Induction and Quantification of NETosis

Neutrophil extracellular traps (NET) are web-like structures composed of nuclear material and neutrophil granular proteins that are released to the extracellular space after specific microbial or sterile inflammatory stimuli. NETosis is a specific type of cell death that ensues when neutrophils extrude NETs. It is important to develop validated standards to induce and quantify NETs in order to accurately compare results between laboratories and advance the understanding of this biological process and its implications in health and disease. This unit presents methods for isolating human neutrophils from peripheral blood and inducing NETs through various types of stimulation that rely on the production of reactive oxygen species from NADPH oxidase or mitochondria. Alternative methods are also described for low- and high-throughput quantification of NETs. © 2016 by John Wiley & Sons, Inc.