We are grateful to the Roney Family Foundation for the support of this study. We acknowledge the contribution of Dr. M. Lucrecia Alvarez to the planning and execution of the experiments described in this work.

注：変更された沈殿法を用いて尿中エキソソームの単離に含まれるステップは、 図1に示されている最初のステップは、遠心分離によって大きな死細胞を除去し、細胞破片を含む。その後の各工程から回収した上清から得られた最終ペレットは、タンパク質、RNA、およびDNAのさらなる抽出の分離溶液に溶解させたエキソソームに対応する。 尿の1.コレクション<ol><li>尿採取の前に、製造業者の説明書に従って、プロテアーゼ阻害剤カクテルを調製し、空の無菌容器にこのカクテルの3.125ミリリットルを加える。プロテアーゼ阻害剤カクテルを含むチューブは、その後、尿収集ボランティアに配布することができる。 NOTE：プロテアーゼ阻害剤を添加する理由は、尿試料中のタンパク質分解を最小にすることである。我々は、特にからプロテアーゼ阻害剤カクテルを使用しましたがシグマ、他の市販のプロテアーゼ阻害剤を置換することができる。 </li><li>最初のボイド尿の約10ミリリットルを収集します。ストレージまたは冷蔵せずにすぐに収集した後に尿を処理します。修正された沈殿法を用いて、二重に尿中エキソソームを分離。他の尿エキソソーム粒子の定量化のためであるが、重複の一つは、DNA、全RNAおよびタンパク質抽出のためにある。 注：24時間の尿サンプルを4℃で保存することによってエキソソームの単離のために収集することができる。 </li></ol>細胞の破片とタム·ホースフォールタンパク質（THP）の2の除去<ol><li> 15ミリリットル遠心管に尿サンプルを転送します。遠心分離機37℃で10分間17000×gで尿10mlの細胞および細胞破片（ 図1）を除去する。 </li><li>ピペットを使用して、さらなる処理のために上清を新しいチューブに移す。分離溶液500μlでペレットを再懸濁（250mMのスクロース、10mMの害しよ最終濃度200 mg / ml）で37℃で：DL-ジチオスレイトール（DTTと10分間インキュベートし、続いてエタノールアミン、pHは7.6）。 NOTE：DTT歩留まりの低下につながる、エキソソームを捕捉THPを分解するために添加される。上澄みの転送中に邪魔されずにペレットを残すように注意してください、そして上清をペレットの明確であることを確認する。 </li><li>渦ペレットサンプル毎に2分間、ペレットが完全に溶解するまで。その後、溶解したペレットに分離溶液500μlを追加します。 </li><li>直ちに、ペレットに分離液を加えた後、37℃で10分間17000×gで遠心する。ピペットで上清およびそれ以前回収した上清と組み合わせること。 </li><li> SDS-PAGE分析のために分離液50μl中にペレットを再懸濁する。 注：PBSまたはTBS、ペレットを再懸濁し、代わり分離溶液を使用することができる。 </li></ol>エキソソームペレットの3.絶縁<ol><li>収集されたsupernの11ミリリットルにatant、ExoQuick-TC試薬の3.3ミリリットルを追加し、少なくとも12時間、4℃でインキュベートする。 NOTE：ExoQuick-TC試薬に対する上清の体積の比は、エキソソーム収量を増加または減少させるように調整することができる。私たちは、追加された試薬の少ないボリュームがエキソソームの小さい収量を生産することを観察した。 </li><li>インキュベーション後、25℃で30分間遠心分離を10,000×gでのサンプル/ ExoQuick-TCミックスを。 </li><li> SDS-PAGE分析のために上清を新しいチューブに移す。 </li><li>エキソソームペレットを、DNA、RNAおよびタンパク質抽出及びCD9 ELISA 11を用いて、エキソソーム粒子の定量化のために-80℃で保存することができる。 NOTE：エキソソームペレットが限ら凍結融解による1年間-80℃で保存することができる。 </li></ol>エキソソームペレットの4バイオマーカーの検出と解析<ol><li>下流の分析のために、DNA / RNA /タンパク質の単離キットおよびRNA単離キを使用してエキソソームペレットからDNA、RNAおよびタンパク質を抽出製造業者の説明書に従ってトン。試料を2μlの260での吸光度と280nmのを測定することにより、ナノドロップ分光光度計を用いて濃度を決定する。 </li><li>のTaqManマイクロRNAアッセイを用いて定量的リアルタイムRT-PCR（定量PCR）を実行するか、miRNA特異的プライマー（我々の場合、我々は、ヒトのmiR-192及びmiR-1207-5p特異的アッセイを使用）。 </li><li> SDS-PAGE分析のために、一次元電気泳動により4〜12％Bis-Trisゲルおよび別個のタンパク質サンプルを使用する。製造業者の説明書に従って銀染色キットを用いてゲルを可視化する。 </li><li>製造業者の指示に従ってPVDF膜を用いたウエスタンブロット分析を行う。ウェスタンブロット検出で101のタンパク質またはTSG101腫瘍感受性遺伝子とタンパク質XまたはALIXとマウスモノクローナル抗体相互作用、アポトーシスに連結された遺伝子-2に対するウサギポリクローナル抗体を使用する。オープンアクセスNIH ImageJソフトウェア（http://rsbweb.nih.gov/ij/）によるバンドの密度を定量化するか、同様のプログラム。 </li><li>製造業者の指示に従ってCD9 ELISAキットを用いて得られた尿中エキソソーム粒子の数を定量化する。 </li></ol>

