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

Masuda, Sakiko; Shimizu, Sakika; Matsuo, Junji; Nishibata, Yuka; Kusunoki, Yoshihiro; Hattanda, Fumihiko; Shida, Haruki; Nakazawa, Daigo; Tomaru, Utano; Atsumi, Tatsuya; Ishizu, Akihiro

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

Masuda, Sakiko; Shimizu, Sakika; Matsuo, Junji et al.

Cytometry Part A, 2017, 91(8), 822-829

Permalink : https://hdl.handle.net/2115/67193

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アクセス権	open access
URI	https://hdl.handle.net/2115/67193
タイトル	Measurement of NET formation in vitro and in vivo by flow cytometry
著者	Masuda, Sakiko ; Shimizu, Sakika ; Matsuo, Junji ; Nishibata, Yuka ; Kusunoki, Yoshihiro ; Hattanda, Fumihiko ; Shida, Haruki ; Nakazawa, Daigo ; Tomaru, Utano ; Atsumi, Tatsuya ; Ishizu, Akihiro
著者名 Masuda, Sakiko
著者名 Shimizu, Sakika
e-Rad_Researcher 50359486 著者名 Matsuo, Junji
著者名 Nishibata, Yuka
著者名 Kusunoki, Yoshihiro
著者名 Hattanda, Fumihiko
著者名 Shida, Haruki
著者名 Nakazawa, Daigo
e-Rad_Researcher 20360901 著者名 Tomaru, Utano
e-Rad_Researcher 20301905 著者名 Atsumi, Tatsuya
e-Rad_Researcher 60321957 著者名 Ishizu, Akihiro
言語	英語
キーワード	neutrophil
	neutrophil extracellular trap
	SYTOX Green
	NETosis
発行日	2017-08-24
収録物名	Cytometry Part A
巻(号)	91(8)
ページ	822-829
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.
資源タイプ	学術雑誌論文
出版タイプ	Version of Record
権利情報	Creative Commons Attribution-NonCommercial 4.0 International
PISSN	1552-4922
EISSN	1552-4930
関連情報 (isIdenticalTo)	出版社版DOI https://doi.org/10.1002/cyto.a.23169