1。ヒト血液からPMN分離抗凝固剤としてEDTAまたはヘパリン（10 U / ml）で約24ミリリットルのヒトの血液を使用してください。 <ol><li> 15ミリリットルファルコンチューブに6ミリリットルHistopaque 1119を追加し、慎重にレイヤ上に5〜7ミリリットルの全血。 </li><li>ブレーキなしで800 × gで20分間遠心する。 </li><li>吸引して廃棄黄色と透明な上部層を、新鮮なファルコンチューブに顆粒球を含む低赤相を転送する。 </li><li> 300 × gで10分間PBSと遠心機でファルコンチューブを充填することにより細胞を洗浄</li><li>その間に2ミリリットル10倍PBSで18 mlのパーコールを混合して100％のパーコール溶液を調製。 1X PBSは85％、80％、75％、70％、65％パーコール4mlの溶液を調製して。 </li><li>降順で互いの上にすべての割合のレイヤー2ミリリットルでPercoll勾配で2ファルコンチューブを準備します。 </li><li>遠心分離上清を除去した後、PBS 4 mlでペレットを再懸濁します沈降した細胞を組み合わせる。 </li><li>グラデーションの各上に再懸濁の慎重にレイヤ2ミリリットル。 </li><li>ブレーキなしで800 × gで20分間遠心する。 </li><li>遠心分離後に末梢血単核細胞でトップ層は65％層の大部分を削除し、新たなファルコンチューブに85％層まで、白残りinterphasesを収集する。 </li><li> PBSと300 × gで10分間、遠心分離でファルコンチューブを充填することにより細胞を洗浄</li><li> PBSの2ミリリットルで上清と再懸沈殿細胞を（通常&gt; 95％がPMNいる）を取り外します。 </li><li>血球計算板を用いて細胞を数える。 </li></ol> 2。アクティベーションPMNは<ol><li>各ウェルに滅菌した13ミリメートル円形のガラスカバースリップ（＃1,5）を置くことによって、24ウェル細胞培養プレートを準備します。 </li><li>シード2ウェルあたり500μLRPMIで× 10 5細胞（ヒト血清アルブミン2％を含む）と37℃CO 2インキュベーターで1時間インキュベート℃に</li><li>一方RPMIに600 nMのPMA溶液を調製し、ウェルあたり100μlの細胞に加える。 37℃までCO 2インキュベーター内で4時間まで15分のインキュベート℃まで</li><li> PBSに溶解した4％（最終濃度）パラホルムアルデヒドで細胞を固定する。 </li></ol> PMAは、ポジティブコントロールとして機能し、これまでNET形成を誘導するための最も強力なエージェントです。また、病原体と他​​の刺激または共培養は、NET誘導に使用することができます。 追加のパラレルサンプルが用意されている場合、時間の経過は、進行している間、NET形成のそれぞれの状態を確認することができます。 SYTOXグリーン（Invitrogen社）のような非透過性のDNA色素は非固定細胞に添加されている場合は、唯一の細胞外DNAが検出されます。新しいネットの形成以来、何らかの形で、各時点ごとに1つのパラレルサンプルを使用する必要がある、SYTOXの存在下で低下している。 3。免疫標識NETの検出ネットがさらに固定後に非常に壊れやすく、大半が準備中に迷子になるそうでなければ、細心の注意を払って操作する必要があります。 <ol><li>慎重にカーブした針で端にそれを持ち上げて、24ウェル培養プレートから添付された細胞とガラスカバースリップを削除し、微細な鉗子でそれをつかむ。 PBSのドロップに逆さまにカバースリップを置く。これは、試験管のスタンドを覆うパラフィルムシートで行うことができます。このような5分間3回洗浄する。 </li><li>トリトンX - 100室温で1分間、細胞を透過性に0.5％の低下で同じようにカバースリップをインキュベートする。 1分間PBSで3回洗浄する。 </li><li>パラフィルムやウェットティッシュで湿度の高い部屋を準備します。カバーは​​、ブロッキングバッファー（5％ロバ血清）のドロップに逆さま伝票はレイと37℃で30分間インキュベート℃に</li><li>ブロッキングバッファーで一次抗体、例えば、MS抗ヒストンとRB抗好中球エラスターゼを、希釈する。 </li><li>一次抗体の低下にブロッキングバッファーから直接湿度の高い部屋でスリップをカバーし、37℃で1時間インキュベート転送℃に</li><li> PBSで5分間3回洗浄する。 </li><li>二次抗体、例えばDKアンチMS Cy2とし、ブロッキング緩衝液でDK抗RBのCy3を、希釈する。 </li><li> 37℃で1時間二次抗体とインキュベートのドロップ℃の上に湿潤なチャンバにカバースリップを転送する</li><li> PBSで5分間3回洗浄する。染色DNAはUV励起または遠赤色励起のためのDRAQ5とを必要とdistで二回洗うヘキスト33342（1μg/ mlの）のいずれかで5分間、あなたの蛍光顕微鏡のオプションによって異なります。水。 </li><li>スライドガラス上にMowiolの20μlのドロップを設定し、逆さまに細胞をカバースリップをマウントします。細胞がカバーガラスとスライドの間にあることがあります。 Mowiolのドロップは、カバースリップがドロップで配置される均質な厚さの薄層を形成することになる。通常は、サンプルを押す必要はありません。あなたが非液浸レンズを使用する場合、試料は、検査の準備ができています。液浸レンズと顕微鏡分析のために、Mowiolは、サンプルの端に固化するまで約1時間のための試料は乾燥させて。 </li></ol> 4。走査型電子顕微鏡のための準備NETS（SEM） <ol><li> 2,5％のグルタルアルデヒドで24ウェルプレートにガラスカバースリップ上に細胞を固定ポスト。 </li><li> 24ウェル培養プレートから細胞を含むガラスカバースリップを外し、水滴に逆さまに置く。このような5分間3回洗浄する。 </li><li> 30分間0.5％OsO4とインキュベートを含む24ウェル細胞培養プレートに戻してカバースリップを転送します。 </li><li> 24ウェル培養プレートから細胞を含むガラスカバースリップを外し、水滴に逆さまに置く。このような5分間3回洗浄する。 </li><li>バック30分で1％タンニン酸とインキュベートを含む24ウェル細胞培養プレートにカバースリップを転送する。 </li><li>繰り返しのステップ4.2から4.4 </li><li>以下のレジメン（5分ごとに）を介して脱水。 <ul><li> 30％エタノール</li><li> 50％エタノール</li><li> 70％エタノール</li><li> 80％エタノール</li><li> 90％エタノール</li><li> 100％エタノール</li><li> 100％エタノール</li><li> 100％エタノール</li></ul></li><li>臨界点乾燥機と乾燥試料中にスリップをカバー転送する。 </li><li>薄層蒸発器を使用して5nmのプラチン/カーボン層を持つ試料のコートの表面</li></ol> 5。代表的な結果： 分離方法は、通常95％より高い純度を持つ無刺激の実行可能な好中球が得られます。刺激後の異なる時点で固定したとき、免疫染色のプロトコルはNETosisの平坦化細胞、核の小葉の喪失、顆粒の消失と核（ヒストンすなわち）と粒状の（すなわちの増加重複につながる核の完全性の間に形態学的変化のシーケンスを示しています。好中球エラスターゼ）染色。このプロトコルは、好中球による病原体の特異的な相互作用を分析する出発点として利用することもできます。この相互作用は、走査型電子顕微鏡のための準備のプロトコルを使用してより詳細に解剖することができます。 NET蛍光 NETコンポーネント（青= DNA、赤=ヒストン、緑=好中球エラスターゼ）について染色刺激好中球のサンプル。画像はNETローカリゼーション、DNAとヒストンの核局在化と好中球エラスターゼのための顆粒状パターンのほかに、表示されます。 SEM PMA刺激非刺激およびPMA -刺激好中球を示す電子顕微鏡写真をスキャン。刺激後、好中球は平らにならすとネットを生成する。 SEMネットと赤痢菌 NETSの中に閉じ込め赤痢菌の高分解能SEM像。