<ol>
	<li>Miranda, K. C., Bond, D. T., McKee, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acids+within+urinary+exosomes/microvesicles+are+potential+biomarkers+for+renal+disease.">Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease.</a> Kidney Int. 78, 191-199 (2010).</li><li>Pisitkun, T., Shen, R. F., Knepper, 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=Identification+and+proteomic+profiling+of+exosomes+in+human+urine.">Identification and proteomic profiling of exosomes in human urine.</a> Proc Natl Acad Sci USA. 101, 13368-13373 (2004).</li><li>Raj, D. A., Fiume, I., Capasso, G., Pocsfalvi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multiplex+quantitative+proteomics+strategy+for+protein+biomarker+studies+in+urinary+exosomes.">A multiplex quantitative proteomics strategy for protein biomarker studies in urinary exosomes.</a> Kidney Int. 81 (12), 1263-1273 (2012).</li><li>Pisitkun, T., Gandolfo, M. T., Das, S., Knepper, M. A., Bagnasco, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+systems+biology+principles+to+protein+biomarker+discovery:+urinary+exosomal+proteome+in+renal+transplantation.">Application of systems biology principles to protein biomarker discovery: urinary exosomal proteome in renal transplantation.</a> Proteomics Clin Appl. 6 (5-6), 268-278 (2012).</li><li>Gonzales, P. A., Zhou, H., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+exosomes+in+urine.">Isolation and purification of exosomes in urine.</a> Methods Mol Biol. 641, 89-99 (2010).</li><li>Zhou, H., Yuen, P. S., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+storage,+preservation,+and+normalization+of+human+urinary+exosomes+for+biomarker+discovery.">Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.</a> Kidney Int. 69, 1471-1476 (2006).</li><li>Thery, C., Amigorena, S., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Prot Cell Biol. 43, 3.22.1-3.22.29 (2006).</li><li>Cheruvanky, A., Zhou, H., Pisitkun, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+urinary+exosomal+biomarkers+using+a+nanomembrane+ultrafiltration+concentrator.">Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator.</a> Am J Physiol Renal Physiol. 292, 1657-1661 (2007).</li><li>Gonzales, P. A., Pisitkun, T., Hoffert, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+proteomics+and+phosphoproteomics+of+urinary+exosomes.">Large-scale proteomics and phosphoproteomics of urinary exosomes.</a> J Am Soc Nephrol. 20, 363-379 (2009).</li><li>Rood, I. M., Deegens, J. K., Merchant, 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=Comparison+of+three+methods+for+isolation+of+urinary+microvesicles+to+identify+biomarkers+of+nephrotic+syndrome.">Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.</a> Kidney Int. 78, 810-816 (2010).</li><li>Alvarez, M. L., Khosroheidari, M., Kanchi Ravi, R., DiStefano, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+protein,+microRNA,+and+mRNA+yields+using+different+methods+of+urinary+exosome+isolation+for+the+discovery+of+kidney+disease+biomarkers.">Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers.</a> Kidney Int. Nov. 82 (9), 1024-1032 (2012).</li><li>Kang, S. W., Kim, Y. W., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+association+of+-667+aquaporin-2+(AQP-2)+A/G+promoter+polymorphism+with+the+incidence+and+clinical+course+of+chronic+kidney+disease+in+Korea.">Study of the association of -667 aquaporin-2 (AQP-2) A/G promoter polymorphism with the incidence and clinical course of chronic kidney disease in Korea.</a> Renal Fail. 29, 693-698 (2007).</li></ol>

