著者らの研究室での作業は、カナダ自然科学工学研究評議会、健康研究のカナダ研究所、およびカナダリサーチチェアプログラムからの補助金によってサポートされています。 JLは、フォンデルシェルシュエンサンテケベックからの資金提供を通じて、GroupeドールエチュードデProtéines Membranaires（GEPROM）からフェローシップでサポートされています。

1。予備：細菌株及び上皮細胞。 細胞や細菌の操作は、層流フードの下に、無菌的に実行されます。 <ol><li>たてE.を隔離大腸菌株2787、2787Δ アイーダ 、およびC600グリセロールストックからの溶原性のブイヨン（LB）寒天プレート（1％トリプトン、0.5％塩化ナトリウム、0.5％酵母エキス、1.5％寒天）上で、37で成長℃、アッセイのばらつきを最小限に抑えるために、それは新たにメッキ菌株を常に使用すると℃の密閉ペトリ皿上にのみ数週間、最大で4℃で菌株を維持することをお勧めします。 </li><li> 10％の熱不活化ウシ血清（熱不活性化が56で行われる℃で30分間）を添加した高グルコースダルベッコ改変イーグル培地（DMEM）で培養HEp - 2細胞（ATCC CCL - 23）。ルーチン培養中に我々はまた、10 U / mlペニシリン、10μg/ mlのストレプトマイシンを追加します。 </li><li> 37 HEp - 2細胞を成長· CO 2 5％を含有する雰囲気中で細胞培養におけるC。 75センチメートル2フラスコで増殖させ、彼らがコンフルエントに達するたびに継代培養された細胞を、維持するために標準的な細胞培養の手順を使用します。彼らが30〜40継代に到達し、それらを破棄するまで、我々は培養細胞を使用してください。 </li><li> 75センチメートル2フラスコでHEp - 2細胞がほぼコンフルエントに達しているときにアッセイを準備し、したがって、接着アッセイのために準備が整いました。 </li></ol> 2。 1日目：接種と上皮細胞の準備<ol><li> 8グラム/ LのNaCl、0.2 g / Lの塩化カリウム、0.2 g / LのKH 2 PO 4、0.21グラム/ LのNa 2 HPO 4：：7H暖かいダルベッコのリン酸が（DPBS緩衝生理食塩水で一度フラスコにHEp - 2細胞を洗う2 O） </li><li>新鮮な暖かい完全培地を添加する前に5分間EDTA - 細胞を、0.05％トリプシンでインキュベートする。新鮮な培地が追加された後、細胞をピペッティングにより再懸濁する。 </li><li> 5分間、2,000 rpmで細胞懸濁液を遠心し、DPBSでペレットを再懸濁し、再度遠心する。 </li><li> 10％の血清を添加したが2 × 10 5細胞/ mlの濃度で、どの抗生物質を含まない新鮮な培地で細胞を再懸濁します。 </li><li> 24ウェルプレートの中央にシードデュプリケートウェルの3セットで細胞懸濁液の1ミリリットル（各系統用）と、細胞培養インキュベーターで一晩インキュベートする。 注：血清を徹底的にdecomplementedするとその抗生物質がこの段階の後に省略されていることが重要です。これを怠ると、感染菌を殺すかもしれない。 </li><li> LBブロス5mlの（1％トリプトン、0.5％塩化ナトリウム、0.5％酵母エキス）で各細菌株の一つ孤立したコロニー（2787年、2787Δ アイーダとC600）を植菌し、37℃で一晩成長°激しく振盪とC（180 rpmで）。 </li></ol> 3。 2日目：細胞の感染<ol><li>彼らは少なくとも90％コンフルエントと汚染されていないことを確かめるために倒立顕微鏡下でHEp - 2細胞を検査する。 </li><li>新鮮な培地1 mlの10％の血清を補充がない抗生物質を含まないを追加、それぞれによく、暖かいDPBSで細胞を洗浄し。セルなしの3つのウエルに新鮮培地を追加することも。これは、各菌株のための接種物中の細菌の総数を決定するために使用されます。 </li><li>細菌培養の600nm（OD 0.600 nm）での光学密度を測定します。 HEp - 2細胞を含むデュプリケートウェルの一組にしてもいずれでもない細胞を含む各細菌培養物のアリコートを追加。通常、我々は10 6コロニー形成単位（cfu）に対応する一晩培養液量を使用してください。 （：セル細菌）これは、5:1の感染の多重度（MOI）を表します。我々が直接私たちの一晩培養を使用していますが、それは（そのような細胞毒のような、例えば）一晩培養中に存在する分泌分子の有害な影響を避けるために、細菌を遠心分離し、DPBSでそれらを再懸濁するために時々お勧めです 注：MOIは、100:1〜1:10の間で異なる場合があります。高いMOI収量より高い変動と背景と細菌はプレートのプラスチックに固執する傾向がある。 MOI下でも高い変動性をもたらします。 MOI選択されると、それは一貫性があるとこのMOIを保つために不可欠です。 </li><li> 37℃で3時間の細胞培養インキュベーターに感染した細胞をインキュベート℃、5％のCO 2。 注：これは、細胞と接触して、すべての細菌を直接持って来るために、低速（1〜2分間など千× g）で簡単にプレートを遠心することも可能です。