仕事はNIH R01 NS071039-01A1研究助成金によって支えられている。

<ol>
	<li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+the+Healthy+Brain.">The Role of Microglia in the Healthy Brain.</a> J. Neurosci. 31, 16064-16069 (2011).</li><li>Graeber, M. B., Li, W., Rodriguez, M. L. <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+microglia+in+CNS+inflammation.">Role of microglia in CNS inflammation.</a> FEBS Lett. , (2011).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Perruisseau-Carrier, C., Jurga, M., Forraz, N., McGuckin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+stem+cell+reprogramming+for+neuronal+induction+and+differentiation.">miRNAs stem cell reprogramming for neuronal induction and differentiation.</a> Mol. Neurobiol. 43, 215-227 (2011).</li><li>McCoy, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+miRNAs+in+cytokine+signaling.">The role of miRNAs in cytokine signaling.</a> Front Biosci. 17, 2161-2171 (2010).</li><li>He, M., Xu, Z., Ding, T., Kuang, D. M., Zheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-155+regulates+inflammatory+cytokine+production+in+tumor-associated+macrophages+via+targeting+C/EBPbeta.">MicroRNA-155 regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta.</a> Cell Mol. Immunol. 6, 343-352 (2009).</li><li>Ponomarev, E. D., Veremeyko, T., Barteneva, N., Krichevsky, A. M., Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-124+promotes+microglia+quiescence+and+suppresses+EAE+by+deactivating+macrophages+via+the+C/EBP-alpha-PU.1+pathway.">MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway.</a> Nat. Med. 17, 64-70 (2011).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546-3791 (2010).</li><li>Gabriely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+21+promotes+glioma+invasion+by+targeting+matrix+metalloproteinase+regulators.">MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.</a> Mol. Cell Biol. 28, 5369-5380 (2008).</li><li>Caffarelli, E., Filetici, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+in+cancer+development.">Epigenetic regulation in cancer development.</a> Front Biosci. 17, 2682-2694 (2011).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J. Neurosci. Methods. 194, 287-296 (2011).</li><li>Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Throughput+MicroRNA+Profiling:+Optimized+Multiplex+qRT-PCR+at+Nanoliter+Scale+on+the+Fluidigm+Dynamic+ArrayTM+IFCs.">High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs.</a> J. Vis. Exp. (54), e2552 (2011).</li></ol>

利害の衝突が宣言されません。

最近大きな関心は、ミクログリアと神経炎症および神経変性に関連する多くの病態におけるこれらの細胞の関与に与えられた。さらに、免疫やがん細胞の分化におけるマイクロRNAの役割は劇的に過去数年間5,10を介して成長してきました。我々は以前通常のCNS ​​におけるミクログリアの非活性化または休止表現型の維持7でのmiR-124の役割を報告しているが、ミクログリアの?...

<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> 1X PBS </td><td> GIBCO </td><td> 14190 </td><td> 7.2から7.4：pHは、実験室で乾燥成分から調製することができる</td></tr><tr><td> 10×PBS </td><td>サーモフィッシャーサイエンティフィック</td><td> SH30258.02 </td><td> 7.2から7.4：pHは、実験室で乾燥成分から調製することができる</td></tr><tr><td> DMEM、FBS </td><td> GBCO </td><td> 11965; 10438 </td><td></td></tr><tr><td> miRVanaキット</td><td>インビトロジェン</td><td> AM1561 </td><td></td></tr><tr><td>パーコール</td><td>シグマ</td><td> P4937-500ミリリットル</td><td> 100％のパーコール100 mlの10×PBSの10mlおよびシグマからパーコールの90ミリリットルを混合して調製されています。 70％パーコールの50ミリリットルです。100％のパーコールの35ミリリットルの混合によって調製し、DMEM 15 mlの 40％の50ミリリットルパーコールは、100％のパーコールの20ミリリットルとの1X PBS 30mlを混合することによって調製された</td></tr><tr><td> 35から55のペプチドをモグ</td><td> AnaSpec株式会社</td><td> 60130から5 </td><td></td></tr><tr><td> CFA </td><td> DIFCO </td><td> 263810 </td><td></td></tr><tr><td>百日咳毒素</td><td>シグマ</td><td> P7208 </td><td></td></tr><tr><td> FcRは、抗体をブロッキング</td><td> BDバイオサイエンス</td><td> 553142 </td><td>クローン2.4G2 </td></tr><tr><td>抗CD11b - PEモノクローナル抗体</td><td> BDバイオサイエンス</td><td> 553311 </td><td>異なる蛍光色素（例えば、AF488）で使用することができます</td></tr><tr><td>抗CD45-APC-Cy7 mAbを</td><td> Biolegend </td><td> 103116 </td><td>異なる蛍光色素（例えばAPC）で使用することができます</td></tr><tr><td> Paraformaldehyde（16％） </td><td> EMS社</td><td> 15710 </td><td> 1X PBSで1：15に希釈し</td></tr><tr><td> 15パーセントTBE-尿素ゲル</td><td>ライフ·テクノロジーズ·インク</td><td> EC68855BOX </td><td></td></tr><tr><td>のTaqMan MicroRNAの逆転写キット</td><td>ライフテクノロジーズ株式会社</td><td> 4366596 </td><td></td></tr><tr><td>のTaqManユニバーサルPCRマスターミックス</td><td>ライフテクノロジーズ株式会社</td><td> 4304437 </td><td></td></tr><tr><td>のTaqMan MicroRNAのアッセイMMU-MIR-124A </td><td>ライフテクノロジーズ株式会社</td><td> 4427975 ID 001182 </td><td></td></tr><tr><td>のTaqMan MicroRNAのアッセイsnoRNA-55 </td><td>ライフテクノロジーズ株式会社</td><td> 4427975 ID 001228 </td><td></td></tr><tr><td>ヌクレアーゼフリー水</td><td>ライフテクノロジーズ株式会社</td><td> AM9937 </td><td>ヌクレアーゼフリー水</td></tr><tr><td> 384ウェルクリア光反応プレート</tD&gt; <td>ライフ·テクノロジーズ·インク</td><td> 4309849 </td><td>別のリアルタイムPCR機器で他のプレート（例えば、96ウェルプレート）で置換することができます</td></tr><tr><td>ダウンスホモジナイザー（15ミリリットル） </td><td>ウィートン科学製品。 </td><td> 358044 </td><td>テフロン/ガラス</td></tr><tr><td> 40μナイロンセルストレーナー</td><td>ファルコン</td><td> 352340 </td><td></td></tr><tr><td>大きな遠心</td><td> Sorval </td><td> RT 6000B </td><td>ローター直径18.5センチメートル</td></tr><tr><td>小型遠心機</td><td>エッペンドルフ</td><td> 5415 R </td><td>ローター直径6.5センチメートル</td></tr><tr><td>リアルタイムPCR装置</td><td>ライフ·テクノロジーズ·インク</td><td> AB 7900 HT </td><td>他の楽器で置換することができる</td></tr><tr><td>フローサイトメーター</td><td> BD Biosciencess </td><td> LSR II </td><td>他の楽器で置換することができる</TD&gt; </tr><tr><td> FACS </td><td> BDバイオサイエンス</td><td> FACSAria </td><td>他の楽器で置換することができる</td></tr><tr><td>光度計分光光度計</td><td>サーモフィッシャーサイエンティフィック</td><td>光度計1000年</td><td></td></tr></tbody></table>

detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation

ミクログリアは、中枢神経系（CNS）1に存在する骨髄系の細胞である。これらの細胞は、例えば、多発性硬化症（MS）2などの神経炎症に関連付けられている多くの疾患の病態に重要な役割を果たしている。正常な中枢神経系におけるミクログリアはマクロファージマーカーCD11bを発現し、CD45などといった活性化マーカーの低レベルを発現させることにより、安静時の表現型を示す。 CNSの病理学的イベント中に、ミクログリアは活性としてCD45のアップレギュレーションおよび他のマーカー3で決定となります。 CNSのミクログリアの表現型や機能に影響を与える要因は十分に研究されていません。マイクロRNA（miRNA）は、細胞の増殖と分化4や5のような炎症の病態として、多くの正常な生理学的プロセスに関与している保存された分子（〜22ヌクレオチドの長さ）の成長の家族です。 miRNAが結合相補的な配列によって、特定の標的遺伝子の発現をダウンレギュレートIRは、mRNAとマクロファージ6とミクログリア7を含む先天性免疫細胞の活性化に重要な役割を果たしている。ミクログリアの表現型、生物学的機能を定義し、マクロファージの他のタイプのミクログリアを区別するためのmiRNA-介在経路を調査するためには、定量的にCNS常駐ミクログリアの異なるサブセット内の特定のマイクロRNAの発現を評価することが重要である。 CNSにおけるmiRNAの発現を測定するための一般的な方法は、全体の神経組織からとin situハイブリダイゼーションの定量PCRが含まれています。しかし、全体の組織ホモジネートから定量PCRは、神経組織の細胞の唯一の5から15パーセントを表すミクログリアにおけるmiRNAの発現の評価をすることはできません。 in situでのハイブリダイゼーションは組織切片内の特定の細胞型におけるマイクロRNAの発現を評価できますが、このメソッドは完全に定量的ではありません。本報告では、定量的およびSENSを記述する通常のCNSから、または実験的自己免疫性脳脊髄炎（EAE）、MSのマウスモデルを用いて、神経炎症時に単離されたミクログリアにおけるリアルタイムPCRによるmiRNAを検出するためのitive方法。記載された方法は、通常のCNSやMS、脳卒中、外傷、アルツハイマー病や脳の腫瘍を含む種々の病状に関連付けられている神経炎症時のミクログリアにおけるマイクロRNAの発現レベルを測定することが有用であろう。

Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

ミクログリアは中枢神経系の防衛と免疫監視の最初の行を提供するマクロファージを常駐しています。 microRNAはマクロファージの活性化と分化を含む多くの生理学的プロセスにおいて重要な役割を果たす調節分子である。本稿では、ミクログリアにおけるマイクロRNAの測定方法を説明します。

ノーマルCNSのリアルタイムPCRによると神経炎症時のミクログリアにおけるマイクロRNAの検出

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

detection-micrornas-microglia-real-time-pcr-normal-cns-during

Research

JoVE Journal

Immunology and Infection

19.3K ビュー.  Harvard Medical School. ミクログリアは中枢神経系の防衛と免疫監視の最初の行を提供するマクロファージを常駐しています。 microRNAはマクロファージの活性化と分化を含む多くの生理学的プロセスにおいて重要な役割を果たす調節分子である。本稿では、ミクログリアにおけるマイクロRNAの測定方法を説明します。

ビデオ: Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

免疫・感染学

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

ハイスループット定量的リアルタイムRT-PCRアッセイのタイプIおよびIIIインターフェロンサブタイプの発現プロファイルを決定するための

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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 ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

のジェノタイピング<em&gt;黄色ブドウ球菌</em&gt;によってリボソームスペーサーPCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

ナノスケールPCRによる動物標本における呼吸器病原体のハイスループット検出

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

リアルタイム揺れる誘起変換用糞便材料の CWD プリオンの検出

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...