ドミニクミルズはブルームズベリー大学博士学生の身分（2007-2010年）のによってサポートされていました。著者は、VDCのモデルを開発する上で彼らの支援のためにルブラGundogduとアブディエルミ両方に感謝したいと思います。

1。特別な0.4μmのフィルタでIECの単分子膜の成長<ol><li> 10％（v / v）のウシ胎児血清、100 U / mlのペニシリン、100μg/mlの/ mlのストレプトマイシンおよび1％（v / v）の内の非必須アミノ酸を補充したダルベッコ改変必須培地で培養のCaco-2細胞（DMEM） 37℃で標準組織培養インキュベーター℃、5％CO 2、95％空気中のC。種子は、0.4μmのあたり4×10 5のCaco-2 IECSは2〜3日ごとにメディアを変え、21日間にわたってフィルタと偏光に成長。 </li><li>ボルト - オーム抵抗計を用いて単層の偏光状態を確認するTEERを測定する。我々の研究では、21日のTEERはのCaco-2これらの0.4μmのフィルタで増殖した細胞は700-800Ωcmで2です。 </li></ol> 2。 C.の調製ジェジュニと共培養アッセイ用細菌培地<ol><li> VDCの共培養アッセイの所望の出発点の前に24時間は、Cの新鮮な血液寒天プレートの準備。 37℃ジェジュニとインキュベート℃の可変雰囲気インキュベーターで微好気条件（85％N 2、5％O 2、10％CO 2）下。 </li><li>同時に、可変雰囲気のインキュベーター中で微好気条件下で37℃でブルセラ菌培養液を30mlのpreinc​​ubate。 </li></ol> 3。アッセイに先立ちVDCハーフチェンバースの準備と滅菌<ol><li>完全に浸すVDC半分室と同様、Oリング、滅菌溶液中のプラグ。 </li><li>無菌の20ml注射器を使用して、半室の両方でガス注入口を通して殺菌液をフラッシュします。これを怠ると、共培養中に、サンプルの汚染が発生することがあります。 </li><li> &gt; 1時間消毒液に浸漬し、すべてのコンポーネントにしておきます。 </li><li>ガス注入口を洗浄するための別の滅菌20ミリリットル注射器を使用して、新鮮な滅菌水ですべてのコンポーネントをすすぐ。 </li></ol> 4。準備細菌接種の<ol><li> Cの半分のプレートを収穫血液寒天プレートからジェジュニの成長は前インキュベートブルセラブロス1mlのに前日用意しました。これは、〜10 10 CFU / mlに得られるはず。 </li><li>細菌のコロニー形成単位（CFUの）をsemiquantifyする600nmで細菌懸濁液の光学密度を測定します。 </li><li>プレインキュベートブルセラブロス4mlの中で希望の接種レベルに細菌懸濁液を調整します。 </li><li>その後接種に存在する細菌のCFUの数を定量化するために48時間変数雰囲気インキュベーター中37℃でインキュベート三重における血液寒天プレート上で適切な希釈のメッキに続いて、この最後の細菌懸濁液からの段階希釈を行います。 </li></ol> 5。 VDCを設定する<ol><li>ベンチの上にVDCフラットの下半分室を配置します。下半分室にOリングを取り付けます。 </li><li> TからIECSを運ぶつのフィルタを切り離す彼のキャリアとは、無菌のPBSを400μlで3回洗浄する。 Oリングが所定の位置に残っていることを確認し、下半分室の上にフィルターを置きます。 </li><li>優しく場所に上半分室を下げる。つの半室が組み立てられた後、リングクランプを使用して一緒にクランプします。上向きに直面している二つの開口部と、作業台の上に水平にVDCを置きます。 </li><li>尖半室に細菌接種4 mlを加え。 </li><li>基底外側半分室に細胞培養培地4 mlを加える。 </li><li>両方の半室の開口部に、エンドピースを置きます。 </li></ol> 6。可変雰囲気インキュベーター内ガスマニホールドにVDCを取り付ける<ol><li>ガスマニホールドに近接変数雰囲気インキュベーターにVDCを置きます。 </li><li>ガスマニホールドに接続されたガス供給レギュレータを開く。 VDCの基底外側半分室のガス入口にマニホールドからチューブを取り付けます。 GAを取り付けSアウトレットパイプは基底外側半室でエンドピースでアウトレットのいずれかに可変雰囲気インキュベータの導出。 </li><li>基底外側半室でエンドピース内の他の出口を閉鎖。これは、側底媒体の放出をもたらすことができるように、過剰な気体圧力を避けるために非常にゆっくりと開くように注意しながら、ガスマニホールドにガスの流れを開く。 </li><li>目的は、すべての2-5秒1バブルのガス流量である。定期的に、実験の過程でガスの流れを確認し、必要に応じて調整します。 </li></ol> 7。共培養後VDCの分解<ol><li>適切な共培養期間後に、ガスマニホールドに接続されたガス供給レギュレータを閉じる。マニホールドでVDC内にガスの流れを遮断する。ガス入口、ガス出口とVDCからストッパーを外します。 </li><li>変数大気インキュベーターからVDCを削除します。 subsequenため-80℃で頂端および基底外側清や店舗°Cを削除するT分析。 </li><li>リングクランプを外し、静かにVDCを離れて取る。半室と無菌6ウェル細胞培養皿への転送からフィルタを削除します。滅菌PBS400μlのでIECS 3回洗浄する。 </li><li>相互作用する細菌の総数の計数のため、IECを溶解し、室温で20分間インキュベートしIECSに0.1％を含有する滅菌PBS400μlの（v / v）のトリトンX-100を加える。その後、細菌CFUの相互作用の数を定量化するために48時間変数雰囲気インキュベーター中37℃でインキュベート三重における血液寒天プレート上で適切な希釈のメッキに続いて、この細胞溶解物から段階希釈​​を行います。 </li><li>侵入した細菌の総数を列挙するために、IECSに150μgの/ mlのゲンタマイシンを含むDMEM細胞培養培地を400μlを加え、37℃で標準的な組織培養インキュベータ中で2時間インキュベート℃のCO 2、5％C、95％細胞外の細菌を殺すために、空気、その後、上記のステップ7.4の場合とフォロー。 </li></ol> 8。共培養後VDCのクリーニング実験の次のラウンドのために滅菌水や店舗でVDCを洗ってください。