<ol>
	<li>Brinkmann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+kill+bacteria.">Neutrophil extracellular traps kill bacteria.</a> Science. 303, 1532-1535 .</li><li>Urban, C. F., Reichard, U., Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps+capture+and+kill+Candida+albicans+yeast+and+hyphal+forms.">Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.</a> Cell Microbiol. 8, 668-676 (2006).</li><li>M, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+amazonensis+promastigotes+induce+and+are+killed+by+neutrophil+extracellular+traps.">Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps.</a> PNAS. 106, 6748-6753 (2009).</li><li>Buchanan, J. T., Simpson, A. J., Aziz, R. K., Liu, G. Y., Kristian, S. A., Kotb, M., Feramisco, J., Nizet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase+expression+allows+the+pathogen+group+A+Streptococcus+to+escape+killing+in+neutrophil+extracellular+traps.">DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps.</a> Curr Biol. 14, 396-400 (2006).</li><li>Fuchs, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+cell+death+program+leads+to+neutrophil+extracellular+traps.">Novel cell death program leads to neutrophil extracellular traps.</a> J Cell Biol. 176, 231-241 (2007).</li><li>Brinkmann, V., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+suicide:+why+neutrophils+die+to+make+NETs.">Beneficial suicide: why neutrophils die to make NETs.</a> Nat Rev Microbiol. 5, 577-582 (2007).</li><li>Aga, E., Katschinski, D. M., van Zandbergen, G., Laufs, H., Hansen, B., Muller, K., Solbach, W., Laskay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+the+spontaneous+apoptosis+of+neutrophil+granulocytes+by+the+intracellular+parasite+Leishmania+major.">Inhibition of the spontaneous apoptosis of neutrophil granulocytes by the intracellular parasite Leishmania major.</a> J Immunol. , 169-898 (2002).</li></ol>

