中性粒细胞胞外陷阱
细胞外
体外
免疫系统
中性粒细胞弹性蛋白酶
体内
共焦显微镜
细胞生物学
化学
共焦
刺激
生物物理学
生物
炎症
生物化学
免疫学
生物技术
神经科学
几何学
数学
作者
Tineke Kraaij,Fredrik C. Wieland,Sylvia W.A. Kamerling,Charles D. Pusey,Hans Ulrich Scherer,René E. M. Toes,Ton J. Rabelink,Cees van Kooten,Y.K. Onno Teng
标识
DOI:10.1016/j.autrev.2016.02.018
摘要
A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.
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