荧光显微镜法显示体外中性粒细胞胞外杀菌网

Koji Tanaka; Tadanobu Shimura; Yuji Toiyama; Yoshinaga Okugawa; Yasuhiro Inoue; Toshimitsu Araki; Yasuhiko Mohri; Akira Mizoguchi; Masato Kusunoki

doi:10.21769/BioProtoc.1550

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Peer-reviewed

Visualization of ex vivo Neutrophil Extracellular Traps by Fluorescence Microscopy

荧光显微镜法显示体外中性粒细胞胞外杀菌网

Koji Tanaka email

发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1550 浏览次数: 11605

评审: Ivan ZanoniAchille BroggiMarco Di Gioia

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参见作者原研究论文

The authors used this protocol in:

Cover of PLOS ONE, featuring study using the protocol.

Nov 2014

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Abstract

Neutrophil extracellular traps (NETs) are extracellular DNAs decorated with nuclear and granular proteins such as histones, neutrophil elastase or myeloperoxidase. They exhibit fibrous mesh-like, web-like, or string-like structures. Here, we describe our protocol regarding visualization of ex vivo NETs released from neutrophils activated by lipopolysaccharide (LPS) using fluorescence microscopy.

Materials and Reagents

Whole blood from wild-type C57/BL6 mice (Japan SLC, Inc.)
Whole blood from human volunteers
LPS (Escherichia coli, serotype 0111:B4) (Sigma-Aldrich, catalog number: L4391 )
Polymorphprep^TM (Axis Shield PoC AS, catalog number: 1114683 )
RPMI 1640 medium (no phenol red) (Life Technologies, catalog number: 32404-014 )
Fetal bovine serum (Life Technologies, catalog number: 12483-020 )
ACK (Ammonium-chloride-potassium) lysing buffer (Lonza, catalog number: 10-548E )
SYTOX Green (Life Technologies, Invitrogen^TM, catalog number: S7020 )

Equipment

Glass Pasteur pipets (Iwaki brand, Asahi Techno Glass Corporation)
96-well plates (TPP Techno Plastic Products AG)
CO₂ Incubator (SANYO)
Fluorescence microscopy (Olympus, model: IX71 )

Procedure

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Tanaka, K., Shimura, T., Toiyama, Y., Okugawa, Y., Inoue, Y., Araki, T., Mohri, Y., Mizoguchi, A. and Kusunoki, M. (2015). Visualization of ex vivo Neutrophil Extracellular Traps by Fluorescence Microscopy. Bio-protocol 5(15): e1550. DOI: 10.21769/BioProtoc.1550.