Name: neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment
Uploaded: 2018-08-03T14:30:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

技術と事務作業、さん j. 篠原と I. Nieda に感謝します。また、松本史朗夫妻、秀典春田、倉科健太郎、高橋一也は、手術室でのサンプルの収集への協力を感謝いたします。この仕事は、学術振興 (17 K 10606) 教育省、科学、スポーツ、日本文化と日本社会科学研究費補助金によって支えられました。

LDN は本研究で登録された患者から得られたし、制度的レビュー ボードの自治医科大学によって承認されました。
1. 腹腔内洗浄と純の検出から LDN の分離
<ol><li>サンプルの収集<ol>
<li>消化管の悪性腫瘍による腹部手術を受けた患者の創傷閉鎖する前に腹腔内に直接通常の滅菌生理食塩水 1000 mL を吹き込みます。 注: サンプルは、年齢や性別に基づくバイアスのない胃切除術、結腸切除術、または食道切除術を受けた患者から得られました。生理食塩水は、コンテナーに転送され、注がれる腹部全体 1 分以内。これは日常的に患者に重大な影響なし術後腹腔洗浄として実行されます。</li>
		<li>少なくとも 1 分間広く腹腔洗浄液。 注: サンプルが均一なので、外科医の手でゆっくりと、注入液攪拌はそれをお勧めします。</li>
		<li>4 50 mL 注射器洗浄液 200 mL を回復します。 注: 時々 ゴム コネクタが流体を取るされます。</li>
	</ol>
</li>
	<li>顆粒球特定 mAb、CD66b16を使用して腹膜の LDN の浄化を実行します。 注: 密度勾配遠心後の中間層には、多くの単核細胞が含まれています、CD66b モノクローナル抗体を用いた多形体好中球の正の選択を行った。<ol>
<li>腹腔洗浄液を 50 mL のチューブに転送します。 注: この手順では、不純物を除去する 100 μ m ナイロン フィルターを通して流体を渡します。</li>
		<li>室温 7 分 270 x g で腹水を遠心分離します。</li>
		<li>5 mL の 0.02 %edta で PBS でペレットを再懸濁します。</li>
		<li>慎重に 3 mL 密度勾配ソリューションに細胞懸濁液 5 mL をオーバーレイします。</li>
		<li>休憩なしの常温 15 分 1,700 × g で遠心分離機します。</li>
		<li>2 〜 3 mL のピペットを使用して中間層 (図 1 a) を含む溶液を収穫し、10 ml の PBS の 0.02 %edta のミックスします。</li>
		<li>RT で 7 分の 400 x g で腹水を遠心し、上澄みを廃棄します。</li>
		<li>破棄した上清で 7 分間 270 x g で 0.02 %edta と遠心分離機の PBS の別の 10 mL を追加します。</li>
		<li>磁気細胞分離キット用のバッファーの 60 μ L 細胞ペレット (1 x 10 の7) を溶かしてください。</li>
		<li>ペレットに Fc ブロックの 20 μ L を加え、4 ° C で 10 分間インキュベート</li>
		<li>反 CD66b の 20 μ L マイクロ ビーズと共役し、4 ° C でさらに 10 分間インキュベートを追加します。</li>
		<li>洗浄し、再 MAC バッファーを 500 μ l 添加の餌を中断します。</li>
		<li>磁気セパレーターの磁場中磁気列に細胞懸濁液を適用し、15 mL のバッファーとカラムを洗浄することにより、流れを含んでいる CD66b(-) 細胞を収集します。</li>
		<li>磁選機から列を削除して、しっかりと列にプランジャーを押すことにより磁気標識 CD66b(+) 細胞をすぐに洗い流します。 注: CD66b(+) セル人口を示す FACS 解析によって決定される好中球のほとんどで構成されています > CD66(+) 分画の 95% は CD11b、CD15、CD16 に肯定的な CD14 の負。</li>
	</ol>
</li>
	<li>ネットの世代<ol>
<li>RT で 7 分間 270 × g で遠心分離後再分離の LDN を中断 (5 x 106) 1 ml の 10% の FCS で RPMI1640。</li>
