111 Garry M. Walsh (ed.), Eosinophils: Methods and Protocols, Methods in Molecular Biology, vol. 1178, DOI 10.1007/978-1-4939-1016-8_11, © Springer Science+Business Media New York 2014 Chapter 11 Eosinophil Shape Change and Secretion Lian Willetts, Sergei I. Ochkur, Elizabeth A. Jacobsen, James J. Lee, and Paige Lacy Abstract The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experi- mental approach for comparing unstimulated cells with those that have been stimulated to undergo che- motaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an enzyme-linked immunosorbent assay technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation. Key words Chemotaxis, Platelet-activating factor, Eotaxin, Chemokine, Confocal microscopy, Secretion, Degranulation, Eosinophil peroxidase 1 Introduction Eosinophil shape change is an important cellular event that precedes the movement and transmigration of cells from the blood to tissue compartments in response to chemotactic stimuli [1–3]. Shape change occurs following chemokine receptor stimulation, which activates actin cytoskeleton remodeling through a plethora of actin-binding proteins [4]. Dynamic actin reorganization is a fundamental process in cell motility and involves the reversible transformation of soluble G-actin to filamentous F-actin [4]. The movement of actin throughout cells allows eosinophils to form leading edges, pseudopods, filopodia, lamellipodia, and uropods, and provides directionality and force to cellular migration. Many chemokines are capable of inducing migration in eosinophils, among which platelet-activating factor (PAF) and CCL11 (eotaxin-1) are among the most potent [5, 6]. Concurrently with