これは、感染を同期させる利点があり、短いインキュベーション時間（わずか15〜30分）が可能。 </li><li>感染細胞から培地を取り出し、冷めないようにDPBSで細胞を3回洗浄。このステップでは、付着細菌は通常、標準的な顕微鏡で見ることが、我々は日常的にこのチェックを実行することができます。 </li><li>細胞を溶解し、付着した細菌をデタッチするにはteriaは、よく細胞を含むそれぞれに1％トリトンの100μlのX - 100を追加します。他の洗剤は（そのようなインスタンスのためのサポニンとして）使用することができます。 </li><li>室温で10分間細胞をインキュベートし、LB培地900μlを加える。 </li><li>静かにアップダウンを繰り返しピペッティングにより懸濁液をホモジナイズする。接種を含む細胞を含まないウェルを同様に穏やかにホモジナイズされています。 注：一部の細菌は、我々は0.05％トリプシンとのインキュベーションにより上皮細胞を切り離し、その場合には、洗剤に敏感すぎかもしれない- EDTAを20分間37℃で付着細菌と一緒に細胞はまた、ピペットチップでウェルから掻き取りすることができます。 </li><li> LB寒天培地上の3希釈（通常1:1,000、1:10,000及び1:100,000希釈）からLBブロスとプレートを100μlを用いて付着した細菌や接種の懸濁液の連続10倍希釈液を調製し、37℃で一晩インキュベート° C 。 </li></ol> 4。 3日目：CFUカウントとデータのプレゼンテーション。 <ol><li>プレート上のコロニーをカウントし、希釈液の各シリーズを平均化することにより、付着した細菌のと接種のcfuの数を計算する。 10から300コロニーの間を持つ唯一のプレートはカウントする必要があります。 </li></ol> 5。代表的な結果： 次の表は、異なる日に行わ3つの実験から付着した細菌や接種のカウントcfuの典型的な結果を示しています。 <img src="/files/ftp_upload/2783/2783table1.jpg" alt="表1" /> 結果は、図1Aに示すように、直接付着したCFUとして報告することができます。接種材料のサイズが原因で成長率またはピペッティングエラーの違いの菌株間で異なるため、それは多くの場合、付着細菌の割合として結果を報告することをお勧めします。図1Bに示すように、付着細菌の割合は、接種のCFUの数によって付着細菌のCFUの数で除して算出することができます。図1Bのすべての列を比較するために反復測定ANOVAとBonferroniのポストテストを行うことにより、我々は2787年と2787Δ アイーダの間に、同様に2787とC600の間に有意差（P &lt;0.05）が、有意差があることがわかります2787Δ アイーダとC600の間に。 <img src="/files/ftp_upload/2783/2783fig1.jpg" alt="図1"/> 図1。付着した細菌のCFU（A）または付着細菌の割合（B）のような結果の表現観察された遵守の割合は、しばしば実験的なセットアップ（および、より少ない程度で、実験上）に非常に依存しています。特に重要なMOIで、洗浄回数は、その割合は、文字通りに解釈されるべきではない。 接種材料中の細菌は、しばしば細胞のウェルの中の細菌の本当の総数と比較して接種のサイズを傾斜、細胞の存在下で細菌よりもはるかに速く成長することができます。この問題を軽減するために、それはどちらにお勧めです：（i）の接種を決定するために細胞と井戸を使用してください：HEp - 2細胞は、追加のウェルに播種し、感染している。アッセイの終わり、ではなく、廃棄の上清と洗浄で、10％トリトンX - 100100μlをウェルに添加する。細胞が溶解されると付着し、非付着細菌を含むライセートを穏やかにピペッティング上下を繰り返すことで均一化される;または（ii）アッセイの終わりに感染した細胞の上清を収集するだけでなく、上清DPBSで洗浄し、これらのプールされた上清中のCFUの数を決定します。これは、非付着菌のCFUの数が得られます。付着細菌の割合は、付着および非付着細菌のcfuの数を加えることにより付着した細菌のCFUの数で除して算出することができます。 アッセイの頑健性を示すために、最近設立された気管子豚の細胞株は、nptr 8から細胞とインキュベートS4074で行わアッセイ、 アクチpleuropneumoniae 7の接着剤ブタ病原性株でこのプロトコルを比較することができます。さておき細菌や細胞の増殖に必要なメディアの違いから、唯一の違いは、感染のために使用される細菌は指数関数的に成長している文化から、ではなく、定常期の一晩培養からのものであるということです。 A.あるpleuropneumoniaeそれはそうでなければ毒素の分泌は、細胞死とバイアスの結果を引き起こす、MOI 10:1を尊重し、感染の3時間を超えていないことも非常に重要です。 Aのほかに、この菌株それはコンフルエントの細胞を使用すると細胞は脱却していないことを視覚的に確認することが重要ですので、pleuropneumoniaeは容易にプラスチックに付着することがあります。