提供するプロトコルでは、かなりの純度で非刺激好中球の分離、NET形成の誘導とNETosis時の形態学的変化の解析が可能になります。つまり、緩く接続されたネットのほとんどが失われます過酷な洗浄条件を避け、慎重に試料を取り扱うときは、別の刺激条件下でのNETの形成（持続時間、刺激）の量を比較することができます。好中球/病原体相互作用、刺激の順序、他の免疫細胞との相互作用：この点において、提供されているプロトコルは、より洗練されたシナリオを分析する手法を確立するための出発点として利用することもできます。

neutrophil extracellular traps: how to generate and visualize them

好中球顆粒球は、末梢血中の白血球の最も豊富に存在するグループです。プロの食細胞として、彼ら巻き込む細菌とその抗菌顆粒はファゴソームと融合するときに細胞内にそれらを殺す。活性化により、彼らは一緒に病原体に結合する細胞外繊維を形成顆粒のタンパク質とクロマチンを解放：私たちは、その好中球が微生物を殺すの追加の方法を持っています。これらの新規な構造、または好中球細胞外トラップは（NETS）、病原性因子を低下させ、細菌1、真菌2および寄生虫3を殺す。ネットの構造骨格はDNAであり、そして彼らはDNasesの存在下で速やかに分解される。従って、DNasesを表現する細菌は、4より病原性があります。 TEM、SEM、免疫蛍光法および生細胞イメージング技術を組み合わせて相関顕微鏡を用いて、我々は、刺激によって、好中球の核は、その形状を失うとEUとヘテロクロマチンが均質化することを示している可能性があります。その後、核膜と顆粒膜は、NETコンポーネントの混合を可能に崩壊する。最後に、NETSは、細胞膜の区切りとしてリリースされています。この細胞死のプログラム（NETosis）は、アポトーシスとネクローシスとは区別され、NADPHオキシダーゼ5による活性酸素種の生成に依存する。 好中球細胞外トラップは、急性炎症のサイトで豊富です。 NETSは、バインドの微生物という、拡散を防ぐ、および病原性因子を分解し、好中球は、さらに彼らの寿命を超えて、その抗菌機能を発揮できるようにするため、病原体を殺すために抗菌剤の高い局所濃度を確保する自然免疫応答の形のように見える。証拠が高まっている、しかし、そのネットもその疾患に関与している自己免疫症候群から不妊6の範囲。 我々は、末梢ヒト血液の7から好中球顆粒球を分離し、NETSを形成するためにそれらを刺激する方法を説明します。また、我々は、光と電子顕微鏡内のネットを視覚化するプロトコルが含まれます。

