著者は大いにピエールBourdoncle、トーマスギルバートとコーチンイメージング施設のルイーズRimbaultを認める。この作品は、INSERM、CNRSとリーグ国立コントル·ルがん（エキップのlabellisée）によってサポートされていました。

Tリンパ球の調製<ol><li> 10,11を説明したように一次マウスまたはヒトT細胞を準備します。 </li><li> 3×10 6細胞/ mlの- 2の密度で、10％ヒト血清ABを含む完全RPMI培地中のヒトT細胞を養う。 注：代わりに、ヒト血清のウシ胎児血清（FCS）を使用すると、培養中のヒトT細胞の大部分の自発分極を誘発することができる。 4培養におけるこれらの細胞を維持 - 5日、さらに、この期間中にいつでもそれらを処理。 </li><li>類似の細胞密度で、10％FCSを補充した完全RPMI培地中でマウスT細胞を維持する。 10ng / mlのIL7の存在下で、37℃でO / N培養した後、次の日すぐに使用するか。 </li><li>完全RPMI中CEMヒトT細胞株を育成10％FCSを補充した。 </li></ol> 2.ケモカイン刺激<ol><li> 10 mMのHEPEを補足した暖かいHBSS培地で実験条件あたり5×10 5細胞を洗浄S。いくつかの実験のために、2μgのpmaxGFP及び8μgのをpcDNA3.1 +（空ベクター対照）、デッドPKCζキナーゼ、またはN末端​​Par6プラスミドで前日初代ヒトT細胞を共トランスフェクトする。 </li><li>温かいHBSS-Hepes緩衝培地中で細胞ペレットを再懸濁し（実験条件0.3mlのxの番号を使用する）。 </li><li> 1.5ミリリットルマイクロ遠心チューブに細胞を配布します。 注：したがって、単一の実験条件に対応する1管は0.3ミリリットルHBSS-HEPES培地中で5×10 5個の細胞が含まれています。いくつかの実験では、500 nMのラトランクリンAまたは媒体（DMSO）を追加し、ケモカイン刺激の前に30分間細胞をインキュベート。 </li><li>各チューブに10から500 / mlのCCL19またはCXCL12ケモカインを追加します。 注： - ヒトT細胞のための200 / mlのケモカイン我々は通常、10を使用しています。 CEM細胞は、CCL19に結合するCCR7受容体を発現しない。そのため、CEM細胞だけCXCL12刺激に反応することができるようになります。 500 n - でまた、より高いケモカ​​イン濃度（300を使用グラム/ ml）をマウスのT細胞を分極する。 </li><li>チューブを数回反転し、それぞれ、一次T細胞またはCEM 8または10分間37℃の水浴中でそれらを培養する。 </li><li>各チューブに2％パラホルムアルデヒド（PFA）及びPBS中10mMのHepesを含む温かい溶液0.3mlを加えて分極処理を停止する。チューブを反転し、5分間37℃でそれらをインキュベートする。 </li></ol> 3.染色は<ol><li>チューブフローサイトメトリーマイクロチューブから細胞を転送します。 </li><li> 1％ウシ血清アルブミン（BSA）、0.5mMのEDTA、PBS中の10mMのHepesを含むRT溶液で完全にチューブを埋める。 </li><li> 4分間460×gでチューブを遠心。 </li><li>上清を捨て、PBS中の5％FCSを含有するRT溶液100μlで細胞を懸濁します。 </li><li>離れて光から、渦それらを、各チューブに、FITC抗HLA-ABCの5μl加えて、室温で30分間、それらをインキュベートする。 </li><li> PBSで5％FCS、Aを含むで細胞を1回洗浄する一度PBSでのみND。 </li><li> RTで：1％のBSA、0.5mMのEDTA、PBS中10mMのHEPESを含む溶液で細胞を洗浄し、10分間インキュベートし、500μlの1％PFAを加える。 </li><li>上清を捨て、0.2％BSA、0.1％サポニン、0.5mMのEDTAおよび10mMのHepes（透過化緩衝液）を含むPBS溶液で完全にチューブを埋める。 </li><li>この培地中で細胞を2回洗浄する。 </li><li>培地を除去し、細胞ペレットを解離するためにそれらをボルテックスするチューブを裏返し。 </li><li> 、各チューブに抗P-ERMの抗体の100倍希釈渦にそれらを、室温で30分間インキュベート：0.25 / mlのTRITCファロイジンおよび/または1を追加します。 </li><li>透過化バッファーで細胞を洗浄。必要に応じて、蛍光二次抗体とインキュベートします。 </li><li> 200μlのPBSに細胞を再懸濁し、マイクロチューブに転送。 注：細胞を取得するための準備ができている。また、チューブあたり200μlの最大総量を維持しながら、最終的に1％に濃縮されたPFAを追加細胞は、さらなる処理のために同じ日に使用されるべきではない場合染色を保存するために集中。 </li></ol> 4.イメージの取得と分析<ol><li>装置のスイッチを入れ（ 特定の試薬​​/機器の表を参照）、それはメーカーの指示に従って、それ自体を校正しましょう。 NOTE：INSPIREソフトウェアは対物レンズ40倍（0.75 NA）で使用した。 IDEAS 3.0ソフトウェアは、報告されたすべての定量化のために使用された。 </li><li>将来の実験のためのテンプレートに保存することができます取得一連のウィンドウを準備します。 RMS勾配値を定量化ヒストグラムを描画します。 RMS勾配ヒストグラム上で選択し「フォーカスでは「集団から、エリアのヒストグラムの「単一セル」の人口をdelimitate。マイクロ遠心チューブが機械内に挿入された後、個々の細胞上にゲートをレーザーパワーを調整し、5000 Tリンパ球を取得する。 </li><li>アイデアでソフトウェアは、取得ファイルを開き、個々のセルに対して得られた明視野画像（チャンネル1）のための勾配RMS値の値が表示されたヒストグラムを作成します。 57より上のRMSの勾配値を示す細胞を考慮RMS勾配ヒストグラムにゲートを描きます。 注：RMS勾配分析において考慮焦点面内にある唯一のTリンパ球を取ることができる。我々は、57よりも優れたRMS勾配値を示す個々の細胞を焦点面にあり、正確に考慮することができることを実験的に決定している。 </li><li>解析/マスクは、メニューでは、2画素の侵食された明視野画像M01、上の形態学のマスクから（M01,2）を侵食すると呼ばれる、新しいマスクを作成します。その後、分析に/むしばむ（M01,2）マスク上の計算されたエリアの新機能を、作成し、メニューを用意しています。前のゲートに選択された細胞から、この新しく作成されたマスクを使用してセルの面積を示すヒストグラムを開きます。 注：によって利用可能な形態学マスクデフォルトでは、セルの実際のサイズよりもはるかに大きいです。これにより、2ピクセルにそれを侵食することがより正確である。 </li><li>キャリブレーションビーズや破片（小エリア）とダブレット（大面積）を除く、個々のT細胞上にゲートを描きます。各取得時にこのゲートを調整します。 注：セルサイズの変動は、ゲートの位置に影響を与える。マウスT細胞はCEM細胞より小さい自体、ヒトT細胞よりも小さいように、各細胞型の領域は、その大きさに比例する。 </li><li>前のステップでゲートしたシングル、焦点の合った細胞を考慮すると、ファロイジンでのRAWマックス·ピクセルの関数として、HLA-ABC染色に直接上限·ピクセルからなるドットプロット（チャネル2、X軸）を開く染色（チャンネル4、Y軸）。無染色または飽和蛍光イベントを除外するためにゲートを作成します。 HLA-ABC（チャネル2）とファロイジン（チャンネル4）染色のために直接上限画素を示すオー​​プンつのヒストグラム。 </li><li>解析/マスクメニュー、レコード生成で2画素の浸食HLA-ABC染色細胞（チャネル2）の形態のマスクから（M02,2）をむしばむと呼ばれるEA新しいマスク、、。その後、分析に/むしばむ（M02,2）で計算真円度パラメータからなる​​新しい機能を作成し、メニューを用意しています。 注：このパラメータは、円からの細胞形状の偏差を測定します。したがって、完全に丸いT細胞は、低円形度を示すことになる細長い極性細胞に対し高い真円度値を有する。 </li><li>その後、以前にゲート細胞から円形の特徴の値を報告します別のヒストグラムを作成します。個々のセルのための円形度の値に応じて合焦細胞集団、直接個々の分布をプロットするために、このヒストグラムを使用する。 </li><li>非刺激細胞のためにヒストグラムの形状を見ると、極性細胞のためにゲートを描き、非刺激細胞のほとんどの真円度の限界値まで最低の真円度の値で始まるプロットされている。ゲーテッド人口（％はゲートで囲われた）の間で偏細胞の割合の統計表示を見てください。 注：我々は、13以下の円形度指数値のため、このゲートを設定している。 </li><li>細胞内のアクチン染色の偏光を見るために、HLA-ABC染色（チャネル2）およびファロイジン染色を比較し、明るい詳細類似R3の値のヒストグラムをプロット（チャンネル4）。 注：この大きな特徴の値は、二以上の染色である重畳。 </li><li>前と同じゲート戦略を使用して、偏アクチン染色を有する細胞のパーセンテージを示す分離された染色のための非刺激細胞上にゲートをプロットする。 </li><li>完了すると、MHCクラスIおよびファロイジン染色、偏光された細胞の割合の分布に偏析を示す細胞のパーセンテージは平均±SD、全てのセルの真円度と類似度指標の平均値をcorrespに示されていると共にonding統計パネル。 </li></ol>

