ビジョン保健研究ネットワーク - この作品は、ヘルスリサーチ（178918）、フォン·ド·RECHERCHEエンサンテケベックのカナダの研究所からの助成金によってサポートされています。

注：男性C57BL / 6マウス（5-7週齢）をチャールズ·リバー·ラボラトリーズインターナショナル社（セント·コンスタント、ケベック州、カナダ）からのものであり、CHUサントジャスティン動物実験委員会によって承認されたプロトコルに従って操作。マウス気管支組織外植片は、上皮細胞上のLMPのアポトーシス促進効果を調査するための主要な気管支上皮細胞の良い情報源を提供する。このプロトコルは、のLMP のインビトロ発生、ならびにLMPで処理した気管支組織外植片におけるアポトーシス上皮細胞を検出するための方法を記載する。このプロトコルは、3つのセクションで構成されています。 1.のLMP産生および特徴注：汚染を防止するために、この実験で使用されるすべての材料は滅菌されているか、オートクレーブしていることを確認してください。特に断りのない限り、無菌状態の下で生物学的安全キャビネット内で室温ですべての手順を実行します。 1.1）刺激とMPのコレクション9 <ol><li> 37℃の水浴1000万CEM T細胞のアリコートを解凍する。 10ミリリットルで希釈し、予め温めた無血清200 GX 5分で15ミリリットル滅菌チューブと遠心分離機で、例えばX-VIVOのような造血中。予め温め培地5mlの上清と再懸濁細胞を吸引除去する。 </li><li>このようなX-VIVOとして予め温め造血媒体15 mlの（懸濁細胞）T75組織培養フラスコに細胞を移し、5％CO 2で37℃で加湿インキュベーター中で4日間インキュベートする。 </li><li> 4日後、100mlの新鮮な培地を含有するT175組織培養フラスコ中にすべての培地および細胞を移す。彼ら200万細胞/ mlの密度に成長するまで、同じ条件下で約72時間細胞をインキュベートし続ける。 </li><li>均等に4 T175フラスコをそれぞれ含む150ミリリットルの新鮮な培地と細胞を2万個/ mlの密度まで（約48時間のインキュベーション）に成長するまで、細胞培養を継続する間にセルを分割する。 </li><li&gt; 5分間200×gでの遠心分離によって各フラスコから細胞を収集し、200万/ mlの細胞密度を維持するために、150ミリリットル新鮮な培地を含む新しいT175フラスコに300×10 6個の細胞を懸濁します。 </li><li>を0.5μg/ mlの最終濃度で培地に（2 mg / mlのでDMSOに溶解）アクチノマイシンDを添加し、24時間インキュベートする。 </li><li> 50ミリリットルコニカルチューブにすべての培養液を移し、5分間750×gで細胞をスピンダウン。大細胞断片を除去するために15分間1500×gで50ミリリットルコニカルチューブと遠心分離機に上清を移し。 </li><li> 50分間12000×gで250ミリリットルボトルと超遠心機に上清を移し。上清を捨て、ペレットを収集します。 </li><li>洗浄50分間12000×gで遠心分離によって50mlチューブ中で40 mlの滅菌PBSにペレットのLMPが富化。二回、この手順を繰り返します。 </li><li>最後の洗浄上清を収集。車両の制御に使用される。 1でのLMPペレットを一時停止mlのPBSと1.5 mlの滅菌マイクロチューブに移す。アリコートとストア（複数の空き融解サイクルを避けるために）-80℃でのLMPを単離した。 </li></ol> FACS分析4を介してMPの1.2）キャラクタリゼーション<ol><li>のCaCl 2なしで2アネキシンバッファのサンプル、1とし、別のを準備します。ヘペスが10mM、NaClが140 mMの、プラスマイナス5のCaCl 2。 </li><li>粒子を除去するために0.22μmのフィルターを用いてアネキシンバッファおよびFACSフローシース流体をフィルタ。 </li><li> FACSチューブに5 mMのCaCl 2をアネキシンバッファー44μlの中のLMPの1μLに希釈する。のCaCl 2なしアネキシンバッファの44μL（陰性対照）でのLMPの1μlの別のチューブを準備します。 </li><li>各チューブにアネキシンV-Cy5での5μlを加え、よく混ぜる。暗所でRTで15分間インキュベートする。各チューブ内FACSフローシース液の400μlのミックスを希釈することにより反応を停止します。 </li><li> BEAを数える7μmの10μlの（20万ビーズ）を追加各管中の内部標準としてのds懸濁液は絶対数を取得する。 </li><li>相対的な大きさのゲート（FSC-H、PMT E00、スケールログ）と相対粒度（SSC-H、PMT 325、ログスケール）のサイズ·キャリブレーション蛍光ビーズを使用して、フローサイトメーター上のドットプロットを確立さ1μm（ゲート1）と7ミクロン（ゲート2）でビーズゲートを数える。 </li><li> 2万カウンティングビーズがゲート2に到達するまでの信号を取得することにより、ドットプロット（ログスケール、PMT 765）アネキシンのための確立されたゲートとFL-4チャネルを使用してFSC-H / SSC-Hプロット上のLMPサンプルを分析します。 </li><li>のCaCl 2を含有アネキシンバッファ内のLMPのアネキシンV陽性の事象を決定し、その後のCaCl 2（ネガティブコントロール）することなく、アネキシンバッファ内のLMPのイベントを引く。 </li><li>次式に基づいてMPの絶対数を計算する。 <img alt="式1" fo:content-width="4in" src="/files/ftp_upload/52651/52651eq1.jpg" /></li></ol> MP Pの1.3）の決定rotein濃度（ブラッドフォードアッセイ） <ol><li> 1.25〜20 / mlのからのタンパク質標準の5連続希釈を準備します。ピペット連で清潔な試験管に各標準と試料液を800μL。各チューブにブラッドフォード色素試薬を200μlを追加します。室温で5分間インキュベートその後、よく混ぜる。 </li><li> 595 nmの吸光度を測定します。標準曲線の線形回帰を使用してのLMPのタンパク質濃度を決定します。 </li></ol> 2.気管支組織外植とのLMPトリートメント注：無菌の作業環境には特に注意してください、そして無菌的に以下の実験に使用される溶液及び培地を調製。 、完全な治癒培地を調製100ミリリットル組織ヒーリング中に（氷上で解凍）血清を用いて組織ヒーリングミディアムサプリメントの1ミリリットルを加え、よく混ぜます。 気管支組織外植片の2.1）の準備<ol><li>培養前に、1cm 2の6領域を傷つけるメスの刃で各100mmの組織培養皿の表面のエッジにそれぞれ。コートはそれぞれの培養皿のコーティング溶液2mlと100mmの組織培養皿に傷、37℃の加湿CO 2インキュベータO / Nで皿をインキュベートする。真空が余剰液を吸引し、組織洗浄​​媒体15mlで料理を埋める。 </li><li>動物のケア倫理委員会によって承認されたプロトコルに従ってCO 2吸入により（5〜7週齢）C57BL / 6マウスを安楽死させる。 </li><li>無菌的には、メス、デュモンスーパーファインピンセット、および外科はさみで肺組織を解剖。慎重に実質および血管を取り除く。該当する場合は、研究室への輸送のための氷冷組織洗浄媒体に肺組織を置きます。 </li><li>さらに組織洗浄培地中に沈め気管支を解剖し、末梢肺組織から1〜2.5ミリメートルの直径を有する気管支の分離。メスで〜5ミリメ​​ートルの厚さの気管支リングにスライス気管支組織。 </li&gt; <li>気管支断片をピックアップし、料理の傷領域上に配置する無菌の湾曲したmicrodissecting鉗子ですくい運動を使用してください。 </li><li>組織洗浄媒体を取り外し、それらを皿に付着させ〜室温で5分間の断片をインキュベート。 </li><li>各ディッシュに完全な治癒中の10ミリリットルを追加し、制御された雰囲気モジュラーインキュベーターチャンバーに配置します。高いO 2混合ガス（70％O 2、25％N 2及び5％CO 2）を有するチャンバを洗い流す。ベンチトップ軌道インキュベーター内室を置き、37℃でそれを振る。培地フラグメント上に断続的に流れることができるように、毎分10サイクルで24時間チャンバーを振る。 </li><li> 24時間インキュベートした後、位相差倒立光学顕微鏡で組織外植片を観察します。完全な、細い髪の動きとその後のLMPの治療のために活発な気管支上皮と気管支植片を選択します。 </li></ol>2.2）のLMPトリートメント<ol><li>解凍成長培地サプリメントを氷上で血清および線維芽細胞阻害剤と次のように完全増殖培地を準備します。血清及び増殖培地100mlに200μlの線維芽細胞阻害剤による成長培地サプリメントを1ml加える。完全に混合する。使用前に10分間37℃で、完全増殖培地を温める。 </li><li>を800μg/ mlの濃度でのLMPストックを調製するためにPBSで新しい無菌のエッペンドルフチュー​​ブに孤立のLMPを希釈する。 </li><li> 12ウェル組織培養プレートの各ウェルに完全な増殖培地を0.5mlを加える。 </li><li>組織培養プレートの各ウェルに、前のプロトコル（2.1節）から湾曲したmicrodissecting鉗子と、選択した気管支植片を転送します。 </li><li>のLMP処理ウェルと対照ウェルを識別するために、適切に培養プレートにラベルを付けます。そして25μlの制御車両（40 / mlの最終濃度）を各ウェルのLMPの治療に25μLのLMPの株式を追加します（LMP<html> S対照ウェルへの生産）。 </li><li>穏やかに振盪しながら37℃で制御された雰囲気モジュラーインキュベーターチャンバー内のインキュベーションを続けます。 </li><li> 24時間後、PBSで外植3回洗浄し、次のステップ（4％パラホルムアルデヒド[PFA]固定）に進みます。 </li></ol> 3.病理組織学的検査 3.1）次のステップに進む前に、次のソリューションを準備<ol><li> 137のNaCl、2.7のKCl、10のNa 2 HPO 4、1.76 mMのKH 2 PO 4、pHが7.4を混合して1×PBS緩衝液を準備します。 </li><li> 、4％PFAを調製400mlの水に20gのPFAを溶解、攪拌しながら60℃で加熱すること。解決策をクリアする10 M NaOHを数滴を追加します。次に1×PBS緩衝液を追加し、7.4に500ミリリットルとpHに音量を調整します。フィルタとアリコート。 -20℃で保存する。 </li><li>以下の脱水や水分補給の試薬を準備します。 100％、90％、70％、50％エタノール及びキシレン。 </li></ol>e_step &quot;&gt; 3.2）植固定および組織節脱パラフィン<ol><li> 4％PFAの1.5ミリリットルで標識されたマイクロ遠心チューブ内の各植片を置き、4℃でO / Nインキュベートする。 1×PBSで2回植片をすすぐ。 </li><li>アルコール系を通して外植片を脱水し（70％エタノール：各3回、30分間、90％エタノール：各2回​​30分間、100％エタノール：3回30分ずつ、次にキシレン：3回20分ずつ）。ドラフト内で室温ですべての手順を実行します。 </li><li>オーブンで58℃のパラフィンに組織外植を埋め込む。ロータリーミクロトームを用いて5μm厚の組織切片を準備します。 </li><li> 56℃の水浴中で切片をフロートし、次いで標識された組織スライド上に切片をマウントする。 1時間65℃で手動染色ラックと乾燥でスライドを置きます。スライドを室温で冷却する。 </li><li>パラフィンを除去するために10分間、キシレンをそれぞれ含む4個の連続染色ディッシュ内のラックを浸し。目、その後、100％、95％のキシレンを除去し、エタノール系列でラックを浸し次に、80％、70％、次いで50％エタノール（各ステップで5分間）エン。エタノールを除去し、5分間水道水でラックをすすぐ。 </li></ol> 3.3）ヘマトキシリン​​およびエオシン（H＆E）染色<ol><li>固定組織切片で作業を続ける。 15分間マイヤーのヘマトキシリン​​で満たされた染色皿にラックを配置。 20分間ヘマトキシリン​​を除去するために水道水でラックをすすぐ。 </li><li> 30秒間の95％エタノール中で30 sec.Place蒸留水に置く。 1分間のエオシンYソリューション染色皿に置きます。 2分ごとに、95％エタノール、100％エタノール、キシレン2変化を通して脱水する。 </li><li>過剰エオシンが除去されることを保証するために、顕微鏡下でクイックチェックを実行します。カバーガラスでカバーした後、各スライド上に封入剤（フィッシャーSP15-100）の2から3滴を置きます。 </li></ol> 3.4）In Situ細胞死検出の場合 ：TUNELアッセイ<ol><li>開始する前に、プロテイナーゼKワーキング溶液を調製：20μg/ mlの中で10mMトリス/塩酸、pHは7.4。 </li><li>セクション3.2（外植固定および組織切片の脱パラフィン）の5〜ステップ1を繰り返します。脱イオンH 2 Oでスライドをすすぐ</li><li> 10分間1X PBSでスライドを浸し。過剰のPBSを排出します。プロテイナーゼK希釈標準溶液をRTで30分間、組織切片をインキュベートする。リンスは、1×PBSで2回スライドする。 </li><li>細胞死検出キットの取扱説明書に記載されているようにTUNELアッセイを実行します。封入剤を使用してマウントし、カバーガラスを使用して手動でカバースリップ。 </li><li>光学顕微鏡下でサンプルを分析します。茶色のアポトーシス細胞を分析するために4.5 Proの画像を使用してください。 </li></ol>