<ol>
	<li>Mills, D. 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=Increase+in+Campylobacter+jejuni+invasion+of+intestinal+epithelial+cells+under+low-oxygen+coculture+conditions+that+reflect+the+in+vivo+environment.">Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.</a> Infect. Immun. 80, 1690-1698 (2012).</li><li>Marteyn, B., 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=Modulation+of+Shigella+virulence+in+response+to+available+oxygen+in+vivo.">Modulation of Shigella virulence in response to available oxygen in vivo.</a> Nature. 465, 355-358 (2010).</li><li>Dorrell, N., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+second+century+of+Campylobacter+research:+recent+advances,+new+opportunities+and+old+problems.">The second century of Campylobacter research: recent advances, new opportunities and old problems.</a> Curr. Opin. Infect. Dis. 20, 514-518 (2007).</li><li>Young, K. T., Davis, L. M., Dirita, 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=Campylobacter+jejuni:+molecular+biology+and+pathogenesis.">Campylobacter jejuni: molecular biology and pathogenesis.</a> Nat. Rev. Microbiol. 5, 665-679 (2007).</li><li>Hu, L., Kopecko, D. J., Nachamkin, I., Szymanski, C. M., Blaser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+17.">Chapter 17.</a> Campylobacter. , 297-313 (2008).</li><li>Everest, P. H., 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=Differentiated+Caco-2+cells+as+a+model+for+enteric+invasion+by+Campylobacter+jejuni+and+C.+coli.">Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli.</a> J. Med. Microbiol. 37, 319-325 (1992).</li><li>Konkel, M. E., Hayes, S. F., Joens, L. A., Cieplak, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+the+internalization+and+intracellular+survival+of+Campylobacter+jejuni+in+human+epithelial+cell+cultures.">Characteristics of the internalization and intracellular survival of Campylobacter jejuni in human epithelial cell cultures.</a> Microb. Pathog. 13, 357-370 (1992).</li><li>Monteville, M. R., Konkel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin-facilitated+invasion+of+T84+eukaryotic+cells+by+Campylobacter+jejuni+occurs+preferentially+at+the+basolateral+cell+surface.">Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface.</a> Infect. Immun. 70, 6665-6671 (2002).</li><li>Friis, L. M., Pin, C., Pearson, B. M., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+culture+methods+for+investigating+Campylobacter+invasion+mechanisms.">In vitro cell culture methods for investigating Campylobacter invasion mechanisms.</a> J. Microbiol. Methods. 61, 145-160 (2005).</li><li>Schuller, S., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerobic+conditions+enhance+type+III+secretion+and+adherence+of+enterohaemorrhagic+Escherichia+coli+to+polarized+human+intestinal+epithelial+cells.">Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.</a> Environ. Microbiol. 12, 2426-2435 (2010).</li><li>Cottet, S., Corthesy-Theulaz, I., Spertini, F., Corthesy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microaerophilic+conditions+permit+to+mimic+in+vitro+events+occurring+during+in+vivo+Helicobacter+pylori+infection+and+to+identify+Rho/Ras-associated+proteins+in+cellular+signaling.">Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.</a> J. Biol. Chem. 277, 33978-33986 (2002).</li></ol>

