Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health (NIH), Bethesda, MD, USA.
Methods Mol Biol. 2022;2543:129-140. doi: 10.1007/978-1-0716-2553-8_11.
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.
中性粒细胞是人类血液中最丰富的白细胞类型，在先天宿主防御中发挥着关键作用。最近的研究揭示了通过多种机制（包括中性粒细胞胞外陷阱（NETs）的形成）在癌症和自身免疫性疾病发病机制中的额外关键作用。进一步研究以扩大对中性粒细胞在健康和疾病中的作用的理解受到限制，因为缺乏技术来客观、可重复和有效地定量经历 NET 形成的中性粒细胞。在本章中，我们描述了一种使用双色、实时内容成像平台（Incucyte™S3（Essen BioScience，Inc.）系统）实时定量 NETting 中性粒细胞的自动化高通量方法，该系统与基于膜完整性的双染料方法相结合，用于成像细胞内和细胞外 DNA。基于核形态和膜完整性的特征差异，该方法也可用于区分不同类型的中性粒细胞死亡。该平台有助于评估中性粒细胞生理学，并开发和测试治疗靶点。
