Measurement of NET formation in vitro and in vivo by flow cytometry.
Measurement of NET formation in vitro and in vivo by flow cytometry.
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DOI：
10.1002/cyto.a.23169
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发表时间：
2017-08
期刊：
影响因子：
--
通讯作者：
Ishizu A
中科院分区：
文献类型：
--
作者：
Masuda S;Shimizu S;Matsuo J;Nishibata Y;Kusunoki Y;Hattanda F;Shida H;Nakazawa D;Tomaru U;Atsumi T;Ishizu A
中性粒细胞胞外陷阱（NET）是细胞外染色质纤维装饰有抗菌蛋白，如髓过氧化物酶（MPO），其从活化的中性粒细胞中挤出。NETosis是中性粒细胞的变态，伴随着DNA的解凝聚和质膜的破裂而形成NET。虽然NET在先天免疫中发挥重要作用，但NET的过度形成可能对宿主有害。到目前为止，已经报道了用于评估NET的各种方法。虽然各有优点，但金本位尚未建立。在这里，我们展示了一个简单的，客观的，定量的方法来检测NET使用流式细胞术。该方法使用质膜不可渗透的DNA结合染料SYTOX绿色。在暴露于NET诱导剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）的人外周多形核细胞中检测到SYTOX绿色阳性细胞。SYTOX绿色阳性细胞的数量增加取决于PMA的暴露持续时间和浓度。此外，证实了SYTOX绿色阳性细胞的MPO和质膜附属DNA的共定位。此外，NET抑制剂二苯基碘鎓可显著减少PMA诱导的SYTOX绿色阳性细胞数量。集体证据表明SYTOX绿色阳性细胞包括形成NET的中性粒细胞。已建立的方法可以检测经历NETosis但不发生早期凋亡的中性粒细胞，其定量等同于另一种基于荧光染色的常用图像分析。此外，体内形成的NET也可通过该方法检测。可以想象，建立的方法将使我们更好地了解NETosis和人类疾病之间的关系。版权所有© 2017作者。Cytometry Part A由Wiley Periodicals，Inc.出版。关于ISAC
Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane‐impermeable DNA‐binding dye, SYTOX Green. SYTOX Green‐positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12‐myristate 13‐acetate (PMA). The number of SYTOX Green‐positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co‐localization of MPO and plasma membrane‐appendant DNA of SYTOX Green‐positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green‐positive cells induced by PMA. The collective evidence suggests that SYTOX Green‐positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well‐used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
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DOI：
10.1084/jem.20100239
发表时间：
2010-08-30
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Li P;Li M;Lindberg MR;Kennett MJ;Xiong N;Wang Y
通讯作者：
Wang Y
影响因子：
5.5
作者：
Ueki S;Tokunaga T;Fujieda S;Honda K;Hirokawa M;Spencer LA;Weller PF
通讯作者：
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影响因子：
82.9
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DOI：
10.1161/atvbaha.111.242859
发表时间：
2012-08
期刊：
Arteriosclerosis, thrombosis, and vascular biology
影响因子：
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作者：
Fuchs TA;Brill A;Wagner DD
通讯作者：
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影响因子：
82.9
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