我々は、開示することは何もありません。

宿主細胞と細菌性病原体の相互作用を研究するためのin vitro細胞培養法での使用が広く多くの研究室で用いられる技術である。例えば、細胞培養アッセイは好気的条件下で行われるしかし、これらは、in vitroモデルにおいて正確にホスト病原体の相互作用が行われたインビボ環境では表していない可能性がある。広く試験管内感染モデルで使用さ...

宿主細胞と細菌性病原体の相互作用は、in vitro細胞培養法での使用広範囲に研究されてきた。例えば、細胞培養アッセイを用いて、宿主細胞、宿主細胞受容体の同定、宿主細胞シグナル伝達経路および宿主細胞の細菌侵入に対する細菌の付着は、すべての多くの重要な観察結果として、詳細に研究されている。しかしながら、細胞培養アッセイは、インビボ環境を代表しないかもしれない好気的条件下で行われる。胃腸感染を研究するために使用されるin vitroモデルの主な制限は、高い酸素レベルを含む培養条件は、一般に、真核細胞の生存を支持することである。しかし腸管腔の状況はほぼ嫌気性になります。非常に低酸素環境での腸内病原体はその発現が変化する好気的条件下で2病原性遺伝子を発現する。このように、データは、標準的な細胞培養モデルを用いて得メートルAYは、宿主細胞と細菌相互作用の不正確な表示を与える。 カンピロバクターは、軽度の下痢から重度の炎症性腸炎に及ぶという症状を、世界的な細菌性急性胃腸炎の主要な原因物質である。 C.の大半合併症のない胃腸炎でジェジュニ感染の結果、しかし、C.ジェジュニは、ギラ ​​ン·バレー症候群（GBS）を含む末梢神経障害でも最も一般的に同定され感染性病原体である。英国では、C.によって引き起こされる腸炎の50万人以上のケースがあると推定されているポンド5.8億の英国経済への予測コストジェジュニ感染毎年。発展途上国では、C.ジェジュニは、子どもの死亡の主要な原因である。紛れもカンピロバクター感染症の重要性と徹底したゲノムベースの分析、C.含む研究の数十年にもかかわらず、 ジェジュニ病因は依然として不十分understですOOD、そのようなサルモネラ 、 大腸菌 、 赤痢菌 、およびコレラ菌などの他の腸の病原体とは対照的である。便利な小さな動物モデルの欠如は、この3つの主な理由です。も広くビトロ感染モデルで使用気性Cを研究するためのより多くの不適切な通性嫌気性菌であり、他の腸内病原体のためのよりジェジュニ 。ながらC.ジェジュニは侵襲的な病原体、Cのメカニズムとして認識されている腸上皮細胞のジェジュニ侵攻（IECS）は依然として不明である4,5。C.ジェジュニ侵入はマイクロフィラメント、微小管、両方またはどちらも5の組み合わせのどちらかに依存することが示されている。このエリアの混乱はおそらくビトロアッセイ条件の通報を使用することによるものである。 異なる細胞培養アッセイの数は、C.の相互作用を調査するために使用されているジェジュニ宿主細胞である。のCaco-2 6、INT 407 7、およびT84 8細胞株は、すべての異なる℃の接着および浸潤能力を研究するために使用されているジェジュニ菌株。しかし、Cの細菌の付着と侵入のレベルIECSとジェジュニは他の腸病原体9に比べ劇的に低くなっています。 Cの共培養IECSとジェジュニは、通常IECSの生存に必要な、大気中の酸素濃度に近い条件下で、CO 2インキュベーター内で行われる。℃でジェジュニ遺伝子の発現は、大気中の酸素条件と比較して腸管腔の低酸素環境において非常に異なるであろう。 垂直方向の拡散室（VDC）システムの使用は、異なる媒体とガス条件1,10,11で細菌および宿主細胞の共培養を可能に開発されている。このシステムは、細菌が解除になり、人間の腸内の条件を模倣非常に低い酸素ながら組織のファンデ条件は、血流からの酸素が供給される。特別0.4μmのフィルターで増殖偏IEC単層をそれぞれ個別細菌ブロスおよび細胞培養培地が充填され（ 図1）とした頂端側底区画を作成VDCに入れた。 VDCは、変数雰囲気のインキュベーターは、37℃で85％のN 2、5％O 2、10％CO 2を含有する°C、Cの最適条件を表すに入れたジェジュニ 。頂端側画分が開いたままと可変雰囲気のインキュベーター内で微好気雰囲気にさらされた、密封された側底区画をしながら、5％CO 2 /圧力の蓄積を防止する出口管と、95％O 2ガス混合物の一定の投与により酸素を供給した。