著者らはこの手法を開発するためのすべてのEIBのメンバーに感謝したいと思います。この研究は、特定領域研究&quot;膜輸送&quot;（HO）、および（KH）、若手研究（SFとKH）、および科学研究&quot;感染現象のマトリックス&quot;（HOによって部分的にサポートされていました）、および革新的な領域研究日本の教育、文化、スポーツ、科学技術省から&quot;細胞内ロジスティクス&quot;（HO）、。

1。細菌細胞の調製<ol><li>ストリークグリセロールは、LB上の蛍光細菌（例えば、ネズミチフス菌の発現GFPなど）アンピシリン100μg/ mlを含む寒天プレートを買い物カゴに追加。 </li><li>新しいLB培地2 mlの一晩LB寒天培地からの培養は、単一のコロニー。 </li><li> 600nmで1.0の光学密度に達するまで新たなLB培地とインキュベート4.5 mlにバクテリア培養液0.5mlを追加。 </li><li>遠心により収穫細菌細胞（3,000 × gで、5分、4℃）。 </li><li>上清を捨て、滅菌リン酸緩衝生理食塩水（PBS）5.0 mlで2回洗浄する。 </li><li> PBS 5mlでバクテリアペレットを再懸濁し、そして接種物として約10 7コロニー形成単位（CFU）を含む懸濁液50μlを使用してください。 </li><li>蛍光標識された細菌を用いた場合には、細菌細胞は、標準的なプロトコルに従って蛍光標識試薬で標識した。 </li></ol> 2。 Anesthesia <ol><li> 5％（v / v）で、小さなプラスチック製ボックス（10 × 10 × 5センチ）埋めることは、空気（：200ml /分の流量）と混合イソフルラン気化。 </li><li>ボックスの16 -弱い齢の雄または雌マウス八への麻酔。 </li><li>麻酔の後に解剖台にマウスを移動します。 </li><li> （ 図1）：連続して2％（V / V）でマウスを麻酔とイソフルランは、空気（200ml /分の流量）と混合気化。これは端末の手順です。 </li></ol> 3。連結したパイエル板のループアッセイ<ol><li>その後、切り込みを入れる腹部の皮膚の1cmと麻酔マウスの腹部腹膜をカットし、パイエル板を含む小腸を取り出してください。 </li><li>血管をしないように注意しながら、糸を縫製して腸をライゲーションする。注：のみバインド腸の一方の側、そして反対側は緩いまま。 </li><li>緩みの上で連結されたパイエル板のループに注射器で細菌懸濁液またはPBS（コントロール）の50μlを注入する腸のIDE（ 図2）。 </li><li>ゆるみ側をバインドし、クリップをマウスの腹部を閉じます。 </li><li> 1時間後、連結したパイエル板のループを削除し、頚椎脱臼によりマウスを安楽死させる。 </li><li>結紮パイエル板ループから税パイエル板。 </li><li>過剰な粘液や細菌を除去するために針に接続された注射器を使用してPBSを1mlによってパイエル板の頂端側に点滅。パイエル板はさらに2回点滅。 </li></ol> 4。全体のマウントの染色とパイエル板の共焦点顕微鏡解析<ol><li> 1時間氷上でBD Cytofix / Cytoperm溶液中でのパイエル板を修正。 </li><li>上で30分間0.1％（w / v）のサポニン、0.2％（w / v）のPBS中のBSAを含むブロッキングバッファー1mlをブロックしてBDパーマ/ 5分間洗浄バッファーを1mlのでパイエル板を3回洗浄し、氷。 </li><li>検出するために試料を200倍の抗マウス希釈GP2モノクローナル抗体（5μg/ ml）を追加するM細胞。 </li><li> 4℃、室温または一晩で2時間℃でインキュベート</li><li>冷PBS 1mlで3回洗浄し、その後DyLight549（20μg/ ml）を持つ試料に結合した200倍希釈した抗ラットIgG抗体を加える。 </li><li> 2時間のためのサンプルを氷上でインキュベートする。 </li><li>冷PBS 1mlで3回洗浄し、その後、F -アクチンを検出するために試料を50倍希釈のAlexa 633共役ファロイジンを追加します。 </li><li> 2時間氷上でインキュベートする。 </li><li>冷PBS 1mlで軽く洗ってください。その後、スライド上に円形のカバーガラス3〜4枚を配置し、スライド上のPBSでグリセロールの30％溶液（ 図3）の試料を埋め込 ​​みます。 </li><li> DM - IRE2共焦点レーザ走査型顕微鏡やDeltaVisionの復元のデコンボリューション顕微鏡で標本を観察。 </li></ol> 5。代表的な結果： GFP - チフス菌とパイエル板のループアッセイ結紮マウスの試験片の例muriumはDeltaVisionの復元のデコンボリューション顕微鏡（ 図4）で観察された。 GFP -、ネズミチフス菌は、GP2 + M細胞によってtranscytosedれる。マウスパイエル板腸管ループアッセイは、M細胞によってtranscytosedすることができる共生や病原性細菌の種類を識別することができます。 <img alt="図1" src="/files/ftp_upload/3225/3225fig1.jpg" /> 図1。気化イソフルラン条件の下でマウスの麻酔。気化イソフルランは、麻酔専用装置（左側）によって提供されました。 <img alt="図2" src="/files/ftp_upload/3225/3225fig2.jpg" /> 図2。結紮パイエル板腸管ループアッセイの概要。蛍光細菌懸濁液は腸の緩み側に連結されたパイエル板のループに注射器で注入した。 <img alt="図3" src="/files/ftp_upload/3225/3225fig3.jpg" /> <strong&gt;図3。試料のマウント。試料は穴あきプラスチックプレート（厚さ1mm）のスライドガラス上に接着剤でバインドされた特殊なスライド上にPBSでグリセロールの30％溶液に包埋した。 <img alt="図4" src="/files/ftp_upload/3225/3225fig4.jpg" /> 図4 GFP -、ネズミチフス菌とパイエル板のループアッセイを結紮マウスの例。 M細胞特異的マーカー5として報告されているGP2は、抗マウスGP2の抗体（赤）で染色した。試料はDeltaVision復元のデコンボリューション顕微鏡で観察した。 GFP発現、ネズミチフス菌は、M細胞の頂端部細胞膜上とapexの下にある細胞質小胞（矢頭）でGP2と共局在していた。上部、根尖ビュー。底面と側面、側面図。スケールバー：10μmである。