この記事の発行手数料はSBIバイオサイエンス社支払われた。

尿からエキソソームの沈殿を含むほとんどのメソッドは、タム - ホースフォールタンパク質（THP）によって形成されたポリマーネットワークの除去に取り組んでいない。このタンパク質は、推定上のトラップが初期低速遠心分離中エキソソーム尿中に多量に存在する。私たちの修正された方法では、エキソソーム降水前にDTTを使用してTHPを減少させた。 図2に示すように、THPのより高い量は、タンパク質の除去を示し、最後の上清およびエキソソームペレットでないTHPで、未処理の尿及びペレットで観察された。ペレット中のタンパク質の定量は、6倍高い収率を示し、そしてペレットのCD9 ELISA定量には、尿1mlあたりエキソソームの収率が（結果は11には示されていない）未修飾の沈殿法の5倍高かった観察された。バイオマーカーアリックス6とTSG101 8はエキソソームペレットで検出された。我々は唯一の集中を使ってこれらのタンパク質を検出することができた未修飾の沈殿法のサンプル。我々はまた、修正された沈殿法11を使用したmiRNA（ すなわち 、のmiR-192及びmiR-1207-5p）とmRNA発現（ すなわち 、TSG101、PDCD6IPとAQP2 12）の最高レベルを観察した。この修正された沈殿法の利点と欠点を表1に示す。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> メリット </td><td> デメリット </td></tr><tr><td>簡単に簡単な手順と実行するための相対的な少し専門知識が必要</td><td>尿中エキソソームから得られたタンパク質の低純度</td></tr><tr><td>超遠心分離工程を必要としない</td><td>による超遠心分離工程が存在しないため、得られた尿エキソソームペレットを超遠心分離法から得られたエキソソームペレットと比較して、純粋でない</td></tr><tr><td> Urinの小さなボリュームを使用するエキソソームの単離のための電子サンプル</td><td>特定の分析のための純粋なタンパク質を得るためには、アフィニティークロマトグラフィーなどのタンパク質精製工程が必要とされる</td></tr><tr><td>非改変沈殿法と比較して尿中エキソソームからmiRNAは、mRNAおよび蛋白質の良い量を得</td><td>非改変沈殿法に比べタム - ホースフォールタンパク質によって捕捉を減らすためにDTTを添加して必要とされるいくつかの追加のステップ</td></tr><tr><td>分析されたバイオマーカーの特異性および感度が得られるものと類似しており、超遠心分離法を用いて分析</td><td></td></tr><tr><td>尿中エキソソームは臨床試験に適した、一度に多数の試料から単離することができる</td><td></td></tr><tr><td>少ないハンズオンタイム超遠心分離法に比べ</td><td></td></tr></tbody></table> 修正されたprecipitaの表1.長所と短所尿中エキソソームを単離するためのション方法。 我々は、沈殿法の我々の修正尿エキソソームを単離するための単純、迅速かつ効率的な方法であり、労働集約的であり、高価な装置を必要とする超遠心法、適切な代替物であると結論付けている。本手法では、全く超遠心分離工程を必要としない、実施が容易であり、さらに、その後の分子解析のための高収量や純エキソソームを提供しながら、より少ない手順が必要です。また、この方法を用いてmiRNAおよびmRNAの収率が高いため、そのまま次の下流のRNAプロファイリングの用途に使用できることを示している。しかし、我々はこの方法の制限は、ショ糖勾配超遠心分離法を用いて得られる限り純粋でない得られたタンパク質の品質は、前下流プロテオーム解析において利用する追加の精製工程を必要とすることに注意。我々は現在、このメタの有用性を評価されている糖尿病性腎症の患者から得られた尿試料を用いて作ろ。