<ol>
	<li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chemokines+and+their+receptors.">The biology of chemokines and their receptors.</a> Annu Rev Immunol. 18, 217-242 (2000).</li><li>Thelen, M., Stein, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chemokines+invite+leukocytes+to+dance.">How chemokines invite leukocytes to dance.</a> Nat Immunol. 9 (9), 953-959 (2008).</li><li>Rougerie, P., Rho Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+masters+of+T+lymphocyte+migration+and+activation.">GTPases: masters of T lymphocyte migration and activation.</a> Immunol Lett. 142 (1-2), 1-13 (2012).</li><li>Sanchez-Madrid, F., del Pozo, 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=Leukocyte+polarization+in+cell+migration+and+immune+interactions.">Leukocyte polarization in cell migration and immune interactions.</a> Embo J. 18 (3), 501-511 (1999).</li><li>Adams, D. 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著者は、彼らが競合する経済的利益を持っていないことを宣言。

イメージングフローサイトメトリーの最近の技術を使用して、ケモカイン刺激によって誘発される細胞および分子事象を分析するための迅速かつ有益なゲーティング戦略が提示されている。ケモカイン刺激及び分極プロセス中の異なるタンパク質の細胞内分布によって誘導される細胞形態の変化：単一の実験から、人は情報の2つの主要なタイプを得ることができる。興味深いことに、証拠は...

ケモカインは、特殊な場所1に細胞を引き付ける小さな可溶性タンパク質である。したがって、それらは、組織中の細胞の正確な位置決め、発達および生理学において重要な機能に関与する。それは効果的な免疫応答を協調して作用する多くの異なる細胞型の作用に依存して、免疫系は、この規則の例外ではない。外来抗原が検出され、中和され得る前に、所定の状態でつの免疫細胞型の特定の位置を制御することによって、ケモカインは、予め求められている。 特に、Tリンパ球において、ケモカインは、T細胞の運動2,3を支持し係合すると、多くの細胞内シグナルを誘発する、特異的な表面受容体（カルシウム上昇、ERKリン酸化のRho GTPアーゼ活性化、インテグリン親和性および細胞骨格の変化の増加）に結合する。細胞レベルでは、人は、ケモカイン刺激により誘発される形態学的変化を観察することができる。細胞形状の変化はT細胞に特に劇的である：血流中に移動するとき、休止T細胞は、ビーズ状の丸い形態を持っている。前方に前縁しかしながら、炎症部位において又はリンパ系器官の近傍でのケモカインの存在の検出は、現在のバイポーラ形からなる典型的な「ハンドミラー」形態を採用するT細胞の形状を変更しようとしているそしてバック4で立ち下がりエッジ、または腹肢、。また、細胞内成分は、マイグレーションを維持するために、偏T細胞のこれらの二つの対向領域に分離することができる。例えば、ケモカイン刺激5と重合アクチン時アクチンフィラメントの重合増加が偏T細胞2の前面に蓄積する。一方、皮質F-アクチン細胞骨格を血漿膜にリンクエズリン - ラジキシン、モエシン（ERM）ファミリーのリン酸化タンパク質のようないくつかのタンパク質は、偏光の腹肢に再局在化するT細胞を6。興味深いことに、本発明者らおよび他のこの分極処理は、T細胞の遊走に必要とされることを示した。確かに、偏光と干渉する任意の治療は、細胞の運動性を阻害します。例えば非定型プロテインキナーゼC（PKC）ファミリー、PKCζおよびPKCιブロックT細胞分極および樹状細胞7のそれらの遊走スキャン処理のメンバーの活性の阻害。 T細胞の分極ものRho GTPアーゼによって調節される。我々は、最近記載Fam65bタンパク質によるRhoAの活性の調節は、T細胞の形態の変化及びトランスウェルアッセイ6中を移動する能力を妨害することを示している。分極は、細胞の運動性の前提条件のステップであるとして、それはケモカイン応答の主要な読み出しとして定量化することができるようにすることが重要である。細胞形状の変化は、以前に手動8を測定した。しかし、定量化のこの種は、非常に時間のかかるであり、その通常は数そう細胞の数が考慮される。 ここでは、新たな方法を迅速にケモカイン刺激にさらされるTリンパ球の形状変化の度合いを定量化するために提示される。イメージングフローサイトメトリー技術（ 具体的な試薬/機器の表を参照）、フローサイトメーターの利点を組み合わせた、使用され、ケモカイン刺激の異なる条件で効率的に偏光した細胞の量を定量する顕微鏡9。一つは、この技術でロバスト測定できる形態変化の定量化に加えて、ケモカインシグナリングの際にいくつかのタンパク質の細胞内局在の変化を評価することも可能である。

