Automatic quantification of in vitro NET formation.
Automatic quantification of in vitro NET formation.
复制标题
DOI：
10.3389/fimmu.2012.00413
复制
发表时间：
2012
影响因子：
7.3
通讯作者：
Zychlinsky A
中科院分区：
文献类型：
--
作者：
Brinkmann V;Goosmann C;Kühn LI;Zychlinsky A
中性粒细胞胞外陷阱(Net)由解聚的染色质组成，染色质中点缀着颗粒状和一些细胞质蛋白。它们是由激活的中性粒细胞形成的，也称为多形核白细胞(PMN)，这是一种活跃的细胞死亡程序的结果，称为网织红细胞增多症。中性粒细胞与病原体(细菌、真菌、寄生虫、病毒颗粒)、活化的血小板或病原体成分共培养可引起网织红细胞增多症。NETotic级联反应的第一步是刺激一个或几个受体，然后激活Raf/MEK/ERK通路，最终导致多聚体NADPH氧化酶复合体的组装和活性氧物种(ROS)的产生。后来，细胞内膜解体，颗粒蛋白中性粒细胞弹性蛋白酶进入细胞核，处理核心组蛋白，也得到高瓜氨酸化。这会导致染色质的解聚和动员。净形成量随刺激程度的不同而不同，这取决于刺激的类型和浓度。可以使用包括荧光显微镜或测量DNA释放在内的各种方法来量化蚊帐。这些方法中的每一种都有特定的缺点：荧光显微镜的分析容易发生主观变化，DNA的释放不能区分由蚊虫或其他形式的细胞死亡所释放的DNA。在这里，我们提出了一个半自动量化网络形成的协议。它依赖于观察到的结果，即抗染色质抗体更容易与网织红细胞增生症细胞和蚊虫细胞核中存在的去凝集染色质结合。将抗染色质抗体的荧光信号与DNA结合染料的信号联系起来，可以自动计算网状中性粒细胞的百分比。这种方法不需要复杂的显微设备，图像是用公共领域软件包进行量化的。
Neutrophil Extracellular Traps (NETs) consist of decondensed chromatin studded with granular and some cytoplasmic proteins. They are formed by activated neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) as the result of an active cell death program, named NETosis. NETosis can be induced by a wide range of stimuli including coculture of neutrophils with pathogens (bacteria, fungi, parasites, virus particles), activated platelets, or pathogen components. The first step of the NETotic cascade is stimulation of one or several receptors followed by activation of the Raf/MEK/ERK pathway that culminates in the assembly of the multimeric NADPH oxidase complex and the production of reactive oxygen species (ROS). Later, intracellular membranes disintegrate, the granular protein Neutrophil Elastase enters the nucleus and processes core histones that also get hypercitrullinated. This leads to decondensation and mobilization of chromatin. The amount of NET formation varies with the degree of stimulation, and this is dependent on the type and concentration of the stimulus. NETs can be quantified using various methods including fluorescence microscopy or measuring DNA release. Each of these methods have specific drawbacks: analysis of fluorescence microscopy is prone to subjective variations, and DNA release does not differentiate between DNA that has been released by NETosis or by other forms of cell death. Here we present a protocol to semi-automatically quantify NET formation. It relies on the observation that anti-chromatin antibodies bind more readily to decondensed chromatin present in the nuclei of cells undergoing NETosis and in the NETs. Relating the fluorescence signals of the anti-chromatin antibody to the signals of a DNA-binding dye allows the automatic calculation of the percentage of netting neutrophils. This method does not require sophisticated microscopic equipment, and the images are quantified with a public-domain software package.