尿中エキソソームは、miRNAの1のために濃縮されるように見える尿排水システムをライニング糸球体足細胞、尿細管細胞と細胞を含む尿空間での正常および病的条件下での全ての細胞型から派生多胞体（MVB）の100nmの小さな内部小胞である-2。多くの研究者が明確な健康な個体の尿から単離されたエキソソーム中のタンパク質だけでなく、腎臓および/ ​​または体系的疾患3-5のものを検出した。エキソソームは、超遠心8-9、または沈殿10-11にナノ膜フィルターを組み合わせ、スクロース6-7の有無にかかわらずそのような二段階の差動超遠心分離などの異なる方法を用いて単離することができる。我々は最近、尿中エキソソームを分離し、miRNAおよびタンパク質バイオマーカー11を検出するための、簡単、迅速、スケーラブルで効果的な方法を開発した。バイオマーカーは、異なる生物学的流体の様々な検出可能であるが、ウルナインは、比較的非侵襲的な方法で大量に得ることができるため、腎臓および尿路の疾患を有する患者のために最も便利な選択である。 尿中エキソソーム6-11を単離するためのいくつかの方法があるが、最も一般的に使用される手順は、30％ショ糖クッションで差超遠心分離に依存する。この方法は効率的であり、この手順を用いて単離し、得られたエキソソームの品質が高レベルである間は、技術自体は、熟練者を必要とし、時間がかかり、いくつかの超遠心分離工程を必要である。例えば濾過、沈殿などの他の方法は、独自の沈殿試薬を使用するものを含む、エキソソームの単離のために開発されている（ すなわち 、ExoQuick-TC：システムBiosciences社;マウンテンビュー、CA）。この試薬を用いて降水量は、高速かつ比較的容易であるが、分離エキソソームの純度は超遠心メタに比べて低いOD。我々は最近、我々の研究室で開発された11 ExoQuick-TCを使用して沈殿法の修正を含む尿中エキソソームの単離のための6つのメソッドを、比較した。我々は、この修正された沈殿法は、タンパク質に、miRNA、およびmRNAのより高い量を得た手順自体は速く、超遠心分離法よりも実装が簡単であることがわかった。ここでは、段階的に私たちの修正された沈殿法の実験的なプロトコルを記述し、エキソソーム単離の他の方法と比較して、この技術の利点と欠点の議論が含まれています。修正された沈殿法は、エキソソームのタンパク質と相関し、そして尿からエキソソーム収率が低下することが知られているタム - ホースフォールタンパク質を除去することによりエキソソーム粒子の初期析出を伴う。我々は、これらの修正が尿からエキソソーム調製物の収率および純度を増加させることを見出した。

<table border="0" cellpadding="0" cellspacing="0" width="736">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning 15ml Centrifuge Tube</td>
			<td style="width:111px;">Corning (Sigma Aldrich)</td>
			<td style="width:119px;">CLS430791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease Inhibitor Cocktail</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:111px;">Sorvall</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DL-dithiothreitol</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">D9779</td>
			<td>used at final concentration of 200 mg/ml</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Novex NuPAGE SDS-PAGE system (4-12 % Bis-Tris Gel 1.0 mm, 10 well</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">NP0321BOX</td>
			<td>For SDS-PAGE analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ECL Advance Western blotting detection kit</td>
			<td style="width:111px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">RPN2135</td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exoquick-TC Reagent</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOTC50A-1</td>
			<td>For exosome isolation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Exosome CD9 ELISA Complete Kit</td>
			<td style="width:111px;">System Biosciences (SBI)</td>
			<td style="width:119px;">EXOEL-CD9-A-1</td>
			<td>For exosome quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AllPrep DNA/RNA/Protein mini kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">80004</td>
			<td>For DNA, RNA, Protein isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rneasy MinElute Cleanup kit</td>
			<td style="width:111px;">Qiagen</td>
			<td style="width:119px;">74204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:111px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>For Protein Quantification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ProteoSilver Sliver Stain Kit</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">PROTSIL1-1KT</td>
			<td>For staining SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xcell SureLock Mini-Cell Electrophoresis System</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For running the SDS-PAGE gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan microRNA assays</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td>For RT-PCR assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qBasePlus v1.5 software</td>
			<td style="width:111px;">Biogazelle NV</td>
			<td style="width:119px;"></td>
			<td>For analysis of RT-PCR assay data</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit polyclonal antibody to ALIX</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mouse monoclonal antibody to TSG101</td>
			<td style="width:111px;">Abcam</td>
			<td style="width:119px;"></td>
			<td>For western blot detection of biomarkers</td>
		</tr>
	</tbody>
</table>

a modified precipitation method to isolate urinary exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