<ol>
	<li>Kline, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+adhesins+in+host-microbe+interactions.">Bacterial adhesins in host-microbe interactions.</a> Cell Host Microbe. 5, 580-580 (2009).</li><li>Cossart, P., Boquet, P., Normark, S., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+microbiology+emerging.">Cellular microbiology emerging.</a> Science. 271, 315-315 (1996).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-405 (1994).</li><li>Pizarro-Cerda, J., Lecuit, M., Cossart, P., Sansonetti, P., 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=.">.</a> Molecular Cellular Microbiology. , 161-161 (2002).</li><li>Srivastava, A., Isberg, R. R., Sansonetti, P., 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=.">.</a> Molecular Cellular Microbiology. , 179-179 (2002).</li><li>Benz, I., Schmidt, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+an+adhesin+(AIDA-I)+involved+in+diffuse+adherence+of+enteropathogenic+Escherichia+coli.">Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.</a> Infect Immun. 57, 1506-1506 (1989).</li><li>Charbonneau, M. E., Berthiaume, F., Mourez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+processing+is+not+essential+for+multiple+functions+of+the+Escherichia+coli+autotransporter+adhesin+involved+in+diffuse+adherence+(AIDA-I).">Proteolytic processing is not essential for multiple functions of the Escherichia coli autotransporter adhesin involved in diffuse adherence (AIDA-I).</a> J Bacteriol. 188, 8504-8504 (2006).</li><li>Jacques, M., Paradis, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesin-receptor+interactions+in+Pasteurellaceae.">Adhesin-receptor interactions in Pasteurellaceae.</a> FEMS Microbiol Rev. 22, 45-45 (1998).</li><li>Auger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+of+Actinobacillus+pleuropneumoniae+with+porcine+lung+and+tracheal+epithelial+cells.">Host-pathogen interactions of Actinobacillus pleuropneumoniae with porcine lung and tracheal epithelial cells.</a> Infect Immun. 77, 1426-1426 (2009).</li><li>Jacques, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipo-oligosaccharides+and+lipopolysaccharides+in+bacterial+adherence.">Role of lipo-oligosaccharides and lipopolysaccharides in bacterial adherence.</a> Trends Microbiol. 4, 408-408 (1996).</li><li>Nataro, J. P., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diarrheagenic+Escherichia+coli.">Diarrheagenic Escherichia coli.</a> Clin Microbiol Rev. 11, 142-142 (1998).</li><li>Jallat, C., Darfeuille-Michaud, A., Rich, C., Joly, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+clinical+isolates+of+diarrhoeogenic+Escherichia+coli:+diffusely+adhering+E.+coli+strains+with+multiple+adhesive+factors.">Survey of clinical isolates of diarrhoeogenic Escherichia coli: diffusely adhering E. coli strains with multiple adhesive factors.</a> Res Microbiol. 145, 621-621 (1994).</li></ol>

利害の衝突は宣言されません。

このプロトコルは、（ゲンタマイシン保護アッセイ3を使用など）の侵入を研究するように変更することができる標準的な細菌付着アッセイを説明します。コロニー形成単位の計数は、ギムザ染色などの顕微鏡下での標準的な可視化技術に依存するアプローチは、との比較により、定量化が可能になります。後者は、密着性の定性的なビューを提供しますが、接着の様々なパターンを?...