Watch this Scientific Journal Video about Neutrophil Extracellular Traps: How to Generate and Visualize Them at JoVE.com

Neutrophil Extracellular Traps: How to Generate and Visualize Them

好中球細胞外トラップ（NETS）は、病原性細菌、真菌、寄生虫を戦うために重要な自然免疫機構です。ここでは、ヒトの血液から好中球顆粒球を分離するやネットを形成するためにそれらを有効にする方法を説明します。我々は、光と電子顕微鏡内のネットを可視化する準備のテクニックを紹介。

好中球細胞外トラップ：それらを生成して可視化する方法

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

neutrophil-extracellular-traps-how-to-generate-and-visualize-them

Research

JoVE Journal

Immunology and Infection

86.0K ビュー.  Max Planck Institute for Infection Biology. 好中球細胞外トラップ（NETS）は、病原性細菌、真菌、寄生虫を戦うために重要な自然免疫機構です。ここでは、ヒトの血液から好中球顆粒球を分離するやネットを形成するためにそれらを有効にする方法を説明します。我々は、光と電子顕微鏡内のネットを可視化する準備のテクニックを紹介。

ビデオ: Neutrophil Extracellular Traps: How to Generate and Visualize Them

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

簡体ヒト好中球細胞外トラップ（のNET）の単離と取扱い

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

免疫・感染学

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

脂質好中球の変化と好中球細胞外トラップのその後の形成を研究する方法

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

共焦点顕微鏡を用いた可視化マクロファージ細胞外トラップ

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis.

Here, we present a protocol for generating neutrophil extracellular traps (NETs) and operating NETQUANT, a fully automatic software option for quantification of NETs in immunofluorescence images.

NETQUANTを用いた好中球細胞トラップの自動画像ベース定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

高スループット アッセイを行い好中球細胞外トラップ形成の定量化

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by neutrophils in response to systemic inflammation or pathogens. Although NETs have previously been shown to augment clot formation and inhibit fibrinolysis in humans and dogs, the role of NETs in cats with cardiogenic arterial thromboembolism (CATE), a life-threatening complication secondary to hypertrophic cardiomyopathy, is unknown. A standardized method to identify and quantify NETs in cardiogenic arterial thrombi in cats will advance our understanding of their pathological role in CATE. Here, we describe a technique to identify NETs in formaldehyde-fixed and paraffin-embedded thrombi within the aortic bifurcation, extracted during necropsy. Following deparaffinization with xylene, aortic sections underwent indirect heat-induced antigen retrieval. Sections were then blocked, permeabilized, and ex vivo NETs were identified by colocalization of cell-free DNA (cfDNA), citrullinated histone H3 (citH3), and neutrophil elastase (NE) using immunofluorescence microscopy. To optimize the immunodetection of NETs in thrombi, autofluorescence of tissue elements was limited by using an autofluorescence quenching process prior to microscopy. This technique could be a useful tool to study NETs and thrombosis in other species and offers new insights into the pathophysiology of this complex condition.

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

蛍光免疫顕微鏡を用いたパラフィン組み込みネコリン動脈血栓における好中球外細胞トラップの同定

Neutrophil extracellular traps (NETs), composed of cell-free DNA (cfDNA) and proteins like histones and neutrophil elastase (NE), are released by ...

