Questo lavoro è stato sostenuto dal Centro nazionale per le risorse di ricerca e il Centro Nazionale per l&#39;avanzamento Sciences traslazionale, National Institutes of Health, attraverso di Grant UL1TR000124 (FW); Felix &amp; Mildred Yip Dotata cattedra e la Famiglia Fondo Barnes (a DTWW), il National Institute of Dental Research &amp; craniofacciale dei National Institutes of Health in premio Numero T90DE022734 (MT). Il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale del National Institutes of Health.

1. Magnetic Exosome Estrazione basata su Bead <ol><li> Pipettare una soluzione ben miscelato di 5 ml di microparticelle magnetiche rivestite di streptavidina in 495 ml di tampone fosfato salino (PBS) in una provetta per risospendere le sfere. Lavare e risospendere le sfere con 500 ml di PBS per tre volte di utilizzare una griglia magnetica. La cremagliera è una matrice di magneti sul lato di un&#39;unità abitativa che può contenere i tubi campione microcentrifuga. <ol><li> Per ogni lavaggio, prima lasciate che i tubi si siedono sulla griglia per 1 min, e quindi utilizzare un puntale per rimuovere con attenzione il buffer di surnatante senza disturbare le perline. </li><li> Posizionare le provette su un rack regolare senza magneti a lato. Aggiungere 500 ml di PBS nei tubi, e utilizzare la pipetta per miscelare la soluzione e perline insieme. Poi mettere i tubi di nuovo sul sostegno magnetico per separare nuovamente le perle dalla soluzione. </li><li> Eseguire questa rimozione di tampone via di magnetizzazione e risospensione in PBS untotale di tre volte. Questo esegue un lavaggio iniziale delle particelle magnetiche. </li></ol></li><li> Risospendere le sfere in 490 ml di tampone PBS, con il tubo posto sulla parte non magnetizzata del rack magnetica. Dispensare 5 ml di anticorpi anti-CD63 umano topo biotinilato a 1,0 mg / ml di brodo di concentrazione nella miscela di perline. Utilizzare la pipetta per mescolare le perline e anticorpi in soluzione. </li><li> Posizionare i tubi microcentrifuga con tallone e miscela di anticorpi biotinilati su un rotatore campione. Impostare i parametri rotatori per il rotatore campione per rotazione reciproca a 90 ° inclinazione per 5 sec e vibrante a 5 ° per 1 sec. Ruotare i tubi miscela campione-bead questi parametri per 30 minuti a RT. </li><li> Rimuovere l&#39;anticorpo non legato dopo coniugazione. <ol><li> Dopo 30 min di rotazione a RT, posizionare i tubi nel rack magnetica per 5 min. </li><li> Eseguire tre lavaggi di perline rimuovendo la fase liquida utilizzando una micropipetta e lavare con 500 μ; L di PBS. Dopo il lavaggio triple, risospendere le sfere in 490 ml di caseina-PBS e posto sulla parte non magnetizzata del rack. </li></ol></li><li> Estrazione Exosome utilizzando perline legate agli anticorpi. <ol><li> Etichettare ogni provetta con ID del campione mirato. Pipettare un campione di 10 ml di siero o saliva nella provetta. Usare la pipetta per mescolare il campione e sfere magnetiche pipettando più volte. </li><li> Posizionare i tubi con campioni e anti-umane perline anticorpi CD63 sui rotatori e ruotare per 2 ore a temperatura ambiente. Utilizzare gli stessi parametri dei rotatori come descritto al punto 1.2. </li><li> Dopo 2 ore di campione rotante, eseguire un lavaggio tripla magnetizzando di perline separati dalla soluzione, eliminando fase liquida con micropipetta, e risospensione perline in 500 ml di tampone Tris-HCl. Le perle risultanti sono oggi legati ai exosomes e sono pronti per il rilascio campo elettrico e misurazione. </li></ol></li></ol> 2. elettrica campo indotto Rilasciato unnd Misura di Exosomal Content <ol><li> Iniziale prerivestimento di elettrodi con GADPH Primer <ol><li> Applicare una plastica bene ad un matrice di elettrodi per evitare contaminazione incrociata dei singoli elettrodi. Per questo esperimento, utilizzare un array di elettrodi 16-sensore con ciascun elettrodo unità nella matrice costituita da un lavoro, banco, ed elettrodo di riferimento