title: EicosaCell: An Imaging-Based Assay to Identify Spatiotemporal Eicosanoid Synthesis.
abstract: Eicosanoids are bioactive lipids derived from enzymatic metabolism of arachidonic acid via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. These lipids are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. The particular interest to determine intracellular compartmentalization of eicosanoid-synthetic machinery has emerged as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. In this chapter, we discuss the EicosaCell protocol, an assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This chapter covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.
authors.affiliation: Laboratório de Inflamação, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. cbmelo@ufrj.biof.br.
authors.forename: Christianne
authors.initials: C
authors.lastname: Bandeira-Melo
authors.affiliation: Laboratório de Inflamação, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.|Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
authors.forename: Ligia Almeida
authors.initials: LA
authors.lastname: Paiva
authors.affiliation: Laboratório de Inflamação, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Natália R T
authors.initials: NRT
authors.lastname: Amorim
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Weller
authors.affiliation: Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil. pbozza@ioc.fiocruz.br.
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Bozza
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015777:Eicosanoids / Q000096:biosynthesis*; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted / Q000379:methods; D042541:Intracellular Space / Q000378:metabolism; D050356:Lipid Metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods; D061848:Optical Imaging; D010588:Phagosomes; D012984:Software; D013194:Staining and Labeling / Q000379:methods
chemical_list: D015777:Eicosanoids
keywords: Biosynthesis; Carbodiimide; Compartmentalization; EDAC; Eicosanoids; Leukotriene; Lipid droplets; Perinuclear; Phagosomes; Prostaglandin