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

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Title: Measurement of NET formation in vitro and in vivo by flow cytometry
Authors: Masuda, Sakiko Browse this author
Shimizu, Sakika Browse this author
Matsuo, Junji Browse this author →KAKEN DB
Nishibata, Yuka Browse this author
Kusunoki, Yoshihiro Browse this author
Hattanda, Fumihiko Browse this author
Shida, Haruki Browse this author
Nakazawa, Daigo Browse this author
Tomaru, Utano Browse this author →KAKEN DB
Atsumi, Tatsuya Browse this author →KAKEN DB
Ishizu, Akihiro Browse this author →KAKEN DB
Keywords: neutrophil
neutrophil extracellular trap
Issue Date: 24-Aug-2017
Journal Title: Cytometry Part A
Volume: 91
Issue: 8
Start Page: 822
End Page: 829
Publisher DOI: 10.1002/cyto.a.23169
Abstract: 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.
Type: article
Appears in Collections:保健科学院・保健科学研究院 (Graduate School of Health Sciences / Faculty of Health Sciences) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 石津 明洋

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