in oro nudo. </li><li> Preparare una miscela scorta di 100 sonde nM DNA, 0.3 M KCl, e pirrolo 10 mM pipettando reagenti azionari in un tubo di acqua ultrapura distillata. Mescolare bene nel vortex. NOTA: Per questo studio, la sonda di DNA selezionata corrispondente al gene di riferimento GAPDH, che è noto per esistere all&#39;interno esosomi. La sequenza di sonda utilizzata è: 5&#39;-Biotin-AGGTCCACCACTGACACGTTG-3 &#39;. Utilizzare questa miscela su tutti gli elettrodi. </li><li> Pipettare 60 ml di miscela monomerica sonda-DNA sulla superficie di ciascun elettrodo d&#39;oro. Esaminate gli elettrodi per garantire che vi sia un&#39;adeguata copertura della lavorazione, counter, e di riferimento elettrodi per la miscela liquida. </li><li> Electropolymerize miscela monomerica sonda per creare uno strato di polimero conduttore sulla superficie dell&#39;elettrodo applicando un&#39;onda quadra (CSW) profilo ciclico campo elettrico alla superficie dell&#39;elettrodo. Questo campo elettrico consiste nell&#39;applicare 350 mV per 9 secondi e subito il passaggio a 950 mV per 1 sec. Applicare questa profilo ciclico onda quadra all&#39;elettrodo per 10 cicli, per un totale di 100 secondi di campo elettrico applicato. </li><li> Risciacquare superficie del sensore 3 volte con acqua distillata e asciugare con azoto gassoso per rimuovere il liquido dalla superficie dell&#39;elettrodo. Assicurarsi che il liquido sia stato completamente rimosso dall&#39;elettrodo. </li></ol></li><li> Exosome Cargo scarico <ol><li> Load 5 ml di 1 micron di una sonda detector in 495 ml di miscela complessa bead-exosome e utilizzare una pipetta per mescolare. NOTA: La sonda rivelatore è una sequenza di DNA iniettore coniugato ad una molecola fluoresceina all&#39;estremità 3 &#39;. Il prob detectore sequenza utilizzata per questo studio corrisponde al GAPDH mRNA trovato all&#39;interno esosomi. La sequenza della sonda rivelatore coniugato con fluoresceina è: 5&#39;-GCAGTGGGGACACGGAAGGCC-fluoresceina-3 &#39;. </li><li> Pipettare 60 ml di sonda e miscela complessa bead-exosome su superficie elettrodo d&#39;oro con un gruppo di magneti sotto. Questo gruppo magnete consiste di sedici 2,54 millimetri di diametro magneti al neodimio allineati per corrispondere agli elettrodi di lavoro del sensore. Figura 2A illustra il posizionamento dei magneti e soluzione bead-exosome. </li><li> Una volta caricato campione sulla superficie dell&#39;elettrodo, applica 20 cicli del campo elettrico CSW con 9 sec a -300 mV e 1 sec a +200 mV (200 sec totale). Il carico exosomal che viene rilasciato si ibridano alle primer sulla superficie dell&#39;elettrodo. Se marcatori superficiali del exosome sono oggetto di indagine, ignorare questa parte dell&#39;esperimento. Figura 2B illustra questo processo. </li> &lt;li&gt; Wash-off gli analiti non legato sulla superficie dell&#39;elettrodo di triplo risciacquo la superficie dell&#39;elettrodo con acqua distillata. Essiccare l&#39;elettrodo con gas azoto. </li></ol></li><li> Reporter anticorpi e lettura <ol><li> Aggiungere 60 ml di 150 unità / ml di anticorpi anti-fluoresceina coniugato con perossidasi di rafano (HRP in 1: 1.000 diluizione) diluito in caseina / PBS. </li><li> Utilizzare un campo elettrico coniugazione spinti a complessi HRP anti-fluoresceina per il panino della sonda. Applicare -200 mV per 1 sec e +500 mV per 1 sec per 5 cicli alla superficie dell&#39;elettrodo. La Figura 2A mostra la cattura e rivelatore complessi sonda sia una proteina e di acido nucleico. </li><li> Triple superficie del sensore di lavaggio con acqua distillata e asciugare con azoto. </li><li> A seguito del dilavamento di anticorpi anti-fluoresceina in eccesso non legato, aggiungere 60 ml di 3,3 &#39;, 5,5&#39;-Tetrametilbenzidina (TMB) substrato. Caricare questo substrato a ogni superficie del sensore con una pipetta multicanale. </li><li> Eseguire lettura amperometrico della corrente misurando la corrente dell&#39;elettrodo a -200 mV per 60 sec utilizzando un