<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

細胞は、偏光と呼ばれるプロセスで、かつ多くのタンパク質の細胞内局在を変化させることによって、彼らの丸い形状を失うことによる刺激ケモカインに応答します。古典的な画像化技術は、これらの現象を研究するために使用されてきた。しかしながら、それらは時間のかかる形態の定量化および細胞の数の染色の共局在続く多くの細胞の手動取得を必要とした。ここでは、迅速かつ強力な方法は、サイトメーターのものと顕微鏡の利点を組み合わせたイメージングフローサイトメトリー技術を用いて細胞数千からなるサンプルにこれらの現象を研究するために記載されている。 MHCクラスI分子および繊維状アクチンのためのCCL19及び染色で刺激したTリンパ球を使用して、ゲーティング戦略を同時に形状変化の程度及びCCL19シグナル伝達によって影響されるマーカーの共局在化の程度を測定するために提示されている。また、このゲーティング戦略はobservに私たちを許可E CXCL12刺激後の偏T細胞中で（フロントで）繊維状アクチンと（後方の）リン酸化されたエズリン - ラディキシンモエシン（リン酸化ERM）タンパク質の分離。 Par6 /非定型PKCシグナル伝達経路の変異体の薬理学的阻害剤と式でアクチン重合の阻害：この手法は、2つの異なる要素の偏光に遮断効果を観察することが有用であった。したがって、証拠は、この技術は、形態学的変化およびタンパク質の再分配の両方を分析するのに有用であることが示されている。

ここで選択された最初の例では、CCL19で刺激したヒト一次Tリンパ球の使用に関する。しかし、同じ戦略は、初代マウスT細胞、T細胞株またはケモカイン刺激に応答する任意の他の細胞型で使用することができる。ここで紹介するゲーティング戦略は集中イベント、単一のセルを選択一連のウィンドウが含まれています。 （ 図1）またはCCL19刺激された（ 図2）Tリンパ?...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

ケモカインシグナリングにより誘導される細胞と分子分極の迅速かつ堅牢な分析

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

rapid-robust-analysis-cellular-molecular-polarization-induced

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.0K ビュー.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

ビデオ: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

の細胞内相互作用の可視化と定量化髄膜炎菌</em共焦点イメージングによるα-アクチニン>と人間

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

免疫・感染学

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

用ドリップ流れと回転円板の原子炉の使用黄色ブドウ球菌バイオフィルム分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

蚊の機能解析のためのカルシウムの生物発光アッセイ（ネッタイシマカ）とダニ（ Rhipicephalus microplus）Gタンパク質共役受容体

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

合成脂質二重膜上でHIV-1エンベロープ誘発性ウイルス学的シナプスとシグナリングのイメージング

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

TCRとそれに関連するシグナル伝達タンパク質のリアルタイムの同時イメージングのための全反射顕微鏡法

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

プロデュース上皮損傷の解析<em&gt;赤痢アメーバ</em&gt;感染症

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISHによるマウスのBリンパ球における染色体異常の迅速分析

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

インフルエンザウイルスAおよびBの急速な分子の検出と分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

解離性個体の親和性の浄化 (BiCAP) によって多蛋白質シグナリング複合体

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.