Quantification of Neutrophils Undergoing NET Formation and Distinguishing Mechanisms of Neutrophil Cell Death by Use of a High-Throughput Method.

Neutrophils, the most abundant white blood cell type in humans, play a crucial role in innate host defenses. Recent studies have revealed additional key roles in the pathogenesis of cancer and autoimmune diseases through multiple mechanisms including the formation of neutrophil extracellular traps (NETs). Further research to expand the understanding of neutrophils' role in health and diseases is limited by lack of techniques to quantify neutrophils undergoing NET formation in an objective, reproducible, and efficient manner. In this chapter, we describe an automated high-throughput method to quantify NETting neutrophils in real time using a two-color, live-content imaging platform, the IncuCyte™S3 (Essen BioScience, Inc.) system, coupled to membrane integrity-dependent dual-dye approach to image intracellular and extracellular DNA. Based on characteristic differences in nuclear morphology and membrane integrity, this method may also be used to distinguish between different types of neutrophil cell death. This platform can help to assess neutrophil physiology and to develop and test therapeutic targets.