本研究では、アレルギー感染症研究所、国立衛生研究所の学内研究部門によってサポートされていました。我々は、これらの研究で使用されているH57のscFvをご提供するためのヨハネスHuppa、マーク·デイビスに感謝しています。 RVは、この手法の開発中に有用な議論のためのキア·ノイマンに感謝します。

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor-proximal+signals+are+sustained+in+peripheral+microclusters+and+terminated+in+the+central+supramolecular+activation+cluster.">T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.</a> Immunity. 25, 117-127 (2006).</li><li>Yokosuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat. Immunol. 6, 1253-1262 (2005).</li><li>Huppa, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463, 963-967 (2010).</li><li>Tolar, P., Pierce, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conformation-induced+oligomerization+model+for+B+cell+receptor+microclustering+and+signaling.">A conformation-induced oligomerization model for B cell receptor microclustering and signaling.</a> Curr. Top. Microbiol. Immunol. 340, 155-169 (2010).</li><li>Treanor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+skeleton+controls+diffusion+dynamics+and+signaling+through+the+B+cell+receptor.">The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor.</a> Immunity. 32, 187-199 (2010).</li><li>Treanor, B., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insight+into+lymphocyte+activation+through+quantitative+imaging+and+theoretical+modelling.">Mechanistic insight into lymphocyte activation through quantitative imaging and theoretical modelling.</a> Curr. Opin. Immunol. 19, 476-483 (2007).</li><li>Sylvain, N. R., Nguyen, K., Bunnell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vav1-mediated+scaffolding+interactions+stabilize+SLP-76+microclusters+and+contribute+to+antigen-dependent+T+cell+responses.">Vav1-mediated scaffolding interactions stabilize SLP-76 microclusters and contribute to antigen-dependent T cell responses.</a> Sci. Signal. 4, ra14-ra14 (2011).</li><li>Purbhoo, 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=Dynamics+of+subsynaptic+vesicles+and+surface+microclusters+at+the+immunological+synapse.">Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse.</a> Sci. Signal. 3, ra36-ra36 (2010).</li><li>Lasserre, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ezrin+tunes+T-cell+activation+by+controlling+Dlg1+and+microtubule+positioning+at+the+immunological+synapse.">Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.</a> EMBO. J. 29, 2301-2314 (2010).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat. Rev. Immunol. 11, 21-33 (2011).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+7:+Total+internal+reflection+fluorescence+microscopy.">Chapter 7: Total internal reflection fluorescence microscopy.</a> Methods Cell. Biol. 89, 169-221 (2008).</li><li>Williamson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existing+clusters+of+the+adaptor+Lat+do+not+participate+in+early+T+cell+signaling+events.">Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events.</a> Nat. Immunol. 12, 655-662 (2011).</li></ol>

