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.

REMARQUE:. Les étapes impliquées dans l&#39;isolement des exosomes urinaires en utilisant le procédé de précipitation modifiée sont illustrés dans la Figure 1 Les premières étapes impliquent l&#39;enlèvement de grandes cellules mortes et les débris cellulaires par centrifugation. Le culot final obtenu à partir du surnageant recueilli à partir de chaque étape subséquente correspond à la exosomes, qui est dissous dans une solution d&#39;isolation pour une extraction supplémentaire de la protéine, d&#39;ARN et de l&#39;ADN. 1. Collecte de l&#39;urine <ol><li> Avant la collecte de l&#39;urine, préparer inhibiteur de protéase cocktail selon les instructions du fabricant et ajouter 3,125 ml de ce cocktail à une prise vide et stérile. Le tube contenant le cocktail d&#39;inhibiteurs de protéase peut alors être distribué aux bénévoles pour la collecte d&#39;urine. REMARQUE: La raison de l&#39;ajout d&#39;un inhibiteur de protease est de réduire au minimum la dégradation des protéines dans l&#39;échantillon d&#39;urine. Bien que nous avons utilisé spécifiquement un cocktail d&#39;inhibiteurs de protéase duSigma, d&#39;autres inhibiteurs de protease disponibles dans le commerce peuvent être substitués. </li><li> Prélever environ 10 ml d&#39;urine de la première miction. Traiter l&#39;urine immédiatement après la collecte sans stockage ou de réfrigération. Isoler les exosomes urinaires en double en utilisant le procédé de précipitation modifié; un des exemplaires est pour l&#39;ADN, l&#39;ARN total et l&#39;extraction des protéines, tandis que l&#39;autre est pour la quantification des particules d&#39;exosomes urinaires. REMARQUE: Un échantillon d&#39;urine de 24 heures peut être recueilli pour l&#39;isolement de exosomes en stockant à 4 ° C. </li></ol> 2. Elimination des débris cellulaires et Tamm-Horsfall Protein (THP) <ol><li> Transfert des échantillons d&#39;urine tubes de 15 ml de centrifugeuses. Centrifugeuse de 10 ml d&#39;urine à 17 000 xg pendant 10 min à 37 ° C pour éliminer les cellules et les débris cellulaires (figure 1). </li><li> Aide d&#39;une pipette, transférer le surnageant dans un nouveau tube pour un traitement ultérieur. Remettre en suspension le culot dans 500 pl de solution d&#39;isolation (250 mM de saccharose, 10 mM qui éprouveanolamine, pH 7,6), suivi par 10 minutes d&#39;incubation avec DL-dithiothréitol (DTT: concentration finale de 200 mg / ml) à 37 ° C. REMARQUE: la TNT est ajouté à dégrader THP, qui emprisonne exosomes, conduisant à une diminution des rendements. Prenez soin de laisser le culot sans être dérangés pendant le transfert du surnageant, et vérifiez que le surnageant est clair de granulés de bois. </li><li> Des échantillons de granulés Vortex toutes les 2 min jusqu&#39;à ce que le culot soit complètement dissous. Ensuite, ajouter 500 ul de solution d&#39;isolation pour le culot dissous. </li><li> Immédiatement après l&#39;addition de solution d&#39;isolation de la pastille, centrifuger à 17 000 xg pendant 10 min à 37 ° C. Déposer le surnageant et de combiner avec le surnageant recueilli plus tôt. </li><li> Reprendre le culot dans 50 ul de solution d&#39;isolation pour l&#39;analyse SDS-PAGE. REMARQUE: PBS TBS ou peuvent être utilisés à la place de la solution d&#39;isolation pour remettre en suspension le culot. </li></ol> 3. Isolement de Exosome Pellet <ol><li> Pour 11 ml de la Supern recueilliesatant, ajouter 3,3 ml de réactif ExoQuick-TC et incuber à 4 ° C pendant au moins 12 h. REMARQUE: le rapport du volume de surnageant de réactif ExoQuick-TC peut être réglée pour augmenter ou diminuer le rendement de exosomal. Nous avons observé que les volumes moindres de réactif ajouté produisent des rendements inférieurs de exosomes. </li><li> Après incubation, centrifuger le mélange échantillon / ExoQuick-TC à 10 000 g pendant 30 min à 25 ° C. </li><li> Transférer le surnageant dans un nouveau tube pour analyse par SDS-PAGE. </li><li> Le culot d&#39;exosomes peut être conservé à -80 ° C pour l&#39;ADN, l&#39;ARN et l&#39;extraction des protéines et la quantification des particules d&#39;exosomes en utilisant ELISA 11 CD9. NOTE: Le culot d&#39;exosomes peut être conservé à -80 ° C pendant 1 an avec congélation et de décongélation limitée. </li></ol> 4. biomarqueurs de détection et analyse des Exosome Pellet <ol><li> Pour les analyses en aval, extraire l&#39;ADN, de l&#39;ARN et de protéines à partir de la pastille de exosomes en utilisant le / ARN / kit d&#39;isolement de protéines de l&#39;ADN et un Ki d&#39;isolement d&#39;ARNt selon les instructions du fabricant. Déterminer les concentrations en utilisant un spectrophotomètre à Nanodrop en mesurant l&#39;absorbance à 260 et 280 nm avec 2 pi d&#39;échantillon. </li><li> Effectuer quantitative en temps réel RT-PCR (qPCR) en utilisant des tests de microARN TaqMan (dans notre cas, nous avons utilisé les droits de miR-192- et miR-spécifiques 1207-5p-tests) ou des amorces spécifiques miARN. </li><li> Pour l&#39;analyse SDS-PAGE, en utilisant 4-12% Bis-Tris gel et les échantillons de protéines séparées par électrophorèse unidimensionnelle. Visualiser gels en utilisant le kit de coloration à l&#39;argent conformément aux instructions du fabricant. </li><li> Effectuer une analyse de buvardage de Western en utilisant des membranes de PVDF selon les instructions fournies par le fabricant. Utilisation anticorps polyclonal de lapin lié à l&#39;apoptose gène-interacting protein 2 ou X Alix et un anticorps monoclonal de souris à gène de susceptibilité à la tumeur 101 protéine TSG101 ou à la détection par transfert de Western. Quantifier la densité des bandes par accès ouvert logiciel NIH ImageJ (http://rsbweb.nih.gov/ij/) ouun programme similaire. </li><li> Quantifier le nombre de particules d&#39;exosomes urinaires obtenus en utilisant un kit ELISA CD9 selon les instructions du fabricant. </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. 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Frais de publication de cet article ont été payés par SBI Biosciences.