尿中エキソソームは、腎上皮細胞から分泌されるタンパク質に、miRNAとmRNAのバイオマーカーを運ぶ。これらのエキソソームの単離および検出は、糖尿病性腎症などの腎疾患の早期検出に有用な診断ツールを提供してもよい。ヒト被験者から採取した尿サンプルからエキソソームを単離するためのいくつかの方法がある。我々は以前、尿エキソソーム分離の6つのメソッドを比較し、タンパク質、mRNA、およびmiRNAレベル11を結果として調査した。ここに私たちの目的は、詳細に我々は尿中エキソソームの効率的な分離のために開発した修正された沈殿法を記述することでした。 <img alt="図1" src="/files/ftp_upload/51158/51158fig1highres.jpg" /> 図1は、修正された沈殿法を用いて、尿中エキソソームの単離に関与する様々なステップの概略的な代表的なフローチャートを記述する。重複エキソソームのOne得られたペレットは、エキソソーム粒子の相対的な定量化を提供してくれCD9 ELISAキットを用いて、CD9を測定する。 260および280nmでの吸光度を測定することにより、ナノドロップ分光光度計を用いて定量したキアゲンDNA / RNA /タンパク質ミニキット収量DNA、RNAおよびタンパク質を用いて処理し、他のペレット。 <img alt="図2" src="/files/ftp_upload/51158/51158fig2highres.jpg" /> 図2は、（参照11から適応）ウェスタンブロット分析を用いてSDS-PAGE分析およびバイオマーカーの検出での結果の変更された再生である。未処理の尿及び第尿ペレットを示す90kDaの100に観察されたに対応するタンパク質バンドのレーンでそれぞれTHPタンパク質の存在と除去。最後の上清およびエキソソームペレットとレーンに、THPに対応するタンパク質バンドが観察されなかったが、前のステップでTHPを除去することを示す、THERイービエキソソームペレットの収率を増加させる。エキソソームペレットの純度および感度にアクセスするには、そのようなアリックスとTSG101のようなバイオマーカーのウェスタンブロット検出を使用した。私たちは、サンプルの低量はバイオマーカー解析およびTHPを含む豊富な可溶性タンパク質の不在のために大幅に純粋なエキソソームペレットを示す使用された場合でも、アリックスとTSG101を検出した。ウェスタンブロットのデンシトメトリー分析を定量化および検出のレベルを測定するために行った（データは示さず）。

Watch this Scientific Journal Video about A Modified Precipitation Method to Isolate Urinary Exosomes at JoVE.com

A Modified Precipitation Method to Isolate Urinary Exosomes

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

尿中エキソソームを単離するために、修正沈殿法

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

a-modified-precipitation-method-to-isolate-urinary-exosomes

Nanyang Technological University

Research

JoVE Journal

Medicine

17.5K ビュー.  Translational Genomics Research Institute (TGen). This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

ビデオ: A Modified Precipitation Method to Isolate Urinary Exosomes

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

老化がん細胞を定量化するための感度の高い方法

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

医学

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

変更されたカフ法を用いたマウス子宮頸心臓移植モデル

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Modified Method for Heterotopic Mouse Heart Transplantion

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents available. A difficulty is presented due to the small size of the animal and the considerable technical challenges of the microsurgery involved in heart transplantation. In particular, a high rate of technical failure early after transplantation may result from recipient death and post-operative complications such as hind limb paralysis or a non-beating heart. Here, the complete technique for heterotopic mouse heart transplantation is demonstrated, involving harvesting the donor heart and its subsequent implantation into a recipient mouse. The donor heart is harvested immediately following in situ perfusion with cold heparinized saline and transection of the ascending aorta and pulmonary artery. The recipient operation involves preparation of the abdominal aorta and inferior vena cava (IVC), followed by end-to-side anastomosis of the donor aorta with the recipient aorta using a single running 10-0 microsuture and a similar anastomosis of the donor pulmonary artery with the recipient IVC. Following the operation the animal is injected with 0.6 ml normal saline subcutaneously and allowed to recover on a 37&#160;&#176;C heating pad. The results from 227 mouse heart transplants are summarized with a success rate at 48 hr of 86.8%. Of the 13.2% failures within 48 hr, 5 (2.2%) experienced hind limb paralysis, 10 (4.4%) had a non-beating heart due to graft ischemic injury and/or thrombosis, while 15 (6.6%) died within 48 hr.