これらの実験で使用されているすべての動物は特定病原体フリーの環境で維持され、実験は、健康動物のケアおよび使用委員会の国民の協会によって承認されました。

ここではTIRFMと人工APCのようなガラスサポート脂質二重層を用いた抗原特異的なマウスの主要T細胞のシグナル伝達を勉強するようにシステムを説明します。技術は、正常にこれらの細胞でGFPタグ融合タンパク質を発現しているに依存しています。 T細胞をトランスフェクションは、常に困難な作業です。一般的に、レトロウイルスやレンチウイルスを用いたエレクトロポレーション、または遺伝子送達が使用されています。一つの技術は他よりも優れたではありません。両方がそれらの限界と利点を持っています。 1）それは、レトロではなくレンチウイルスのために要求されるように細胞が活発に分裂することを要求され​​ない、2）の発現レベルは細胞がイメージングの前にインキュベートされる時間を変えることによって制御することができます：我々は、エレクトロポレーションは、次のような利点があります見つける。最大の欠点は、我々はそれが非常に困難な初代培養細胞のサイズが大きいタンパク質を発現することを見つけることです。我々は、エレクトロポレーション後、私達に非常に優れた細胞の生存を与える方法を提案した。結果としてそれがapplicabです。siRNAを介した遺伝子サイレンシングを達成するためにそれを使用するル。 また、ここで色収差補正されており、異なる波長ごとに個別のアライメントを必要としないカスタマイズされた2チャンネル同時取得TIRF顕微鏡を説明します。これらの機能はあるが、市販されている。私たちのシステムは、全反射顕微鏡分野の専門家によって発行され、商業の選択肢よりも安価であるの原則に基づいています。私たちのデザインの一つの可能​​な批判は、我々は、異なる励起波長の入射角度と同じを使用していることです。これは、異なる励起波長の異なる全反射侵入深さをもたらすでしょう。我々は、エバネッセント波は指数関数的に減衰するフィールドであり、全反射、被写界深度は、波長の線形関数であるため、これらの影響は小さいと考えています。ガラス表面に近い蛍光体は、2つの波長間でのレーザー強度の差を体験しません。 PEの近くに蛍光体のためにnetrationの深さは、強度の違いは、2つの波長488と640nmの1.3倍、二つの波長488と561 nmの1.15倍となります。同じシステムは、全反射照明を利用して超解像技術と組み合わせて使用​​することができます。 また、カスタム構築されている二つのカメラシステムを記載している。 TIRFシステムと同様に、類似した装置も市販されている。我々のシステムは、波長の異なる組み合わせでの使用を許可し、フィルタとdichroicsを変更する柔軟性を提供します。我々のシステムの欠点は、2つの画像を整列するために必要な回転の1程度である。この問題は、ピエゾ駆動アライメントの回転度を提供しているカメラスタンドを使用することで解決できます。我々はまた、成功したカメラが異なっていて、別のピクセルサイズを持っているでは、この顕微鏡に2つのカメラシステムを導入しています。一つ一つのチャネルの感度と大きなdyを必要に応じてこのようなシステムが有用であろう別のnamic範囲は、どちらも同じカメラではほとんど利用できません。私たちは私たち16μmのピクセルサイズを持つPhotometricsクワント-EMのカメラで12.9μmの有効画素サイズを与える2x2ビニングでPhotometrics HQ-2カメラを使用していました。 180ミリメートルの焦点距離チューブレンズはクワント-EMに使用されたと145ミリメートルの焦点距離チューブレンズはHQ-2カメラを使用しました。 HQ-2はMetamorphソフトウェアを使用して制御されたとクワント-EMは、外部トリガ·モードで別のコンピュータにマイクロ·マネージャー·ソフトウェアを介して制御されていました。外部トリガは、Metamorphのconfigure照明の設定で追加のシャッターとして実装された測定·コンピューティングのDACボードのデジタル出力を使用してクワント-EMカメラに提供されていました。チューブレンズの焦点距離の比は、有効画素サイズの比率と一致しています。代表的なアラインメントを図5に示します。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>マウスT細胞のAmaxaのNucleofectorキット</td><td>ロンザ社</td><td> VPA-1006 </td><td></td></tr><tr><td> PureLink HiPureプラスミドミディプレップキット</td><td>ライフテクノロジーズ</td><td> K2100-05 </td><td>エンドトキシンフリーのDNAプレップ用</td></tr><tr><td> AmaxaのNucleofectorデバイス</td><td>ロンザ社</td><td> AAD-1001 </td><td></td></tr><tr><td>組換えマウスIL-2 </td><td>ぺプロテック株式会社</td><td> 212から12 </td><td></td></tr><tr><td> Fluoresbrite Multifluorescentミクロスフェア0.20μm </td><td>ポリサイエン株式会社</td><td> 24050から5 </td><td></td></tr><tr><td>ラボ·テックII 8ウェルチャンバーカバーガラス</td><td>サーモフィッシャーサイエンティフィック、ヌンク</td><td> 155409 </td><td></td></tr></tbody></table>

a tirf microscopy technique for real-time, simultaneous imaging of the tcr and its associated signaling proteins

