147 Bernhard F. Gibbs and Franco H. Falcone (eds.), Basophils and Mast Cells: Methods and Protocols, Methods in Molecular Biology, vol. 1192, DOI 10.1007/978-1-4939-1173-8_11, © Springer Science+Business Media New York 2014 Chapter 11 Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques Chris H. Bridts, Vito Sabato, Christel Mertens, Margo M. Hagendorens, Luc S. De Clerck, and Didier G. Ebo Abstract The basis of flow cytometric allergy diagnosis is quantification of changes in expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332–339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also up-regulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201–210, 2008). Here, we describe two flow cytometry-based protocols which allow detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation. Key words Basophil activation tests, CD203c, CD63, HistaFlow 1 Introduction Basophil activation assays have predominantly focused on histamine and leukotriene release assays. However, time-consuming proce- dures were needed to isolate cells, to incubate with activators, and to quantify (sometimes labile) mediators. Two decades ago, the dis- covery of the markers CD63 (a.k.a. gp53) and subsequently CD203c (a.k.a. ENPP-3) facilitated the development of a novel technique to analyze and quantify allergen-specific in vitro activa- tion of basophils: the so-called basophil activation test (BAT) [2]. Briefly, the BAT relies on a flow cytometric identification and quantification of various specific activation markers on the surface membrane or activated proteins inside the cells. These changes can be detected and quantified on a single-cell level using specific monoclonal antibodies coupled to fluorochromes. Practically, baso- phils are identified by specific markers such as CCR3/CD3 [3], CD123/HLA-DR [4], or IgE/CD203c [5]. Of these markers, only CD203c is lineage specific. Subsequently, after activation with