<table border="1"><tbody><tr><th>試薬</th><th>会社</th><th>カタログ番号</th></tr><tr><td>高グルコースDMEM </td><td> GIBCO -インビトロジェン</td><td> 12430-054 </td></tr><tr><td> DDPBS </td><td> GIBCO -インビトロジェン</td><td> 14190-144 </td></tr><tr><td>ウシ成長血清</td><td> Hyclone </td><td> SH3054 </td></tr><tr><td>ペニシリン/ストレプトマイシン</td><td> GIBCO -インビトロジェン</td><td> 15140-148 </td></tr><tr><td> 24ウェルプレート</td><td>コー​​ニング</td><td> 3337 </td></tr><tr><td>トリトンX - 100 </td><td>フィッシャーサイエンティフィック</td><td> BP151 - 100 </td></tr></tbody></table>

in vitro assay of bacterial adhesion onto mammalian epithelial cells

感染症を引き起こすために、細菌はそのホストを植民地化する必要があります。細菌性病原体は、細胞1をホストするために添付ファイルを促進することができる様々な分子や構造を表現する。これらのアドヘシンは、宿主細胞表面の受容体や細菌と宿主との間のブリッジとして機能する可溶性タンパク質との相互作用に依存しています。接着前の侵略および/または毒素の分泌に重要な第一歩であり、したがってそれは、細菌性病因で検討する重要なイベントです。さらに、付着した細菌は、しばしば絶妙に微調整された細胞応答を誘導する、の研究は&quot;細胞の微生物学2&#39;のフィールドに出産を与えている。細菌宿主細胞上の接着とその侵略のための堅牢なアッセイは、したがって、細菌性病因の研究において重要な役割を果たし、長い多くの先駆者の研究室3,4で使用されている。これらのアッセイは、現在、細菌性病因に取り組む多くのラボで実践されています。 ここで、我々は、特定のアドヘシンの寄与を示す標準の遵守のアッセイを説明します。我々は、 大腸菌の菌株2787年5、拡散粘着（AIDA）に関与するautotransporterアドヘシンを発現するヒト病原性の菌株を使用してください。コントロールとして、我々 は 、AIDA遺伝子、2787Δ アイーダ （F.バーサウムとM.モーレス、未発表）、およびE.の商用実験室株を欠く変異株を使用大腸菌 、C600（New England Biolabs社）。細菌は一般的に使用されるHEP - 2のヒト上皮細胞株からの細胞に付着して残っている。このアッセイは、以下の広範囲に6日までに記載されている。

Watch this Scientific Journal Video about In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells at JoVE.com

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

このプロトコルは、培養細胞に付着している細菌のコロニー形成単位の数を数えるに構成された単純な細菌の接着アッセイです。アッセイは堅牢であり、アドヘシンの独立は学び、数多くのバリエーションが細菌性病因に取り組む多くのラボで使用されています。

哺乳類上皮細胞上に細菌付着のインビトロアッセイ

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

in-vitro-assay-of-bacterial-adhesion-onto-mammalian-epithelial-cells

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Immunology and Infection

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

25.2K ビュー.  Groupe de Recherche sur les Maladies Infectieuses du Porc GREMIP, Faculte de medecine veterinaire. このプロトコルは、培養細胞に付着している細菌のコロニー形成単位の数を数えるに構成された単純な細菌の接着アッセイです。アッセイは堅牢であり、アドヘシンの独立は学び、数多くのバリエーションが細菌性病因に取り組む多くのラボで使用されています。

ビデオ: In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

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 ...

免疫・感染学

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

腸管上皮細胞の腸内細菌の侵入<em&gt;インビトロ</em&gt;劇的垂直拡散室モデルを用いた強化され

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

インビトロ 病原体誘導性好中球トランス上皮移動を評価するコーカルチャーアッセイ

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

ヒト好フローチャンバー接着アッセイ

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Tリンパ球におけるインテグリン活性化の研究のための静的な接着アッセイ

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

哺乳動物細胞への機能性細菌エフェクターのエレクトロポレーション

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Tリンパ球接着分子の相互作用の研究のための接着性の下でせん断応力の測定

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...