgements

This work was supported by the COST Action BM1007 Mast Cells and Basophils—Targets for Innovative Therapies.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department ImmunologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  2. 2.Department Dermatology/AllergologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  3. 3.Medway School of PharmacyUniversity of KentKentUK

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