Morphological and Compositional Analysis of Neutrophil Extracellular Traps Induced by Microbial and Chemical Stimuli

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of neutrophil extracellular traps (NETs). This study analyzes the morphological and compositional changes in NETs induced by microbial and chemical stimuli using standardized in vitro methodologies for NET induction and characterization with human cells. The procedures described here allow the analysis of NET morphology (lytic or non-lytic) and composition (DNA-protein structures and enzymatic activity), and the effect of soluble factors or cellular contact on such characteristics. Additionally, the techniques described here could be modified to evaluate the effect of exogenous soluble factors or cellular contact on NET composition.
The applied techniques include the purification of polymorphonuclear cells from human peripheral blood using a double density gradient (1.079-1.098 g/mL), guaranteeing optimal purity and viability (&#8805; 95%) as demonstrated by Wright's staining, trypan blue exclusion, and flow cytometry, including FSC versus SSC analysis and 7AAD staining. NET formation is induced with microbial (Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans) and chemical (phorbol myristate acetate, HOCl) stimuli, and the NETs are characterized by DNA-DAPI staining, immunostaining for the antimicrobial peptide cathelicidin (LL37), and quantification of enzymatic activity (neutrophil elastase, cathepsin G, and myeloperoxidase). The images are acquired through fluorescence microscopy and analyzed with ImageJ.

Presented here is a protocol for the induction and analysis of in vitro neutrophil extracellular traps (NETs). Quantification of DNA, cathelicidin (LL37), and enzyme activity yielded data that show the variability in the composition and morphology of NETs induced by microbial and chemical stimuli under similar controlled conditions.

微生物・化学刺激による好中球細胞外トラップの形態・組成解析

Neutrophils function as the first line of cellular defense in an innate immune response by employing diverse mechanisms, such as the formation of ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

改変サンドイッチELISAを用いた好中球細胞外トラップからのミエロペルオキシダーゼ-DNAおよび好中球エラスターゼ-DNA複合体の定量(英語)

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

好中球細胞外トラップを可視化するための染色とイメージング

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in autoimmune diseases and sterile inflammation. They are web-like structures composed of double-stranded DNA filaments, histones, and antimicrobial proteins. Once released, NETs can trap and kill extracellular pathogens in blood and tissue. Furthermore, NETs participate in homeostatic regulation by stimulating platelet adhesion and coagulation. However, the dysregulated production of NETs has also been associated with various diseases, including sepsis or autoimmune disorders, which makes them a promising target for therapeutic intervention. Apart from electron microscopy, visualizing NETs using immunofluorescence imaging is currently one of the only known methods to demonstrate NET interactions in tissue. Therefore, various staining methods to visualize NETs have been utilized. In the literature, different staining protocols are described, and we identified four key components showing high variability between protocols: (1) the types of antibodies used, (2) the usage of autofluorescence-reducing agents, (3) antigen retrieval methods, and (4) permeabilization. Therefore, in vitro immunofluorescence staining protocols were systemically adapted and improved in this work to make them applicable for different species (mouse, human) and tissues (skin, intestine, lung, liver, heart, spinal disc). After fixation and paraffin-embedding, 3 &#181;m thick sections were mounted onto slides. These samples were stained with primary antibodies for myeloperoxidase (MPO), citrullinated histone H3 (H3cit), and neutrophil elastase (NE) according to a modified staining protocol. The slides were stained with secondary antibodies and examined using a widefield fluorescence microscope. The results were analyzed according to an evaluation sheet, and differences were recorded semi-quantitatively.
Here, we present an optimized NET staining protocol suitable for different tissues. We used a novel primary antibody to stain for H3cit and reduced non-specific staining with an autofluorescence-reducing agent. Furthermore, we demonstrated that NET staining requires a constant high temperature and careful handling of samples.

著者スポットライト:ヒトおよびマウス組織における好中球細胞外トラップイメージング 

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

ヒトおよびマウス組織における好中球細胞外トラップの免疫蛍光イメージング

Neutrophil extracellular traps (NETs) are released by neutrophils as a response to bacterial infection or traumatic tissue damage but also play a role in ...