The authors gratefully acknowledge Drs James Roth, William Nauseef and Kayoko Kimura and Mr Tom Skadow for assistance with development of the canine neutrophil protocols. RDG is supported by a Wellcome Trust Fellowship ref: WT093767MA.

すべての実験は、アイオワ州立大学の施設内動物管理使用委員会から倫理的な許可を得て行われました。 1.採血<ol><li>バキュテイナー抗凝固剤に直接橈側または頸静脈、伏在から血液の9ミリリットルを描く（ 例えば、エチレンジアミン四酢酸（EDTA）、1.8ミリグラムのK 2 EDTA / mlの血液）、またはEDTAを含有する血液管への即時転送にシリンジに。穏やかにロールまたは血液と抗凝固剤の適切な混合を確実にするために、血液チューブを反転させます。 </li></ol> 2.好中球の分離<ol><li> 50ミリリットルコニカルチューブでは、無菌の室温等容積の血液を混ぜて3パーセント穏やかに反転することにより、デキストラン-500（0.9％滅菌生理食塩水で作製）。室温で18〜20分間、直立したままにしておきます。それはもはや下位赤色層による汚染なしに収集されなくなるまで新鮮なチューブに淡黄色の上層を転送します。目中の赤血球E元のチューブを廃棄することができます。 </li><li> 4℃で10分間、500×gで上清を遠心。オフ描画し、上清を捨てます。 10mLの室温の滅菌リン酸緩衝生理食塩水（PBS）中に手動で再懸濁細胞ペレット。そのボルテックスは、このプロトコルのいずれかの段階で再懸濁好中に使用すべきではありません注意してください。手動でオフに描くのではなく、上清を捨てることも、細胞収率を最大化するためのプロトコル全体で推奨されています。 </li><li> 50ミリリットルコニカルチューブに室温ポリスクロースおよびジアトリゾ酸ナトリウム細胞分離液（ 例えば 、ヒスト1077）の10ミリリットルを追加します。慎重に細胞分離溶液上に細胞含有液を層。室温に平衡化されていない細胞分離液を使用して、細胞収率を低下させることができることに留意されたいです。 </li><li>ブレーキをオフにして、室温で30分間、300×gで遠心分離します。 </li><li>好中球は勾配の最下層にあるように、オフ描き、uppeを捨てます血漿および血小板、単核細胞の狭ホワイトバンドと密度勾配媒体の透明層の上層からなるR 3層。 </li><li>残りの赤血球を溶解するために、室温の水10ml中の好中球ペレットを再懸濁します。 </li><li> 30秒後、滅菌室温1.8％の塩化ナトリウムの10ミリリットルを追加することで、張性を回復し、穏やかに転倒混和します。 </li><li> 4℃で5分間、500×gで遠心分離します。上清をオフに描画し、廃棄します。 </li><li>ペレットがまだ赤の場合、溶解工程を繰り返します。ペレットは白色である（または溶解が既に2回行っている場合）、穏やかに無菌PBS 10ml中に細胞を再懸濁した場合。 </li><li>自動化されたカウンターまたは血球計数器を用いて細胞を数えます。典型的な収量は、血液1mlあたり少なくとも10 6細胞です。 </li><li> 4℃で5分間、500×gで遠心分離します。上清をオフに描画し、廃棄します。 </li><li>無菌の室温PBSで所望の濃度に細胞を再懸濁。フォー1mlあたり5×10 6細胞で再懸濁、セクション2に記載のDNA放出アッセイrを。 </li><li>必要であれば、細胞の小容積を直接転送したり、スライドガラスに細胞遠心を使用。細胞は「製造業者の指示に従って迅速な固定および染色キットを用いて乾燥し、染色することができます。好中球の単離に成功した場合、有核細胞の顕微鏡で調べ、少なくとも95％は、最小限の赤血球の混入と顆粒球（好中球、好塩基球、好酸球）でなければなりません。 </li></ol> 3. DNA放出アッセイ<ol><li>好中球の単離後、直ちにDNA放出アッセイを設定します。 </li><li>細胞凝集は、光顕微鏡によるストック溶液の審査に存在する場合、すぐにアッセイを設定する前に、無菌の70μmの滅菌フィルターを通して細胞懸濁液を渡します。 </li><li>滅菌96ウェルプレートの各試験ウェルに、5×10 4細胞/ウェルを追加。アゴニストおよびロズウェルパークメモ赤及び100~200μlの最終体積0.5％の熱不活化ウシ胎仔血清を補充したフェノールなしリアル研究所-1640（RPMI）培地。好中球のストック溶液を、等量のPBSで置換したアゴニスト当たり少なくとも二つのブランクウェルを含みます。各アゴニストのためのブランクウェルを設定すると、蛍光アッセイでアゴニストによるアゴニストまたは干渉のDNAの混入を排除する上で重要であることに注意してください。 </li><li>二酸化炭素（4％）インキュベーター内で30分間39℃でインキュベートします。 </li><li>所望の濃度および細胞不透過性核酸色素でアゴニストを追加します。細胞不透過性色素の最適濃度が異なる核酸色素の間で異なることに注意してください。アゴニストは培地の等量で置換された非刺激対照を各実験に含まれるべきです。 注：これは、ウェル間の一貫性の条件を維持するために、培地の同様の量のアゴニストを希釈することが望ましいです。 200の最終的なウェルボリューム＆＃181; lは正常に使用されており、これが変更されている場合、十分ボリュームはすべての条件のために同一のままであるべきです。ホルボール-12-ミリステート-13-アセテート（PMA）（最終濃度≥0.1μM）または血小板活性化因子（PAF）（最終濃度≥31μM）を陽性対照として使用することができます。アゴニストは重複し、少なくとも測定されるべきです。 </li><li> 39℃でインキュベートします。 2時間後に、または所望の間隔でマイクロプレートリーダーを用いて蛍光を測定します。最適な時間間隔が使用アゴニストとともに変化することに注意してください。 注：波長が用いられる色素に依存します。 </li><li>平均蛍光を計算する前に、ブランクの蛍光を引きます。 </li><li>個人間およびプレート間の比較を可能にするために、非刺激ウェルの平均蛍光によって刺激されたウェルの平均蛍光を分割することにより、蛍光の変化倍率を計算します。 </li></ol>