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	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., 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=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., 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=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, S., 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=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. 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著者らは、開示することは何もない。

MPは、細胞間のクロストークのアクティブなメディエーターであり、それらの研究は、科学の多くの分野において有望である。11本研究は、アポトーシスT細胞株由来のLMP のインビトロでの大規模生成のための詳細なプロトコルを提示した。これらのMPは、リンパ球分子の大きなレパートリーを発現し、生物学的細胞および組織の恒常性の調節に関与している。しかし、異なる?...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

<table border="0" cellpadding="0" cellspacing="0" width="828">
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generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

細胞間コミュニケーションにおける細胞膜由来の小胞の生物学的役割への関心が近年増加している。微粒子（MPは）は0.1μm〜1μmの直径の範囲の、小胞のような一種類であり、そして典型的に活性化またはアポトーシスを起こして、真核細胞の細胞膜から脱落。ここでは、D.のLMPアクチノマイシンで刺激しアポトーシスCEM T細胞からTリンパ球由来の微粒子（のLMP）の生成が多段階分画遠心法によって単離し、フローサイトメトリーを使用して特徴づけられる記述する。このプロトコルはまた、マウス初代呼吸気管支組織外植片から派生気管支上皮細胞上のLMPのアポトーシス促進効果を実証するための、その場で細胞死検出法を提案する。本明細書に記載の方法は、in vitroでのアポトーシスリンパ球からのLMPの豊富な量を単離するための再現可能な手順を提供する。のLMPは、導出このように様々な疾患モデルの特性を評価するために使用され、薬理学および毒性試験のためにすることができる。気道上皮は、外部環境と下層組織との間に保護物理的および機能的なバリアを提供することを考慮すると、気管支組織外植片ではなく、不死化上皮細胞系の使用は、気道管組織を必要とする研究のための有効なモデルを提供する。

のLMPは（FACS）分析およびMPの97％（≤1μm）をアネキシンV-Cy5での陽性（ 図1Aおよび1B）であった1μmのビーズを使用してゲート制御を蛍光活性化細胞ソーティングによって、アネキシンV染色10で特徴付けた。典型的には、のLMPの約2.5mgが、このプロトコルに従って得た。 C57BL / 6マウスからの気管支組織外植片は、車両とのLMP処理を行った。気管支セクションの組織病理学的?...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

細胞膜小屋微粒子（MPS）を単離し、それらの病態生理学的効果は、様々なモデルで研究することができるアクティブな生物学的な小胞である。ここでは、Tリンパ球（のLMP）由来のMPを生成する気道上皮細胞に対するアポトーシス促進効果を実証するための方法を記載する。

気道上皮細胞上でのアポトーシス促進効果のリンパ球微粒子および検出の生成

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

generation-lymphocytic-microparticles-detection-their-proapoptotic

Research

JoVE Journal

Immunology and Infection

8.9K ビュー.  University of Montréal. 細胞膜小屋微粒子（MPS）を単離し、それらの病態生理学的効果は、様々なモデルで研究することができるアクティブな生物学的な小胞である。ここでは、Tリンパ球（のLMP）由来のMPを生成する気道上皮細胞に対するアポトーシス促進効果を実証するための方法を記載する。 

ビデオ: Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

免疫・感染学

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

M細胞による細菌の取り込みを評価するパイエル板腸管ループアッセイを連結したマウスのアプリケーション

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

呼吸器病原体に対する宿主細胞応答を研究するために偏ヒト気道上皮Calu-3細胞の液体に覆われた文化の確立<em&gt;インビトロ</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

ヒト胸腺からの骨髄樹状細胞と上皮細胞の単離

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

赤血球上の抗体誘導性補体活性化の定量的検出のための方法

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

網膜色素上皮細胞に貪食アッセイのためにブタまたはウシの感光体外側セグメントの大規模精製

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表現型および慢性リンパ球性脈絡髄膜炎ウイルスに感染したマウスから単離された活性化制御性T細胞の機能解析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

体内上部気道免疫メディエーター レベルの定量化のための粘膜内層流体の非侵襲的サンプリング

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

分離と機能研究の好中球由来の微粒子のキャラクタリゼーション

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...