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, 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=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <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+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

利害の衝突は宣言されません。

<html>細菌懸濁液と連結パイエル板のインキュベーション時間は、M細胞への細菌の混入を観察するために1時間通常です。 4時間のインキュベーションの場合には、細菌は多くの場合、パイエル板のT細胞領域に検出されます。気化イソフルラン吸入麻酔は、マウスを安定に保つことができるように、細菌懸濁液と連結パイエル板のインキュベーション時間は、パイエル板のT細胞領域に移動する?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント（省略可能） </th></tr><tr><td> LB寒天アンピシリン 100 </td><td> SIGMA </td><td> L5667 </td><td></td></tr><tr><td> Cy3のモノ反応 染料パック</td><td> GEヘルスケア</td><td> PA23001 </td><td></td></tr><tr><td>のAlexa Fluor ® 350 カルボン酸、 スクシンイミジルエステル</td><td>インビトロジェン</td><td> A10168 </td><td></td></tr><tr><td>吸入麻酔 装置</td><td>信濃製作所</td><td> SN - 487 </td><td></td></tr><tr><td>固定と 透過化 ソリューション</td><td> BD </td><td> 554722 </td><td></td></tr><tr><td>抗マウス 糖タンパク質2 抗体を</td><td> MBL </td><td>D278 - 3 </td><td> 200倍希釈（5μg/ml） </td></tr><tr><td>ヤギ抗ラットIgG、 F（ab&#39;）2断片 具体的</td><td>ジャクソン イムノ</td><td> 112-505-006 </td><td> 200倍希釈（20μg/ml） </td></tr><tr><td>のAlexa Fluor ® 633 ファロイジン</td><td>分子プローブ</td><td> A22284 </td><td> 50倍希釈</td></tr><tr><td>共焦点レーザー 走査型顕微鏡</td><td>ライカマイクロシステムズ</td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision 修復 デコンボリューション 顕微鏡</td><td>アプライドプレシジョン</td><td> DeltaVisionコア</td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