それは主要組織適合遺伝子複合体（pMHC）抗原提示細胞（APC）の表面に発現したタンパク質によって結合した抗原ペプチド断片によって従事しているときにシグナルがT細胞受容体（TCR）を介して開始されます。 TCR複合体はCD3二量体（εδとεγヘテロとホモ二量体ζζ1）を関連付ける非共有結合的にそのリガンド結合TCRαβヘテロで構成されています。受容体の関与すると、CD3ζ鎖はLckは、Srcファミリーキナーゼによってリン酸化されています。そして、これはLckがによってリン酸化と活性化されるSykの家族キナーゼ、ZAP70の採用につながります。その後、ZAP70は、さまざまなシグナル伝達分子が多数の2を含む近位シグナル伝達複合体の形成を開始し、アダプタータンパク質LATとSLP76をリン酸化する。 カルシウムは、この複雑な最終的に結果の形成​​とRas依存TRAnscription因子の活性化とT細胞の分化2に上昇を与える遺伝子発現プログラムの複雑な一連の結果開始。 TCR信号（および分化の結果の状態）は、抗原の効力とクロストークco-stimulatory/co-inhibitoryと、ケモカイン、および3-4のサイトカイン受容体を含む他の多くの要因によって変調されています。さまざまな刺激条件下での近位シグナル伝達複合体の空間的および時間的な組織を研究することは、従って、TCRシグナル伝達経路だけでなく、他のシグナル伝達経路によるその調節を理解する鍵となります。 以前は5-6を説明したようにT細胞の細胞膜でTCRによって開始されたシグナル伝達を研究するための一つの非常に有用なモデルシステムは、ガラスでサポートされている脂質二重層である。彼らは人工的なAPCのようにT-サービングセルに抗原pMHC複合体、接着性、共刺激分子を提示するために利用することができる。撮像することによってT細胞と相互作用する内部全反射蛍光顕微鏡（TIRFM）を使用して、脂質二重層は、我々は7-8ガラスと細胞表面との間の空間の中では100nmに励起を制限することができます。これは、私たちは主に画像細胞膜で起こるシグナル伝達イベントをすることができます。我々はイメージングにTCR複合体にシグナル伝達タンパク質の募集に興味を持っているように、我々はH57抗体の蛍光灯のFab（フラグメントの抗原結合）断片（ハイブリドーマから精製されたH57-597で標識された、前記TCR 2つのカメラTIRFイメージングシステムを記述する、ATCC、ATCC番号：HB-218）TCRβのために固有のものであり、GFPでタグ付けシグナル伝達タンパク質は、同時に、リアルタイムでイメージングすることができます。この戦略は、T細胞の両方とTCRで行われているシグナル伝達事象の非常に動的な性質のために必要である。この画像診断法は、画像、単一のリガンド9月11日と同様に活性化受容体へのシグナル伝達分子の募集に研究を許可していると12月16日 in-situで生化学を勉強するための優れたシステムです。

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

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

それゆえ、それが関与するタンパク質の空間的および時間的挙動を調べることが重要であるシグナリングを理解するために、細胞膜内や細胞内の場所にどちらかのタンパク質の棲み分けが大幅にシグナル伝達の結果に影響を与えることができる1調節機構である。ここではT細胞のシグナル伝達を研究するための全反射顕微鏡ベースのシステムを説明しますが、広く適用可能である。

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 ...

a-tirf-microscopy-technique-for-real-time-simultaneous-imaging-tcr

Research

JoVE Journal

Immunology and Infection

23.7K ビュー.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. それゆえ、それが関与するタンパク質の空間的および時間的挙動を調べることが重要であるシグナリングを理解するために、細胞膜内や細胞内の場所にどちらかのタンパク質の棲み分けが大幅にシグナル伝達の結果に影響を与えることができる1調節機構である。ここではT細胞のシグナル伝達を研究するための全反射顕微鏡ベースのシステムを説明します...

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

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

ロイコトリエンBのリアルタイムイメージング 4介在細胞の移行とβ-アレスチンとBLT1の相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

免疫・感染学

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

複合体形成シグナリングT細胞のリアルタイムのライブイメージング

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

好中球遊出の下で生理的流れの間に内皮細胞 - 細胞接合のリアルタイムイメージング

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

における骨髄細胞ダイナミクスのリアルタイムイメージング<em&gt;のApc<sup&gt;最小/ +</sup</em&gt;腸腫瘍ディスク共焦点顕微鏡スピニングすることにより

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

リアルタイムPCRによるT細胞受容体切除サークル（TRECs）とK-削除換え切除サークル（KRECs）の同時定量

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

解析<em&gt;腸炎エルシニア</em&gt;エフェクター転β-ラクタマーゼエフェクター融合を用いて宿主細胞に

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

リアルタイム イメージングと二相循環フロー システムを用いた菌バイオ フィルム開発の定量

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

画像処理プロトコル、拡散の解析と蛍光顕微鏡による膜受容体のクラスター サイズ

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...