Jeffery Unity, Gray Robert D, LeVine Dana N
Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University.
MRC Centre for Inflammation Research, University of Edinburgh.
J Vis Exp. 2016 Nov 21(117):54726. doi: 10.3791/54726.
Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.
中性粒细胞胞外陷阱是由DNA、组蛋白和中性粒细胞蛋白组成的网络结构，在受到感染和炎症刺激时释放。尽管是固有免疫反应的一个组成部分，但中性粒细胞胞外陷阱与一系列疾病过程有关，包括自身免疫和血栓形成。本方案描述了一种使用微孔板荧光测定法分离犬中性粒细胞并定量中性粒细胞胞外陷阱的简单方法。使用传统静脉穿刺技术采集血液。利用葡聚糖沉降法和针对犬血液优化的密度梯度条件分离中性粒细胞。在96孔板的孔中附着一段时间后，用佛波醇-12-肉豆蔻酸酯-13-乙酸酯或血小板活化因子等诱导中性粒细胞胞外陷阱形成的激动剂处理中性粒细胞。通过一种细胞不可渗透的核酸染料的荧光来测量DNA释放。该测定法是一种简单、廉价的定量中性粒细胞胞外陷阱释放的方法，但必须用其他方法确认是中性粒细胞胞外陷阱形成而非其他细胞死亡原因。