<ol>
	<li>Schiffman, J. D., Breen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+oncology:+what+dogs+and+other+species+can+teach+us+about+humans+with+cancer.">Comparative oncology: what dogs and other species can teach us about humans with cancer.</a> Philos Trans Rl Soc B Biol Sci. 370, 1673 (2015).</li><li>Williams, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+canine+leucocyte+antigens:+a+significant+advance+in+canine+immunology.">Studies of canine leucocyte antigens: a significant advance in canine immunology.</a> Vet J. 153 (1), 31-39 (1997).</li><li>Eckhart, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplication+of+the+caspase-12+prodomain+and+inactivation+of+NLRC4/IPAF+in+the+dog.">Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.</a> Biochem Biophys Res Commun. 384 (2), 226-230 (2009).</li><li>Eckhart, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+mammalian+caspases+reveals+an+important+role+of+gene+loss+in+shaping+the+human+caspase+repertoire.">Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.</a> Mol Biol Evol. 25 (5), 831-841 (2008).</li><li>Brinkmann, V., et al. <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 (5663), 1532-1535 (2004).</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=Neutrophil+extracellular+traps:+is+immunity+the+second+function+of+chromatin.">Neutrophil extracellular traps: is immunity the second function of chromatin.</a> J Cell Biol. 198 (5), 773-783 (2012).</li><li>Chuammitri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+heterophil+extracellular+traps+(HETs):+novel+defense+mechanism+of+chicken+heterophils.">Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.</a> Vet Immunol Immunopathol. 129 (1-2), 126-131 (2009).</li><li>Pali&#263;, D., Ostoji&#263;, J., Andreasen, C. B., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+cast+NETs:+neutrophil+extracellular+traps+are+released+from+fish+neutrophils.">Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.</a> Dev Comp Immunol. 31 (8), 805-816 (2007).</li><li>Poirier, A. C., Schmitt, P., Rosa, R. D., Vanhove, A. S., Kieffer-Jaquinod, S., Rubio, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+histones+and+DNA+traps+in+invertebrate+immunity:+evidences+in+Crassostrea+gigas.">Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.</a> J Biol Chem. 289 (36), 24821-24831 (2014).</li><li>Robb, C. T., Dyrynda, E. A., Gray, R. D., Rossi, A. G., Smith, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invertebrate+extracellular+phagocyte+traps+show+that+chromatin+is+an+ancient+defense+weapon.">Invertebrate extracellular phagocyte traps show that chromatin is an ancient defense weapon.</a> Nat Commun. 5, 4627 (2014).</li><li>Kaplan, M. J., Radic, M. <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:+double-edged+swords+of+innate+immunity.">Neutrophil extracellular traps: double-edged swords of innate immunity.</a> J Immunol. 189 (6), 2689-2695 (2012).</li><li>Menegazzi, R., Decleva, E., Dri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killing+by+neutrophil+extracellular+traps:+fact+or+folklore?.">Killing by neutrophil extracellular traps: fact or folklore?.</a> Blood. 119 (5), 1214-1216 (2012).</li><li>Parker, H., Albrett, A. M., Kettle, A. J., Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+associated+with+neutrophil+extracellular+traps+is+active+and+mediates+bacterial+killing+in+the+presence+of+hydrogen+peroxide.">Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide.</a> J Leukoc Biol. 91 (3), 369-376 (2012).</li><li>Martinod, K., Wagner, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis:+tangled+up+in+NETs.">Thrombosis: tangled up in NETs.</a> Blood. 123 (18), 2768-2776 (2014).</li><li>Pratesi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+from+patients+with+rheumatoid+arthritis+target+citrullinated+histone+4+contained+in+neutrophils+extracellular+traps.">Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.</a> Ann Rheum Dis. 73 (7), 1414-1422 (2014).</li><li>Nauseef, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+neutrophils+from+venous+blood.">Isolation of human neutrophils from venous blood.</a> Methods Mol Biol. 1124, 13-18 (2014).</li><li>Gray, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+conventional+protein+kinase+C+(PKC)+is+critical+in+the+generation+of+human+neutrophil+extracellular+traps.">Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.</a> J Inflamm (Lond). 10 (1), 12 (2013).</li><li>Jeffery, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dogs+cast+NETs+too:+Canine+neutrophil+extracellular+traps+in+health+and+immune-mediated+hemolytic+anemia.">Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia.</a> Vet Immunol Immunopathol. 168 (3-4), 262-268 (2015).</li><li>McCracken, J. M., Allen, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+neutrophil+apoptosis+and+lifespan+in+health+and+disease.">Regulation of human neutrophil apoptosis and lifespan in health and disease.</a> J Cell Death. 7, 15-23 (2014).</li></ol>

