Laboratory of Immunophysiology, GIGA Institute, Liège University, Liège, Belgium.
Faculty of Veterinary Medicine, Liège University, Liège, Belgium.
Methods Mol Biol. 2022;2506:281-295. doi: 10.1007/978-1-0716-2364-0_20.
Neutrophil extracellular traps (NETs) have the ability to regulate many aspects of asthma pathology. NETs can be detected either in bronchoalveolar lavage fluids (BALF) or in lung biopsies. Here, we describe methods to quantify NETs in BALF, namely the quantification of cell-free DNA, or of myeloperoxidase (MPO) or neutrophil elastase (NE) complexed with cell-free DNA. We also explain how to detect NETs in lung biopsies by two distinct techniques. The first technique is based on quantification of the citrullinated form of histone 3 (Cit-H3 , a specific component of NET) by western blot on tissue protein extracts. The second technique is based on the visualization of extracellular structures composed of MPO co-localizing with Cit-H3 in tissue sections by confocal microscopy. Finally, we describe a method allowing for quantification of NET volume in lung sections.
中性粒细胞胞外诱捕网 (NETs) 具有调节哮喘病理学多个方面的能力。可以在支气管肺泡灌洗液 (BALF) 或肺活检中检测到 NETs。在这里，我们描述了定量 BALF 中 NETs 的方法，即定量游离 DNA，或与游离 DNA 结合的髓过氧化物酶 (MPO) 或中性粒细胞弹性蛋白酶 (NE)。我们还解释了如何通过两种不同的技术在肺活检中检测 NETs。第一种技术基于组织蛋白提取物中组织蛋白 3 的瓜氨酸化形式 (Cit-H3，NET 的特定成分) 的 Western blot 定量。第二种技术基于通过共聚焦显微镜在组织切片中外周结构的可视化，该结构由 MPO 与 Cit-H3 共定位组成。最后，我们描述了一种允许定量肺切片中 NET 体积的方法。
