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...

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

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

Universidad de Cartagena UDEC

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

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

エクソソームおよび人間の血液からの分離を最適化するためにタンパク質の消化が、限外濾過、サイズ排除クロマトグラフィー

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

生化学

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, and as the disease progresses, the patient develops symptoms of dilated cardiomyopathy, arrhythmia, and congestive heart failure. The DMDMDX mutant mice do not express dystrophin, and are commonly used as a mouse model of DMD. In our recent study, we observed that intramyocardial injection of wide type (WT)-myogenic progenitor cells-derived exosomes (MPC-Exo) transiently restored the expression of dystrophin in the myocardium of DMDMDX mutant mice, which was associated with a transient improvement in cardiac function suggesting that WT-MPC-Exo may provide an option to relieve the cardiac symptoms of DMD. This article describes the technique of MPC-Exo purification and transplantation into hearts of DMDMDX mutant mice.

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

精製と筋前駆細胞移植派生エクソソーム デュシェンヌ型筋ジストロフィー マウスの心機能を改善するために

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

遺伝学

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1陽性個体の血漿からエキソソームの単離

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

免疫・感染学

精密な生体流体分析のためのEV分離の強化

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

著者スポットライト: Advancing EVtrap for High-Throughput Proteomics in Disease Biomarker Discovery 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

医学

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含有エキソソームの単離と解析

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

限外ろ過遠心分離法を用いたマウスの気管支肺胞洗浄液細胞小胞の分離

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

尿中エキソソームを単離するために、修正沈殿法

要約

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この動画の章

エクソソームおよび人間の血液からの分離を最適化するためにタンパク質の消化が、限外濾過、サイズ排除クロマトグラフィー

精製と筋前駆細胞移植派生エクソソームデュシェンヌ型筋ジストロフィーマウスの心機能を改善するために

HIV-1陽性個体の血漿からエキソソームの単離

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

RNA含有エキソソームの単離と解析

限外ろ過遠心分離法を用いたマウスの気管支肺胞洗浄液細胞小胞の分離

エキソソームの定量化とサイズの測定のための革新的方法

尿中エキソソームを単離するために、修正沈殿法

要約

さらに動画を探す

この動画の章

エクソソームおよび人間の血液からの分離を最適化するためにタンパク質の消化が、限外濾過、サイズ排除クロマトグラフィー

精製と筋前駆細胞移植派生エクソソーム デュシェンヌ型筋ジストロフィー マウスの心機能を改善するために

HIV-1陽性個体の血漿からエキソソームの単離

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

プロテオミクスおよびリン酸化プロテオミクス解析のための生体液からの細胞外小胞の化学的親和性に基づく単離(英語)

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

RNA含有エキソソームの単離と解析

限外ろ過遠心分離法を用いたマウスの気管支肺胞洗浄液細胞小胞の分離

エキソソームの定量化とサイズの測定のための革新的方法

精製と筋前駆細胞移植派生エクソソームデュシェンヌ型筋ジストロフィーマウスの心機能を改善するために