		<li>6 ウェル ポリ-L-リジン コーティング プレート (直径 6 cm) 5% CO2の 37 ° C で 2 時間の LDN を文化します。</li>
		<li>5 μ M の最終的な集中に蛍光グリーンの対比染色 (核や染色体を染色する膜不透過性色素) を追加します。</li>
		<li>すぐにネット (蛍光顕微鏡下で LDN から追放するものと細胞外 DNA コンポーネント) の形態を観察します。</li>
	</ol>
</li>
</ol>2. 赤い蛍光細胞リンカー染付腫瘍細胞の染色
<ol><li>ひと癌細胞株 MKN45、NUGC、および OCUM 1 を準備します。</li>
	<li>1 x 10 の7セル PBS + 15 の mL の管、室温 7 分 270 × g で遠心分離で 0.02 %edta を洗う</li>
	<li>ペレット、染色色素の溶液 1 mL を追加し、そっとピペットします。</li>
	<li>赤蛍光細胞リンカー色素染色溶液 1 mL に 4 μ L を溶解し、2.2 の段階から細胞懸濁液と混ぜます。</li>
	<li>室温 4 分のペレットを孵化させなさい</li>
	<li>DMEM の 4 つの mL を追加 (10 %fbs) 染色反応を停止します。</li>
	<li>7 分 270 × g を遠心し、上清を捨てます。</li>
	<li>2.6 および 2.7 の手順を 2 回繰り返します。</li>
	<li>蛍光顕微鏡で確認 (励起 = 551 nm、出力 = 567 nm) 腫瘍細胞が赤を染色します。</li>
</ol>3. 癌細胞接着のネットの解析
<ol><li>1 ml 0.1 %bsa 添加 RPMI 1640 の赤蛍光染色腫瘍細胞 (1 x 106) を再停止します。</li>
	<li>手順 1.3 で説明したように網を作り出した LDN 文化に腫瘍細胞を追加します。</li>
	<li>回路網に接触する腫瘍細胞を可能にする、37 ° C で 5 分間インキュベートします。 注: 長い潜伏癌細胞接着の LDN またはプレートを誘導します。</li>
	<li>メディアを取り出して、皿を旋回を prewarmed メディア (0.1 %bsa + RPMI 1640) 2 mL を追加して優しく洗浄します。</li>
	<li>手順 3.4 では洗濯の手順を 2 回繰り返します。 注: ネットから弱アタッチ プレート、網自体の除去を避けるためにできるだけ優しく洗浄を行う必要があります。</li>
	<li>核と染色体 5 μ G/ml 網の可視化のための最終的な集中での汚損のため緑の蛍光染料を追加します。</li>
	<li>網を観察し、適切なフィルターを使用して腫瘍細胞を添付 (緑、励起 = 504 nm、発光 = 523 nm; 赤、励起 = 551 nm、出力 = 567 nm)。</li>
	<li>ネットで閉じ込められている腫瘍のセルを表示する数字をマージします。 注意: いくつかの実験で DNA 分解酵素追加されました LDN 文化 (最終濃度 100 U/mL) に 5 分 5 分の共同培養前に、。</li>
</ol>4 閉じ込められた腫瘍細胞の時間経過ビデオ解析
<ol><li>35 mm で腹膜の LDN の文化はラウンド被覆ポリ L リジン手順 1.3 で説明した料理です。</li>
	<li>核と 1.3 の手順で説明されているようにネットを可視化する染色体の汚損のため緑の蛍光染料を追加します。</li>
	<li>インキュベーションの 1 分後にメディアを取り出し、0.1 %bsa + RPMI 1640 2 mL を加えます。</li>
	<li>メディアを取り出し、無染色 1 × 106 MKN45 セル中断 0.1 %bsa + RPMI 1640 を追加します。右で 5 分間インキュベートします。</li>
	<li>手順 3.4 で優しく洗浄することにより任意の未接続の腫瘍細胞を除去し、100 u/mL ペニシリンおよび 100 μ g/mL ストマイ添加 10 %fcs を DMEM を追加します。</li>
	<li>全体ビューのセルの観測システムで培養皿をマウントし、腫瘍細胞が網によって閉じ込められている適切なフィールドを選択します。</li>
	<li>2 日間をさらに共培養し、フィールドの連続撮影通常の光と MKN45 細胞と網をそれぞれ検出する蛍光の下 6 分毎に進みます。</li>
	<li>各 timepoint でイメージを重ね合わせ、画像ビューアーのソフトウェアを使用して、時間経過のビデオを作成します。</li>
</ol>

