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Measurement of NET formation in vitro and in vivo by flow cytometry


Cytometry a_91(8)_822-829.pdf548.12 kBPDF見る/開く

タイトル: Measurement of NET formation in vitro and in vivo by flow cytometry
著者: Masuda, Sakiko 著作を一覧する
Shimizu, Sakika 著作を一覧する
Matsuo, Junji 著作を一覧する
Nishibata, Yuka 著作を一覧する
Kusunoki, Yoshihiro 著作を一覧する
Hattanda, Fumihiko 著作を一覧する
Shida, Haruki 著作を一覧する
Nakazawa, Daigo 著作を一覧する
Tomaru, Utano 著作を一覧する
Atsumi, Tatsuya 著作を一覧する
Ishizu, Akihiro 著作を一覧する
キーワード: neutrophil
neutrophil extracellular trap
発行日: 2017年 8月24日
誌名: Cytometry Part A
巻: 91
号: 8
開始ページ: 822
終了ページ: 829
出版社 DOI: 10.1002/cyto.a.23169
抄録: 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 Greenpositive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Greenpositive 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.
資料タイプ: article
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 石津 明洋


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