title: A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release.
abstract: Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.
authors.affiliation: Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University.
authors.forename: Unity
authors.initials: U
authors.lastname: Jeffery
authors.affiliation: MRC Centre for Inflammation Research, University of Edinburgh.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Gray
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University; dnlevine@iastate.edu.
authors.forename: Dana N
authors.initials: DN
authors.lastname: LeVine
mesh_terms: D000818:Animals; D001681:Biological Assay; D004285:Dogs; D065206:Extracellular Traps; D005453:Fluorescence; D009504:Neutrophils