La plupart des procédés impliquant la précipitation des exosomes à partir d&#39;urine ne abordent pas le retrait du réseau polymère formé par la protéine de Tamm-Horsfall (THP). Cette protéine est présente en grandes quantités dans l&#39;urine, où il pièges putativement exosomes pendant la centrifugation à basse vitesse initiale. Dans notre méthode modifiée, nous avons réduit THP utilisant la TNT avant la précipitation exosomes. Comme le montre la Figure 2, une plus grande quantité de THP a été observée dans l&#39;urine non transformés et pastille, sans THP dans le surnageant final et exosomes pastille, indiquant une élimination de la protéine. La quantification des protéines dans le culot a indiqué un rendement six fois plus élevée, et sur ​​CD9 ELISA quantification de la pastille, on a observé que le rendement de exosomes par ml d&#39;urine a été cinq fois plus élevé que le procédé de précipitation non modifié (résultats non montrés 11). Les biomarqueurs Alix 6 et 8 TSG101 ont été détectés dans le culot d&#39;exosomes; nous ne avons pu détecter ces protéines en utilisant des concentréséchantillons dans le procédé de précipitation non modifié. Nous avons également observé les plus hauts niveaux de miARN (c.-à-miR-192 et miR-1207-5p) et expression de l&#39;ARNm (c.-à-TSG101, PDCD6IP et AQP2 12) en utilisant le procédé de précipitation modifiée 11. Les avantages et les inconvénients de ce procédé de précipitation modifiée sont indiqués dans le tableau 1. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Avantages </td><td> Inconvénients </td></tr><tr><td> Étapes simples facile et nécessite peu d&#39;expertise par rapport à effectuer </td><td> Faible pureté des protéines obtenues à partir des exosomes urinaires </td></tr><tr><td> Ne nécessite pas une étape d&#39;ultracentrifugation </td><td> En raison de l&#39;absence des étapes d&#39;ultracentrifugation, le culot urinaire exosomes obtenu ne est pas aussi pur que par rapport à la pastille obtenue à partir d&#39;exosomes méthodes ultracentrifugation </td></tr><tr><td> Utilise petit volume de Urine échantillon pour l&#39;isolement d&#39;exosomes </td><td> Pour obtenir la protéine pure pour analyse spécifique, les étapes de purification des protéines telles que la Chromatographie d&#39;affinité seraient nécessaires </td></tr><tr><td> Obtenu bonne quantité de miARN, ARNm et des protéines urinaires d&#39;exosomes par rapport aux non-modifiée méthode de précipitation </td><td> Quelques étapes supplémentaires nécessaires avec l&#39;addition de la TNT pour réduire la provocation par la protéine de Tamm-Horsfall par rapport aux non-modifiée méthode de précipitation </td></tr><tr><td> La spécificité et la sensibilité des biomarqueurs analysés étaient similaires à celles obtenues et analysées en utilisant la méthode d&#39;ultracentrifugation </td><td></td></tr><tr><td> Urinaires exosomes peuvent être isolées à partir de nombreux échantillons à la fois, convenable pour les essais cliniques </td><td></td></tr><tr><td> Moins mains sur le temps par rapport aux méthodes ultracentrifugation </td><td></td></tr></tbody></table> Tableau 1. Avantages et inconvénients de precipita modifiéeméthode de tion pour l&#39;isolement de exosome urinaire. Nous concluons que notre modification de la méthode de précipitation est un moyen simple et efficace, rapide d&#39;isoler les exosomes urinaires et est une alternative appropriée à la méthode de l&#39;ultracentrifugation, qui est beaucoup de travail et nécessite un équipement coûteux. Notre méthode ne requiert aucune étape d&#39;ultracentrifugation, est facile à réaliser et nécessite moins d&#39;étapes, tout en offrant un rendement élevé ou exosomes purs pour les analyses moléculaires ultérieures. Nous montrons aussi que les rendements des miARN et ARNm en utilisant cette méthode sont élevés, et donc peuvent être directement utilisés dans les applications suivantes aval ARN de profilage. Toutefois, nous notons que la limitation de ce procédé est la qualité de la protéine obtenue, qui ne est pas aussi pur que celui obtenu en utilisant les procédés d&#39;ultracentrifugation à gradient de saccharose, et nécessite des étapes supplémentaires de purification avant l&#39;utilisation dans les analyses protéomiques aval. Nous évaluons actuellement l&#39;utilité de cette method en utilisant des échantillons d&#39;urine prélevés sur des patients atteints de néphropathie diabétique.