potenziostato elettrochimico capace di misurazione simultanea di 16 canali. Figura 2C è un esempio di profilo di corrente durante la lettura. </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52439/52439fig2highres.jpg" /> Figura 2. Componenti del Metodo Efirm. (A) Metodo di estrazione esosomi da BIOFLUID con CD63 anti-uomo rivestito microparticelle magnetiche e quindi lo scarico exosome carico con onde quadre ciclici applicate al complesso particelle exosome. (B) Schema di elettrodi biosensore di rilevazione dei bersagli di RNA / DNA / proteine ​​dal exosome rilasciato. (C) Esempio rappresentativo di lettura amperometrica dalla metodologia Efirm, dove più consistente attuale corrisponde to livelli elevati di una biomolecola. Questa cifra è da Wei et al. 14 <a href="https://www.jove.com/files/ftp_upload/52439/52439fig2large.jpg" target="_blank">, cliccate qui per vedere una versione più grande di questa figura.</a>

<ol>
	<li>Rabinowits, G., Ger&#231;el-Taylor, C., Day, J. M., Taylor, D. D., Kloecker, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+MicroRNA:+A+Diagnostic+Marker+for+Lung+Cancer.">Exosomal MicroRNA: A Diagnostic Marker for Lung Cancer.</a> Clinical Lung Cancer. 10 (1), 42-46 (2009).</li><li>Thakur, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+DNA+in+exosomes:+a+novel+biomarker+in+cancer+detection.">Double-stranded DNA in exosomes: a novel biomarker in cancer detection.</a> Cell Research. 24 (6), 766-769 (2014).</li><li>Lau, C. S., Wong, D. T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Exosome-like+Microvesicles+and+Salivary+Gland+Cells+Interplay+Alters+Salivary+Gland+Cell-Derived+Exosome-like+Microvesicles+In.">Breast Cancer Exosome-like Microvesicles and Salivary Gland Cells Interplay Alters Salivary Gland Cell-Derived Exosome-like Microvesicles In.</a> Vitro. PLoS ONE. 7 (3), e33037 (2012).</li><li>Bala, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+in+exosomes+indicate+hepatocyte+injury+and+inflammation+in+alcoholic,+drug-induced,+and+inflammatory+liver+diseases.">Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases.</a> Hepatolog. 56 (5), 1946-1957 (2012).</li><li>Dear, J. W., Street, J. M., Bailey, 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=Urinary+exosomes:+A+reservoir+for+biomarker+discovery+and+potential+mediators+of+intrarenal+signalling.">Urinary exosomes: A reservoir for biomarker discovery and potential mediators of intrarenal signalling.</a> Proteomics. 13 (10-11), 1572-1580 (2013).</li><li>L&#228;sser, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+saliva,+plasma+and+breast+milk+exosomes+contain+RNA:+uptake+by+macrophages.">Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages.</a> Journal of Translational Medicine. 9 (1), 9 (2011).</li><li>Palanisamy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructural+and+Transcriptomic+Analyses+of+Human+Saliva+Derived+Exosomes.">Nanostructural and Transcriptomic Analyses of Human Saliva Derived Exosomes.</a> PLoS ONE. 5 (1), e8577 (2010).</li><li>Camussi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes/microvesicles+as+a+mechanism+of+cell-to-cell+communication.">Exosomes/microvesicles as a mechanism of cell-to-cell communication.</a> Kidney International. 78 (9), 838-848 (2010).</li><li>Lau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+pancreatic+cancer-derived+exosomes+in+salivary+biomarker+development.">Role of pancreatic cancer-derived exosomes in salivary biomarker development.</a> The Journal of Biological Chemistry. 288 (37), 26888-26897 (2013).</li><li>Th&#233;ry, C., Amigorena, S., Raposo, G., 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> Current Protocols in Cell Biology. 3 (22), (2001).</li><li>Park, N. 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David Wong è co-fondatore di RNAmeTRIX Inc., una società di diagnostica molecolare. PeriRx LLC sublicenza proprietà intellettuali relative alla diagnostica molecolare da RNAmeTRIX. David Wong è un consulente di PeriRx.