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

異マウスハートTransplantion用に変更する方法

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

下部尿路薬理学を評価するための方法として膀胱平滑筋収縮ストリップ

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

変更されたベルイストローム法を用いたヒト骨格筋生検手順

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

非経た栄養のためのオリーブオイルベースの3チャンバーバッグの安全性、有効性、および送達を評価する臨床試験

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

ラット腎臓からそのまま糸球体を分離する効率的なふるい分け法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

Ultrasonography of the Adult Male Urinary Tract for Urinary Functional Testing

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting in a significant economic and quality of life burden. Transgenic and other mouse models have been developed to recreate various aspects of this multifactorial disease; however, methods to accurately quantitate urinary dysfunction and the effectiveness of new therapeutic options are lacking. Here, we describe a method that can be used to measure bladder volume and detrusor wall thickness, urinary velocity, void volume and void duration, and urethral diameter. This would allow for the evaluation of disease progression and treatment efficacy over time. Mice were anesthetized with isoflurane, and the bladder was visualized by ultrasound. For non-contrast imaging, a 3D image was taken of the bladder to calculate volume and evaluate shape; the bladder wall thickness was measured from this image. For contrast-enhanced imaging, a catheter was placed through the dome of the bladder using a 27-gauge needle connected to a syringe by PE50 tubing. A bolus of 0.5 mL of contrast was infused into the bladder until a urination event occurred. Urethral diameter was determined at the point of the Doppler velocity sample window during the first voiding event. Velocity was measured for each subsequent event yielding a flow rate. In conclusion, high frequency ultrasound proved to be an effective method for assessing bladder and urethral measurements during urinary function in mice. This technique may be useful in the assessment of novel therapies for BPH/LUTS in an experimental setting.

We describe the use of high frequency ultrasound with contrast imaging as a method to measure bladder volume, bladder wall thickness, urine velocity, void volume, void duration, and urethral diameter. This strategy can be used to assess voiding dysfunction and treatment efficacy in various mouse models of lower urinary tract dysfunction (LUTD).

尿機能検査のための成人男性尿路の超音波検査

The incidence of clinical benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) is increasing due to the aging population, resulting ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

マウスにおける心筋梗塞の誘導のための改良簡便法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, preserving the vasal vessels during MVE might better simulate the normal physiological structure and be meaningful for patients who have undergone varicocelectomy. Nevertheless, preserving the vasal vessels might elevate the risk of increasing the tension of anastomosis, affecting the patency rate and leading to delayed postoperative bleeding. Therefore, we developed a novel vessel-sparing MVE to make it safer. Here is a summary of the improvements to the procedure. 1) The retrograde dissociation of the vasal vessels on the proximal testicular side was adopted as the main method, and the anterograde dissociation of the vasal vessels on the distal testicular side was adopted as a supplement to dissociate the vasal vessels to be preserved. This improvement ensures the blood supply to the vas deferens that will be used for anastomosis and also provides longer vasal vessels, which reduces the tension of anastomosis. 2) By fixing the vas deferens to be anastomosed and the broken end of the vas deferens, the free vasal vessels get fixed, which resolves the problem of transmission of vas tension to the vasal vessels and reduces the risk of vasal vessel hemorrhage. 3) Dissociation of the vas deferens after opening the tunica vaginalis increases the mobilization of the vas deferens, which also makes the new procedure easier to complete. The evaluation of the outcomes of this new procedure showed that no significant postoperative complications occurred in the patients, and the patency rate was no different from that of the conventional procedure. Therefore, this new, improved procedure can be considered safe, with satisfactory postoperative results.

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

修正血管温存顕微手術精巣上体切開術

Microsurgical vasoepididymostomy (MVE) is the main surgical treatment for epididymal obstruction. The vasal vessels are ligated during MVE. However, ...