私たちの腸の内側には共生細菌の膨大な数で生息しています。消化管の粘膜表面は、継続的に、時には病原体にさらされています。腸管関連リンパ組織（GALT）は、これらの微生物1、2への粘膜免疫応答の誘導に重要な役割を果たす。粘膜免疫応答を開始するには、粘膜の抗原は、パイエル板などの組織リンパ濾胞に上皮バリアを介して腸管内腔から運ばれている必要があります。この抗原のトランスは、特殊な上皮M細胞3、4によって媒介される。 M細胞は積極的に高分子や微生物を貪食非定型上皮細胞である。樹状細胞（DC）とマクロファージ、分解のためにリソソームへの標的抗原とは異なり、M細胞は主に内部化された抗原をtranscytose。 M細胞を介してこの積極的な高分子のトランスサイトーシスは、基盤となる組織リンパ濾胞に抗原を提供dは、抗原特異的粘膜免疫応答を開始するために不可欠であると考えられている。しかし、M細胞がこの抗原取り込みを促進する分子機構はほとんど不明です。我々は以前、特に腸の中のM細胞の頂端部細胞膜上に発現する糖タンパク質2（GP2）は、 大腸菌やサルモネラ血清型ネズミチフス菌（S.ネズミチフス菌を含む共生と病原性腸内細菌のサブセットのためのtranscytotic受容体として機能することを報告している）、FimH、細菌の外膜5上のI型線毛の成分を認識すること。ここで、我々は、マウスのパイエル板腸管ループアッセイのアプリケーションM細胞による細菌の取り込みを評価するための方法を提示する。このメソッドは、以前に6,7を説明したマウス腸管ループアッセイの改良版です。改良点は以下のとおりである：1。イソフルランは、麻酔剤として使用した。 2。約1cm結紮腸管ループincludINGパイエル板が設置されました。 3。 M細胞に取り込ま細菌は、蛍光の蛍光標識試薬によってまたは緑色蛍光タンパク質（GFP）などの過剰発現蛍光タンパク質で標識した。 4。パイエル板を覆う濾胞関連上皮におけるM細胞は、抗GP2抗体でホールマウントimmunostainigにより検出した。 5。 M細胞による蛍光細菌のトランスは、共焦点顕微鏡分析により観察した。マウスパイエル板腸管ループアッセイは、どのようなM細胞によってtranscytosed共生や病原性細菌の答えを供給することができる、とM細胞を介して粘膜免疫系を刺激する方法の分子機構を理解するのにつながる可能性があります。

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

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

パイエル板を覆う特殊な濾胞関連上皮におけるM細胞は腸管関連リンパ系組織における粘膜免疫監視に重要な役割を果たす。ここでは、M細胞による細菌のトランスサイトーシスのために評価方法を説明<em&gt; in vivoで</em&gt;。このメソッドは、免疫系におけるM細胞の機能を理解する方法を提供する。

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 ...

application-mouse-ligated-peyers-patch-intestinal-loop-assay-to

Research

JoVE Journal

Immunology and Infection

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

19.4K ビュー.  RIKEN Research Center for Allergy and Immunology. パイエル板を覆う特殊な濾胞関連上皮におけるM細胞は腸管関連リンパ系組織における粘膜免疫監視に重要な役割を果たす。ここでは、M細胞による細菌のトランスサイトーシスのために評価方法を説明 in vivoで。このメソッドは、免疫系におけるM細胞の機能を理解する方法を提供する。

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

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 ...

免疫・感染学

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 ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS媒介細菌競争を監視するには、Visualアッセイ

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

腸間膜リンパダクトカニューレを挿入ラットモデル：腸のリンパ医薬品輸送の評価への応用

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

牛にエフェクターおよびメモリーT細胞応答を評価するための長期培養インターフェロンγ酵素結合免疫アッセイの応用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Fcγ受容体媒介食作用を評価するために、単球単層アッセイの使用

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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 Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

新規In Vitro細胞移動を評価する癒しのアッセイを傷

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

2 光子レーザー顕微鏡を用いた細胞膜修復法

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.