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著者は、彼らは競合する金銭的な利益があることを宣言します。

以前の研究は、10、生体内で純基板11循環腫瘍細胞をトラップできることを報告しています。転移性乳癌細胞は、好中球を刺激しターゲット器官17の腫瘍細胞の成長を支援するネットの形成を誘導する示されています。さらに、術後の洗浄液から LDN の短期的な文化が効率的にさらに刺激15せず腫瘍細胞をわ?...

循環血液における多形核好中球は通常単核細胞から密度グラデーション製法によって区切られます。ただし、CD11b(+)、CD15(+)、CD16(+)、および CD14(-) の表現型を持つ低密度好中球 (LDN) として知られているいくつかの好中球、単核細胞と共同浄化されました。LDN の相対数は、大幅自己免疫疾患1、2敗血症3がん4,5など様々 な病態で増加します。以前の研究では、LDN が好中球6の表現型と機能別のクラスであることを示しています。循環血液で LDN は通常の密度好中球2,7より好中球細胞外トラップ (網) を生成する可能性が高いことに注意してください。ネットは核酸、ヒストンやプロテアーゼ、粉粒体、ゾル性細胞質蛋白質から成る web のような構造であり、彼らは効率的にわなに掛けることができる病原体8を破壊して。
最近では、ネットをキャプチャだけでなく、微生物も血小板、血栓形成9および腫瘍転移10,11に役立つ腫瘍細胞を循環する示されています。しかし、ネットと血小板または腫瘍細胞間接着剤の相互作用の背後にある分子メカニズムはまだ明らかではありません。最近では、体外で接着アッセイは、骨髄性白血病細胞 (K56212) および肺癌細胞 (A54913) が β3 と β 1 インテグリンを介してネットに接続するを明らかにしました。著者には、好中球由来及びホルボール 12-ミリスタート 13-アセタート (PMA) 接着基板14によって活性化ネット ストックが使用されます。かどうかの生産網と同じ分子構造を保持"由来の無細胞ネット証券「高速遠心分離によって分離されたこのアッセイは、好中球の不在で NET コンポーネントと現実の相互作用の検出を使用できますが、それは議論の余地vivo。最近、我々 は腹部手術に含まれる大規模な網を生成、腹膜転移15を引き起こす腫瘍細胞に接続されている多くの成熟した LDN 後その腹腔洗浄液を発見します。本研究では、物理的な操作をすることがなくそのままネットへの腫瘍細胞の接着を調べた正常に。ネットと無料の腫瘍細胞間接着の相互作用を検出する技術の詳細を紹介します。

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>GE Healthcare, SWEDEN</td><td>17-1440-02</td><td></td></tr><tr><td>StraightFrom&amp;trade; Whole Blood CD66b MicroBeads</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-104-913</td><td></td></tr><tr><td>Fc block</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-059-901</td><td></td></tr><tr><td>MACS Rinsing Solution</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-091-222</td><td></td></tr><tr><td>MACS BSA Stock Solution</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-091-376</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-041-306</td><td></td></tr><tr><td>MACS Magnetic Separator</td><td>Miltenyi Biotec, Bergisch Gladbach, Germany</td><td>130-042-501</td><td></td></tr><tr><td>SYTOX green nucleic acid stain 5mM solution in DMSO</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>S7020</td><td></td></tr><tr><td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>P9691</td><td></td></tr><tr><td>Diluent C</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>CGLDIL</td><td></td></tr><tr><td>RPMI1640 Medium</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>R8758</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium-high glucose (DMEM)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>D5796</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>D8537</td><td></td></tr><tr><td>0.5mol/l-EDTA Solution (pH 8.0)</td><td>nacalai tesque, Japan</td><td>06894-14</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, USDA-approved regions</td><td>gibco by life technologies, Mexico</td><td>10437-028</td><td></td></tr><tr><td>Bovine Serum Albumin lyophilized powder, &amp;ge;96% (agarose gel electrophoresis)</td><td>Sigma-Aldrich, St Louis, MO, USA</td><td>A2153</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies Japan</td><td>15140-122</td><td></td></tr><tr><td>Plasmocin Prophylactic</td><td>InvivoGen, San Diego, CA-USA</td><td>ant-mpp</td><td></td></tr><tr><td>DNase I</td><td>Worthington, Lakewood NJ)</td><td>LS002138</td><td></td></tr><tr><td>Poly-L-Lysine-Coated MICROPLATE 6Well</td><td>IWAKI, Japan</td><td>4810-040</td><td></td></tr><tr><td>Poly-L-Lysine-Coated MICROPLATE 24Well</td><td>IWAKI, Japan</td><td>4820-040</td><td></td></tr><tr><td>fluorescein stereomicroscope</td><td>BX8000, Keyence, Osaka Japan</td><td>BZ-X710</td><td></td></tr><tr><td>Whole view cell observation system</td><td>Nikon, Kanagawa, Japan</td><td>BioStudio (BS-M10)</td><td></td></tr><tr><td>MKN45 human gastric cancer cell line</td><td>Riken, Tukuba Japan</td><td>N/A</td><td></td></tr><tr><td>NUGC-4 human gastric cancer cell line</td><td>Riken, Tukuba Japan</td><td>N/A</td><td></td></tr><tr><td>OCUM-1 human gastric cancer cell line</td><td>Osaka City University, Japan</td><td>N/A</td><td>Gift from Dr. M.Yashiro</td></tr></tbody></table>

neutrophil extracellular traps generated by low density neutrophils obtained from peritoneal lavage fluid mediate tumor cell growth and attachment

