一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化
Summary
中性粒细胞胞外陷阱（英语教师）是DNA，组蛋白和中性粒细胞的蛋白质网络。虽然先天免疫反应的一个组成部分，母语有牵连的自身免疫和血栓形成。这个协议描述了犬嗜中性粒细胞的分离和使用微板荧光测定教师的量化的简单方法。
Abstract
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.
Introduction
有超过7000万只宠物狗在美国独自1。为尊贵的家族成员，这些动物经常收到尖端的医疗护理。同样，因为他们分享我们的环境，狗可以提供深入了解人类疾病1的发病机理和治疗。然而，无论是在人类医学成兽医治疗或反之亦然翻译的发现，它彻底表征即使在高度保守的系统种类的变化，例如先天免疫应答是重要的。的犬和人先天免疫系统之间的差异例子包括中性粒细胞的狗2高表达CD4;缺乏胞质鞭毛传感器IPAF犬3功能同系物的和在食肉动物4胱天蛋白酶1/4混合的表达。
中性粒细胞胞外陷阱（英语教师）是先天免疫5的相对最近发现的组成部分。篮网是网络S IN响应释放到大范围炎性或传染性刺激6的DNA的核和颗粒状蛋白质。网状结构已被证实在许多物种包括鸡7，鱼8，软体动物9和acoelomates 10，但也有物种变体。例如，嗜中性粒细胞的小鼠更响应缓慢NETosis刺激比人类嗜中性粒细胞，形成弥漫性减弱英语教师11。有一个庞大的身躯从多个物种蚊帐坑害微生物和更具争议可以直接参与杀灭病原体12,13证据。然而，.NET组件也增强了组织的损伤，促进血栓形成，并作为自身抗原14,15。教师的有益和有害作用之间的平衡可能不同的疾病和不同物种之间变化，这表明，调查无论是在物种和感兴趣条件母语是重要的。
在这里，我们描述了诱导和中性粒细胞犬测量母语释放一个简单的协议。此方法类似于那些用于分离嗜中性粒细胞16和在其他物种中诱发NETosis，但如激动剂浓度和温育时间条件已为犬嗜中性粒细胞进行了优化。类似的NET定量的DNA释放测定也已在其它物种中描述，但这里介绍的方法也为狗8,17,18优化。
Protocol
Representative Results
Discussion
提出的DNA释放法是外DNA一个容易量化分析。该方法改编自用于评估其他物种NET形成类似的技术，但离心速度，激动剂浓度和温育时间已被改变，以优化使用的方法与犬粒细胞8,17,18。类似的调整可以作出适应其他物种的方法。当与其他方法测量NETosis如流式细胞术或图像分析相比，该法是简单又便宜。该方法也并不需要对.NET组件抗体，所以非常适合在狗使用为谁商业化生产的，经过验证的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.
Materials
| Plastic Whole Blood tube with spray-coated K2EDTA | BD | 367835 | |
| Histopaque-1077 | Sigma-Aldrich | 10771 | |
| Dextran-500 | Accurate chemical and scientific corp. | AN228410 | |
| Phosphate buffered saline | ThermoFisher scientific | 20012043 | |
| Fetal bovine calf serum, heat inactivated | ThermoFisher scientific | 10100139 | |
| RPMI Media 1640, without phenol red or L-glutamine | ThermoFisher scientific | 32404-014 | Should be free from phenol red |
| 96 well flat bottomed sterile polystyrene plate | Falcon | 353072 | |
| Phorbol 12-myristate 13-acetate | Sigma-Aldrich | P1585 | |
| Platelet activating factor | Sigma-Aldrich | P4904 | |
| SYTOX Green Nucleic Acid Stain | ThermoFisher scientific | S7020 | |
| Synergy 2 Multi-Mode Reader | BioTek | NA |
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