Come i risultati indicano, CD63 anti-umana rivestite nanoparticelle magnetiche sono in grado di catturare specificamente piccole particelle che hanno una dimensione che varia 70-100 nm. Questa particella catturata è coerente con il profilo precedentemente osservata di esosomi. Inoltre, l&#39;utilizzo del CSW bassa tensione in seguito alla cattura delle particelle è indicata per rimuoverli dalla superficie tallone e causare profili degradazione DNA simile a quello di un metodo tradizionale tampone di lisi base per il r...

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>. The disclosures were updated.

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>.

Errata: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Ricerca Exosome è un settore emergente della ricerca che esamina microvescicole lipidiche che portano RNA 1, 2 DNA e proteine ​​3 cargo. Precedenti indagini exosome biologia hanno portato alla identificazione di esosomi in biofluidi come il sangue 4, 5 urine, latte materno 6, e 7 saliva. Studi hanno dimostrato che esosomi giocano un ruolo in diverse vie cellulari, meditazione remoto comunicazione tra diversi sistemi del corpo 8. A causa del ruolo esosomi giocano nella comunicazione intercellulare, si ipotizza che possano pacchetto obiettivi biomolecole (proteine, RNA e DNA) correlati con stati patologici. In vitro 3 e modello animale 9 studi sembrano confermare questa ipotesi. In indagando contenuti exosomal per la scoperta di biomarcatori, è necessario sviluppare una metodologia per l&#39;isolamento selettivo exosome da biofluidi, expulsi indottosu di carico da esosomi, e la quantificazione di biomolecole exosome. Nella misura di questo lavoro, exosomes saranno definite come una struttura avente un diametro di circa 70-100 nm e possedendo superficie marcatore CD63. I ricercatori in genere prima purificano exosomes di ultracentrifugazione 10 e poi elaborare il contenuto exosomal attraverso l&#39;utilizzo di kit tampone di lisi. L&#39;utilizzo di metodi tampone di lisi richiede tempi di incubazione che vanno da minuti a ore. Questo processo può potenzialmente danneggiare exosome carico e portare a campione degrado. Ad esempio, salivare RNA exosome rilasciato tramite tampone di lisi nell&#39;ambiente extracellulare circostante possiede una emivita di sotto 1 min, rendendo la misurazione di exosomal post-RNA Lysis Buffer un compito particolarmente difficile senza l&#39;aggiunta di reagenti stabilizzazione 11. L&#39;effetto composto di aggiungere vari reagenti per la lisi e la stabilizzazione può introdurre agenti che complicano e interferire con la analysis di contenuti exosomal. Un approccio alternativo può essere utile per lo scarico rapidamente contenuti exosomal e conservare in modo sicuro il carico per la caratterizzazione. In questo lavoro, si propone l&#39;utilizzo di un campo elettrico non uniforme per il rilascio di contenuti exosomal. Campi elettrici sono stati conosciuti per portare la capacità di polarizzare e disturbare il doppio strato lipidico che forma membrane cellulari. Il nostro lavoro sperimentale esplora l&#39;uso di non-uniforme onde quadre ciclici (CSW) per interrompere la struttura microvesicle di esosomi e rilasciando merci trasportate. Questo metodo utilizza tensioni in diverse centinaia di millivolt gamma, il che significa che la maggior parte delle biomolecole non saranno interrotte. Abbiamo dimostrato che l&#39;utilizzo di un&#39;onda ciclica quadrati è in grado di comandare il rilascio di salivare exosome contenuti mRNA nell&#39;ambiente fluido circostante. Questa versione del contenuto exosomal è perfettamente integrato con un sistema di elettrodi che può essere usato per quantificare i