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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 Koji Tanaka
TS Tadanobu Shimura
YT Yuji Toiyama
YO Yoshinaga Okugawa
YI Yasuhiro Inoue
TA Toshimitsu Araki
YM Yasuhiko Mohri
AM Akira Mizoguchi
MK Masato Kusunoki

发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1550 浏览次数: 12079

评审: Ivan ZanoniAchille BroggiMarco Di Gioia

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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

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

Equipment

  1. Glass Pasteur pipets (Iwaki brand, Asahi Techno Glass Corporation)
  2. 96-well plates (TPP Techno Plastic Products AG)
  3. CO2 Incubator (SANYO)
  4. Fluorescence microscopy (Olympus, model: IX71 )

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

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分类

免疫学 > 免疫细胞功能 > 嗜中性粒细胞

细胞生物学 > 细胞成像 > 荧光

细胞生物学 > 细胞成像 > 活细胞成像

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