著者らは、開示することは何もありません。

提示されたDNA放出アッセイは、細胞外DNAのために容易に定量化できるアッセイです。この方法は、同様の他の種でNET形成を評価するために使用される技術が、遠心分離速度、アゴニスト濃度から適応され、インキュベーション時間は、イヌ好中球8,17,18で使用するための方法を最適化するために変更されています。同様の調整は、他の種のための方法を適合させることができます。こ?...

米国だけ1万人の70以上のペットの犬があります。大切な家族のように、これらの動物は、多くの場合、エッジ医療を切断受けます。彼らは私たちの環境を等しく共有しているため、犬は人間の病気1の病因と治療への洞察を提供することができます。しかし、獣医学的治療またはその逆に人間の医学の発見を翻訳するかどうか、徹底的にあっても、そのような先天性免疫応答のような高度に保存されたシステムでは、種のバリエーションを特徴づけることが重要です。イヌとヒトの先天性免疫系の間の違いの例としては、犬の好中球2に高発現CD4を含みます。犬3と肉食4におけるカスパーゼ1/4ハイブリッドの発現の細胞質フラジェリンセンサーIPAFの機能的ホモログが存在しません。 好中球細胞外トラップ（ネット）、先天性免疫5の比較的最近発見されたコンポーネントです。ネットネットワークであります炎症性または感染性の刺激6の広い範囲に対応して放出されたDNA、核および粒状タンパク質の秒。 NETのような構造は、ニワトリ7、魚8、軟体動物9および10を acoelomates含む多くの種を越えて実証されているが、種のバリエーションがあります。例えば、マウス好中球は、ヒト好中球よりNETosisの刺激に応答速度が遅く、少ない拡散のNET 11を形成します 。ネットは直接病原体12,13を殺害に関与している可能性がより多くの論争の微生物を捕捉し、複数の種からの多くの証拠があります。しかし、NETコンポーネントはまた、組織の損傷を高める14,15を自己抗原として血栓症や行動を推進しています。 NETの有益と有害な影響のバランスは、種と関心の状態の両方でネットを調査することが重要である示唆し、異なる疾患と異なる種の間で異なる場合があります。 ここでイヌの好中球によるのNETの放出を誘導し、測定するための単純なプロトコルを記述します。この方法は、好中球16を分離し、他の種にNETosisを誘導するために使用されるものと同様であるが、そのようなアゴニスト濃度およびインキュベーション時間などの条件は、イヌ好中球のために最適化されています。同様のNET定量DNA放出アッセイは、他の種に記載されているが、ここで提示された方法はまた、イヌ8,17,18のために最適化されています。

<table><tbody><tr><td>Plastic Whole Blood tube with spray-coated K&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>BD</td><td>367835</td><td></td></tr><tr><td>Histopaque-1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Dextran-500</td><td>Accurate chemical and scientific corp.</td><td>AN228410</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>ThermoFisher scientific</td><td>20012043</td><td></td></tr><tr><td>Fetal bovine calf serum, heat inactivated</td><td>ThermoFisher scientific</td><td>10100139</td><td></td></tr><tr><td>RPMI Media 1640, without phenol red or L-glutamine</td><td>ThermoFisher scientific</td><td>32404-014</td><td>Should be free from phenol red</td></tr><tr><td>96 well flat bottomed sterile polystyrene plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Phorbol 12-myristate 13-acetate</td><td>Sigma-Aldrich</td><td>P1585</td><td></td></tr><tr><td>Platelet activating factor</td><td>Sigma-Aldrich</td><td>P4904</td><td></td></tr><tr><td>SYTOX Green Nucleic Acid Stain</td><td>ThermoFisher scientific</td><td>S7020</td><td></td></tr><tr><td>Synergy 2 Multi-Mode Reader</td><td>BioTek</td><td>NA</td><td></td></tr></tbody></table>

a simple fluorescence assay for quantification of canine neutrophil extracellular trap release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

このプロトコルを使用して、ポジティブコントロール、PMAおよびPAFで好中球を刺激した後、蛍光の強い倍数変化があるはずです。非刺激細胞（範囲2.0から5.8、N = 5匹の犬）18と比べて蛍光が平均4.0倍の増加で1時間の結果を得るために31μMPAFと図1B、イヌ好中球の刺激に示すように。 18：0.1μM（ 図1A）でPMAは2時間までの蛍光の増?...

