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Measurement of NET formation in vitro and in vivo by flow cytometry
This item is licensed under:Creative Commons Attribution-NonCommercial 4.0 International
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 | SYTOX Green | NETosis |
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. |
Rights: | https://creativecommons.org/licenses/by-nc/4.0/ |
Type: | article |
URI: | http://hdl.handle.net/2115/67193 |
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)
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Submitter: 石津 明洋
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