好中球リリース好中球細胞外トラップ (網) を取り込んで微生物を破壊することができますをアクティブ化します。最近の研究では、ネットがさまざまな病気プロセス、自己免疫疾患、血栓症、腫瘍の転移などに関与していることを示唆しています。ここで、網に付着後成長無料腫瘍細胞の捕捉中にネット活動を検出する詳細な培養技術を紹介します。第 1 に、開腹症例から術後腹腔洗浄液から低密度好中球 (LDN) を集めました。LDN の短期養殖緑蛍光核と染色体の対比染色に視覚化された大規模なネット形成で起因しました。ひと胃癌細胞株ネットと NUGC 4、OCUM 1、MKN45 の共同培養後多くの腫瘍細胞は網によって閉じ込められました。その後、添付ファイルは、DNase I. 低速度撮影ビデオは網によって閉じ込められている腫瘍細胞が死んでいなかったこと明らかに取り付けた網の劣化によって完全に廃止されたが、代わりに連続培養における旺盛します。これらのメソッドは、ネットとセルと材料の様々 なタイプの接着剤の相互作用の検出に適用可能性があります。

2 時間の文化で腹膜灌流液の示した文字列の構造から派生した CD66b(+) LDN の緑色蛍光色素で染色核と染色体 (図 1 b) CD66b(-) 単核細胞がいない中 (図 1)。ただし、前処理は LDN 文化 100 U/mL DNase 私は、特徴的な構造は破壊 (図 1)、彼らが好中球から追放された細胞外 DNA で構成されていたことを示します。LDN ...

Watch this Scientific Journal Video about Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment at JoVE.com

Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

ひと低密度好中球 (LDN) 術後の腹腔洗浄液から回収が大規模な好中球細胞外トラップ (網) を生成し、その後成長無料腫瘍細胞を効率的にトラップの方法を紹介します。

低密度の好中球によって生成された好中球細胞外トラップは仲介する腹腔洗浄液から得られた腫瘍細胞の増殖と添付ファイル

Activated neutrophils release neutrophil extracellular traps (NETs), which can capture and destroy microbes. Recent studies suggest that NETs are involved ...

neutrophil-extracellular-traps-generated-low-density-neutrophils

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.7K ビュー.  Jichi Medical University. ひと低密度好中球 (LDN) 術後の腹腔洗浄液から回収が大規模な好中球細胞外トラップ (網) を生成し、その後成長無料腫瘍細胞を効率的にトラップの方法を紹介します。

ビデオ: Neutrophil Extracellular Traps Generated by Low Density Neutrophils Obtained from Peritoneal Lavage Fluid Mediate Tumor Cell Growth and Attachment

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

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 ...

免疫・感染学

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 ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

抗腫瘍特性を有する好中球の単離とキャラクタリゼーション

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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 ...

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 ...

デュアルダイライブセル解析によるNET形成の定量化

Real-Time High Throughput Technique to Quantify Neutrophil Extracellular Traps Formation in Human Neutrophils

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that neutrophils play in the pathogenesis of cancer, autoimmune diseases, and various acute and chronic inflammatory conditions by contributing to the initiation and perpetuation of immune dysregulation through multiple mechanisms, including the formation of neutrophil extracellular traps (NETs), which are structures crucial in antimicrobial defense. Limitations in techniques to quantify NET formation in an unbiased, reproducible, and efficient way have restricted our ability to further understand the role of neutrophils in health and diseases. We describe an automated, real-time, high-throughput method to quantify neutrophils undergoing NET formation using a live cell imaging platform coupled with a membrane permeability-dependent dual-dye approach using two different DNA dyes to image intracellular and extracellular DNA. This methodology is able to help assess neutrophil physiology and test molecules that can target NET formation.

著者スポットライト:Quantifying Neutrophil extracellular traps in disease and drug screening using dual-color live-cell imaging

We present an automated high-throughput method to quantify neutrophil extracellular traps (NETs) utilizing the live cell analysis system, coupled with a membrane permeability-dependent dual-dye approach.

低密度の好中球によって生成された好中球細胞外トラップは仲介する腹腔洗浄液から得られた腫瘍細胞の増殖と添付ファイル

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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

簡体ヒト好中球細胞外トラップ（のNET）の単離と取扱い

抗腫瘍特性を有する好中球の単離とキャラクタリゼーション

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

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

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

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

ヒト好中球における好中球細胞外トラップ形成を定量化するリアルタイムハイスループット技術(英語)

ヒト好中球における好中球細胞外トラップ形成を定量化するリアルタイムハイスループット技術(英語)

ヒト好中球における好中球細胞外トラップ形成を定量化するリアルタイムハイスループット技術(英語)