これらの条件下でのCaco-2細胞の生存および単層の完全性が経上皮エレメントを監視することによって確認されたのCaco-2細胞単層と頂端コンパートメント内低酸素条件下での頂端と基底外側区画の物理的な分離の生存を実証するため、24時間以上の単層にわたるctrical抵抗（TEER）。変数雰囲気インキュベーター（微好気条件）で、標準的な細胞培養CO 2インキュベーター（好気的条件）で維持のVDCで単層のTEERは異なる大気条件1の下で細胞単層の完全性を示す有意な差は認められなかった。微好気条件下では、タイトジャンクションが存在し、均等に好気性条件1の下で維持された細胞に類似オクルディン染色パターンでセルの枠線との間で分配さ残った。 Cの相互作用VDC内のCaco-2とT84細胞とジェジュニは、細菌の相互作用（接着と浸潤）と浸潤を評価することによって調べた。二つの異なるC.ジェジュニ野生型straiNSは、1を使用しました。Cでジェジュニ 11168Hは、元のシーケンス株NCTC11168のhypermotile誘導体である。 11168H株はひよこ植民モデルではるかに高い植民地化レベルを示し、したがって、宿主 - 病原体相互作用の研究のために使用するのに適した菌株と考えられている。 81から176を分離人間であり、ほとんどの侵襲広く研究されている実験室株の一つである。C.ジェジュニ株は微好気性の条件のどちらかまたは下VDCの頂端の区画に追加されました。我々は相互作用と侵略の両方に高いレートはCのために記録された観察微好気条件1の下ジェジュニ 。増加したC.ジェジュニ相互作用は微好気条件1の下での細菌数の増加によるものではなかった。このデータは、VDCの先端コンパートメントにおける低酸素環境が細菌-宿主相互作用を強化し、示していると我々の仮説をサポートするCの侵襲的な性質ジェジュニは INCRですこれらの条件下で緩和。これは、侵襲的な細菌性病原体を研究するVDCモデルの使用の最初の報告書だったとより密接に生体状況で模倣するという条件の下でのin vitro感染アッセイで行うの意義を強調しています。 VDCモデルは、多くの異なる細菌種のホスト病原体の相互作用を研究するために使用することができる。

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Speciality vertical diffusion system for use with Snapwell inserts</td>
			<td>Harvard Apparatus</td>
			<td>66-0001</td>
			<td>Manifold &#38; six Snapwell chambers</td>
		</tr>
		<tr>
			<td>Caps</td>
			<td>Harvard Apparatus</td>
			<td>66-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Harvard Apparatus</td>
			<td>66-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Harvard Apparatus</td>
			<td>66-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Snapwell filters (pore size 0.4 &#956;m)</td>
			<td>Corning Costar</td>
			<td>3407</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Volt-Ohm resistance meter</td>
			<td>Millipore</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>WPA Lightwave II spectrophotometer</td>
			<td>Biochrom</td>
			<td>80-3003-72</td>
			<td></td>
		</tr>
	</tbody>
</table>

enteric bacterial invasion of intestinal epithelial cells in vitro is dramatically enhanced using a vertical diffusion chamber model