Watch this Scientific Journal Video about A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release at JoVE.com

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

好中球細胞外トラップ（ネット）DNA、ヒストンおよび好中球のタンパク質のネットワークです。先天性免疫応答の成分が、ネットは自己免疫および血栓症に関与しています。このプロトコルは、イヌ好中球の単離およびマイクロプレート蛍光アッセイを用いてのNETの定量化のための単純な方法を記載しています。

イヌ好中球細胞外トラップリリースの定量化のためのシンプルな蛍光アッセイ

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

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JoVE Journal

Immunology and Infection

8.0K ビュー.  Iowa State University. 好中球細胞外トラップ（ネット）DNA、ヒストンおよび好中球のタンパク質のネットワークです。先天性免疫応答の成分が、ネットは自己免疫および血栓症に関与しています。このプロトコルは、イヌ好中球の単離およびマイクロプレート蛍光アッセイを用いてのNETの定量化のための単純な方法を記載しています。 

ビデオ: A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

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

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

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. ...

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 ...

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.

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)

Neutrophils are myeloid-lineage cells that form a crucial part of the innate immune system. The past decade has revealed additional key roles that ...

さまざまなシグナル伝達経路をスクリーニングするための好中球機能アッセイ

A Set of Screening Techniques for a Quick Overview of the Neutrophil Function

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as chemotaxis, reverse migration, phagocytosis, degranulation of cytotoxic enzymes and metabolites, and release of DNA as neutrophil extracellular traps (NETs). Neutrophils not only have tightly regulated signaling themselves, but also participate in the regulation of other components of the immune system. As fresh neutrophils are terminally differentiated, short-lived, and highly variable among individuals, it is important to make the most of the collected samples. Researchers often need to perform screening assays to assess an overview of the many neutrophil functions that may be affected by specific conditions under evaluation. A set of tests following a single isolation process of normal density neutrophils was developed to address this need, seeking a balance between speed, comprehensiveness, cost, and accuracy. The results can be used to reason and guide in-depth follow-up studies. This procedure can be carried out in an average time of 4 h and includes the evaluation of cell viability, reactive oxygen species (ROS) production, real-time migration, and phagocytosis of yeast on glass slides, leaving enough cells for more detailed approaches like omics studies. Moreover, the procedure includes a way to easily observe a preliminary suggestion of NETs after fast panoptic staining observed by light microscopy, with a lack of specific markers, albeit enough to indicate if further efforts in that way would be worthwhile. The diversity of functions tested combines common points among tests, reducing the analysis time and expenses. The procedure was named NeutroFun Screen, and although having&#160;limitations, it balances the aforementioned factors. Furthermore, the aim of this work is not a definite test set, but rather a guideline that can easily be adjusted to each lab&#39;s resources and demands.

著者スポットライト:NeutroFunスクリーンプロトコルによる好中球機能評価における速度、コスト、精度のバランス 

This protocol features a set of neutrophil functional assays to be used as a screening method to cover functions from different signaling pathways. The protocol includes an initial and simple evaluation of cell viability, purity, reactive oxygen species production, real-time migration, phagocytosis, and a preliminary suggestion of neutrophil extracellular traps.

好中球機能の概要を把握するための一連のスクリーニング技術

Neutrophils are known as one of the first lines of defense in the innate immune response and can perform many particular cellular functions, such as ...

イヌ好中球細胞外トラップリリースの定量化のためのシンプルな蛍光アッセイ

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この動画の章

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

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

NETQUANTを用いた好中球細胞トラップの自動画像ベース定量

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

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)

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)

好中球機能の概要を把握するための一連のスクリーニング技術

イヌ好中球細胞外トラップリリースの定量化のためのシンプルな蛍光アッセイ

要約

さらに動画を探す

この動画の章

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

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

体外蛍光顕微鏡による犬の好中球細胞外トラップ形成: ダイナミックな定量的分析

NETQUANTを用いた好中球細胞トラップの自動画像ベース定量

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

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)

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)

好中球機能の概要を把握するための一連のスクリーニング技術

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