Basic techniques to study FcεRI signaling in mast cells

Abstract

Mast cells are the crucial effector cells for allergic reactions. They are activated through the aggregation of the high-affi nity IgE receptor (FcεRI) with allergen and allergen-specifi c IgE. Tyrosine phosphorylation of FcεRI subunits and various signaling proteins is an initial triggering event, leading to the activation of several signaling pathways in mast cells. Much has been learned from analysis of mast cells derived from gene-targeted mice. Therefore, in this chapter we will fi rst describe how to generate mast cells from mouse bone marrow cells and how to correct the genetic defect by retroviral transduction. Then we will describe how to assess early activation events by measuring several protein-tyrosine kinases (PTKs) and serine/ threonine kinases (PS/TKs) such as Akt (protein kinase B), protein kinase C (PKC), and JNK. As signal transduction is highly dependent on protein-protein interactions, we will describe experimental details of co-immunoprecipitation methods that are used to confi rm such interactions.