Exosomes urinaires sont de petites vésicules internes de 100 nm de corps multivésiculaires (MVB) provenant de tous les types de cellules dans des conditions normales et pathologiques dans l&#39;espace urinaire y compris podocytes glomérulaires, les cellules des tubules rénaux et les cellules qui tapissent les systèmes de drainage urinaire qui semblent être enrichi pour miARN 1 -2. De nombreux chercheurs ont détecté des protéines distinctes dans exosomes isolés à partir de l&#39;urine d&#39;individus sains ainsi que ceux atteints d&#39;insuffisance rénale et / ou de maladies systématiques 3-5. Les exosomes peuvent être isolés en utilisant différentes méthodes telles que deux étapes ultracentrifugation différentielle avec ou sans saccharose 6-7, combinant filtre nanomembrane avec ultracentrifugation 8-9, 10-11 ou précipitation. Nous avons récemment développé une méthode simple, rapide, évolutive et efficace pour isoler les exosomes urinaires et de détecter les miARN et des biomarqueurs protéiques 11. Bien que les biomarqueurs peuvent être détectés dans une variété de différents fluides biologiques, urine est le choix le plus pratique pour les patients atteints de maladies du rein et des voies urinaires, car il peut être obtenu en grandes quantités d&#39;une manière relativement non invasive. Bien qu&#39;il existe plusieurs méthodes pour isoler les exosomes urinaires 6-11, le procédé le plus couramment utilisé dépend ultracentrifugation différentielle avec un coussin de saccharose à 30%. Bien que cette méthode soit efficace et la qualité des exosomes isolés résultant de cette procédure est élevée, la technique elle-même requiert un personnel qualifié et prend beaucoup de temps, ce qui nécessite plusieurs étapes d&#39;ultracentrifugation. D&#39;autres méthodes, telles que la filtration et la précipitation, ont été développés pour l&#39;isolement d&#39;exosomes, y compris une qui utilise un réactif de précipitation propriétaire (ce est à dire, ExoQuick-TC: Biosciences système; Mountain View, CA). Bien que les précipitations en utilisant ce réactif est rapide et relativement facile, la pureté des exosomes isolés est faible par rapport à la meth ultracentrifugationod. Récemment, nous avons comparé les six procédés pour l&#39;isolement d&#39;exosomes urinaires, y compris une modification de la méthode de précipitation au moyen ExoQuick-TC que nous avons développé dans notre laboratoire 11. Nous avons constaté que cette méthode de précipitation modifiée a donné de plus grandes quantités de protéines, de miARN et ARNm et la procédure elle-même était plus rapide et plus facile à mettre en œuvre que ultracentrifugation. Ici, nous décrivons le protocole expérimental de notre méthode de précipitation modifiée de manière progressive, et inclure une discussion sur les avantages et les inconvénients de cette technique par rapport à d&#39;autres méthodes d&#39;isolement de exosomes. Le procédé de précipitation modifié implique une précipitation initiale de particules d&#39;exosomes, suivie de l&#39;élimination de la protéine de Tamm-Horsfall, qui est corrélée avec des protéines exosomales, et connu pour réduire le rendement des exosomes à partir d&#39;urine. Nous avons constaté que ces modifications ont augmenté le rendement et la pureté de la préparation de exosomal de l&#39;urine.

