Abstract
We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the ε-sterile transcript synthesis by polymerase chain reaction or Northern blot, the ε excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.
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Acknowledgments
We are grateful to Dr. Honjo, Osaka University Medical School, Japan, for the kind gift of the IgE cDNA. Our work is supported by grants from the Canadian Institute of Health Research and the Multiple Sclerosis Scientific Research Foundation. J.F.G. is a Canada Research Chair recipient.
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Pène, J. et al. (2006). Detection of ε Class Switching and IgE Synthesis in Human B Cells. In: Krishnaswamy, G., Chi, D.S. (eds) Mast Cells. Methods in Molecular Biology, vol 315. Humana Press. https://doi.org/10.1385/1-59259-967-2:319
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DOI: https://doi.org/10.1385/1-59259-967-2:319
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