宿主細胞と細菌性病原体の相互作用は、in vitro細胞培養法での使用広範囲に研究されてきた。例えば、細胞培養アッセイは好気的条件下で行われるしかし、これらは、in vitroモデルにおいて正確にホスト病原体の相互作用が行われたインビボ環境では表していない可能性がある。我々は、異なる媒体とガス条件下で細菌および宿主細胞の共培養を可能にする感染のインビトロのモデルを開発した。垂直拡散室（VDC）のモデルは、細菌が非常に低酸素ながら組織の条件の下になり、人間の腸内での条件が血流からの酸素が供給される模倣。 VDCにSnapwellを挿入で栽培偏腸管上皮細胞（IEC）単層を配置すると、別個の頂端と基底外側の区画を作成します。基底外側区画は、細胞培養培地で充填密封し、酸素ワットで灌流されている微好気条件下でのオープンとインキュベート保ち、頂端区画はスープで満たされているhilst。両方のCaco-2およびT84 IECSは、細胞生存または単層の整合性に明らかな悪影響することなく、これらの条件下で、VDCに維持することができる。異なるCで行わ共培養実験頂端側画分における微好気条件でジェジュニ野生株とVDCモデルにおける異なるIEC線は再現性好気培養条件と比較して、相互作用の数（約10倍）の増加および細胞内（略100倍）細菌をもたらす1。 VDCモデルで作成した環境は、より密接にCで遭遇する環境を模倣人間の腸内ジェジュニとより密接に生体現実に模倣するという条件の下でのin vitro感染アッセイを行うことの重要性を強調しています。私たちは、VDCモデルの使用は、相互作用の新たな解釈は賭けることができますことを提案細菌性病原体と宿主細胞を予期する。

Cで行われる共培養実験ジェジュニ野生型株および頂端側画分における微好気条件でVDCモデルにおけるIECSは時間内に好気培養条件と比較して、相互作用の数の増加（約10倍）および細胞内（略100倍）細菌を実証している依存性の様式1。この観察は、2つの異なるC.を用いて再現可能であったジェジュニは、野生型株（11168H及び81-176）および2つの異なるIEC線（?...

Watch this Scientific Journal Video about Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model at JoVE.com

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

好気的条件下で細菌の付着および浸潤を調べるための細胞培養アッセイを行うと、通常の典型的である<em生体内で&gt;</em&gt;環境。垂直拡散室モデルは、ヒト病原体の相互作用の研究を可能にします<em&gt;カンピロバクター</emもっと下の腸上皮細胞と&gt;<em生体内で&gt;</em条件&gt;のような、強化された細菌の侵入になる。

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 ...

enteric-bacterial-invasion-intestinal-epithelial-cells-vitro-is

Research

JoVE Journal

Immunology and Infection

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

13.7K ビュー.  London School of Hygiene & Tropical Medicine. 好気的条件下で細菌の付着および浸潤を調べるための細胞培養アッセイを行うと、通常の典型的である環境。垂直拡散室モデルは、ヒト病原体の相互作用の研究を可能にしますカンピロバクターのような、強化された細菌の侵入になる。

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

免疫・感染学

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

マウスでイメージ細菌感染にルシフェラーゼを用いた

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

細菌性病原体によるヒト細胞の浸潤

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

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 ...

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 ...

Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. 
We found the optimum fatty acid concentrations to be between 1-5 ng/&mu;l, and that for cholera toxin to be &lt; 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

ヒト腸管上皮細胞およびBALB / cマウスのマクロファージを用いた耐容脂肪酸とコレラ毒素の濃度の測定

The positive role of fatty acids in the prevention and alleviation of non-human and  human diseases have been and continue to be extensively documented. ...

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 ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

リステリア単球遺伝子を用いた感染マウスにおける培養および心臓コロニー形成における心臓細胞の細菌浸潤の評価

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳動物細胞の細菌攻撃後のストレス顆粒形成の免疫蛍光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

高スループット赤痢菌-特定の殺菌試験

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...