<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.

Exosomes urinaires portent protéines, miARN et ARNm biomarqueurs sécrétées par les cellules épithéliales rénales. L&#39;isolement et la détection de ces exosomes peuvent fournir un outil diagnostique utile pour la détection précoce des maladies rénales telles que la néphropathie diabétique. Il existe plusieurs méthodes pour l&#39;isolement d&#39;exosomes à partir d&#39;échantillons d&#39;urine prélevés chez des sujets humains. Nous avons déjà comparé six méthodes d&#39;isolement de l&#39;urine exosomes et étudié les protéines, les niveaux d&#39;ARNm et des miRNA 11 résultant. Notre objectif ici est de décrire en détail la méthode de précipitation modifiée, nous avons développé pour l&#39;isolement efficace des exosomes urinaires. <img alt="Figure 1" src="/files/ftp_upload/51158/51158fig1highres.jpg" /> La figure 1 décrit le schéma fonctionnel représentant schématiquement diverses étapes impliquées dans l&#39;isolement d&#39;exosomes urinaires en utilisant le procédé de précipitation par modification du duplicata Un exosomes.culot obtenu est mesurée pour CD9 utilisant le kit ELISA CD9, qui nous fournit la quantification relative des particules d&#39;exosomes. L&#39;autre pastille traitée à l&#39;aide Qiagen ADN / ARN / protéine Mini Kit rendements ADN, d&#39;ARN et de protéines, qui ont été quantifiées en utilisant Nanodrop spectrophotomètre en mesurant l&#39;absorbance à 260 et 280 nm. <img alt="Figure 2" src="/files/ftp_upload/51158/51158fig2highres.jpg" /> La figure 2 est la reproduction modifiée des résultats sur l&#39;analyse SDS-PAGE et détection de biomarqueurs en utilisant une analyse de transfert de western (adapté de référence 11). Dans les colonnes avec de l&#39;urine non transformés et premier culot urinaire, une bande de protéine correspondant à 90 à 100 kDa a été observée indiquant la présence et l&#39;élimination des protéines THP respectivement. Alors que dans les pistes avec surnageant final et exosomes pastille, la bande de protéine correspondant à THP n&#39;a pas été observée, ce qui indique l&#39;élimination du THP dans les étapes précédentes, therEby augmenter le rendement des exosomes pastille. Pour accéder à la pureté et la sensibilité des granules exosomes, détection Western blot de biomarqueurs tels que TSG101 Alix et a été employée. Nous avons détecté Alix et TSG101 même lorsque de faibles quantités d&#39;échantillon ont été utilisés en précisant culot exosomes sensiblement pur pour l&#39;analyse des biomarqueurs et l&#39;absence de protéines solubles abondantes, notamment THP. L&#39;analyse densitométrique des transferts de Western a été effectuée pour quantifier et mesurer le niveau de détection (données non présentées).

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.

Un procédé de précipitation modification pour isoler les exosomes urinaire

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

Vidéo: 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.

Une méthode sensible pour quantifier les cellules sénescentes du cancer

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

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Médecine

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.

Murin col de l&#39;utérus Cœur, la transplantation en utilisant une technique de modification de manchette

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.

Une méthode modifiée de Heterotopic souris coeur la transplantation

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.

Vessie muscle lisse bande de contractilité comme une méthode pour évaluer urinaires inférieures Pharmacologie

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.

Procédures Muscle squelettique humain biopsie aide de la technique de modification Bergström

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.

Un essai clinique évaluant l'innocuité, l'efficacité et la livraison de sacs à trois chambres à base d'huile d'olive pour la nutrition parentérale

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.

Une méthode efficace de tamisage pour isoler Intact glomérules du rein de Rat adulte

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).

Ultrasonographie de l'appareil urinaire masculin adulte pour l'essai fonctionnel urinaire

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.

Méthode simple modifiée pour l’induction d’un infarctus du myocarde chez la souris

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.

Vasoépididymostomie microchirurgicale émustipuleuse modifiée

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