livelli di espressione biomarker 12,13. Questo metodo proposto consente una rapida, sensibile, e tampone di lisi analisi prive di contenuti exosome. <img alt="Figura 1" src="/files/ftp_upload/52439/52439fig1highres.jpg" /> Figura 1. Panoramica Efirm Workflow.. Il metodo Efirm è sostanzialmente divisa in tre principali fasi che sono necessarie per la purificazione e esosomi analisi. Questo metodo di rilascio e di analisi dei contenuti exosomal basato CSW viene utilizzato in combinazione con microsfere magnetiche specifici CD63 per l&#39;isolamento exosome. Queste perle di CD63-affinità consentono l&#39;isolamento selettivo di esosomi da campioni salivari (e altri fluidi biologici). Dopo l&#39;incubazione e l&#39;estrazione dei esosomi utilizzando le perle magnetizzati, le perle sono migrati al sistema sensore elettrochimico per la CSW basato rilascio contenuto e parte l&#39;analisi dell&#39;esperimento. Figura 1 fornisce una panoramica del lavoroflusso del metodo Efirm.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helios 16-Channel Reader System with Chip Interface&#160;</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td style="width:132px;">&#160;RS-1000-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16X Sensor Chip, Bare Gold, pack of 5 chips</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td>SC1000-16X-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Biotinylated anti-human CD63 Antibody</td>
			<td style="width:157px;">Ancell, USA</td>
			<td style="width:132px;">215-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dynabeads MyOne Streptavidin T1</td>
			<td>Invitrogen, USA</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Neodynium Magnetics ( 1/10&#34; dia. x 1/32&#34; thick)</td>
			<td style="width:157px;">K&#38;J Magnetics, USA</td>
			<td>DH101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Ultrapure Distilled Water</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Mettler Toldeo 3M KcL Solution</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>1911512</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Pyrrole</td>
			<td style="width:157px;">Sigma-Aldrich, USA</td>
			<td style="width:132px;">W338605-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Anti-Fluorescein-POD, Fab fragments</td>
			<td style="width:157px;">Roche, Germany</td>
			<td>11426346910</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:347px;">3, 3&#8242;, 5, 5&#8242; tetramethylbenzidine substrate (TMB/H2O2, low activity)&#160;</td>
			<td style="width:157px;">Neogen, Usa</td>
			<td style="width:132px;">330175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Phosphate Buffered Saline Solution</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td align="right" style="width:132px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Casein/PBS</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>37532</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of exosomal biomarker by electric field-induced release and measurement (efirm)

Esosomi sono strutture microvescicolare che svolgono un ruolo di mediazione nella comunicazione intercellulare. È interessante studiare il carico interno di esosomi per determinare se trasportano malattie biomarcatori discriminatori. Per l&#39;esecuzione di analisi exosomal, è necessario sviluppare un metodo per l&#39;estrazione e l&#39;analisi exosomes da biofluidi bersaglio senza danneggiare il contenuto interno. Rilascio elettrico campo indotto e misura (Efirm) è un metodo per estrarre specificamente esosomi da biofluidi, scaricando il loro carico, e testare il loro contenuto di RNA / proteine ​​interna. L&#39;utilizzo di un CD63 specifici anticorpi microparticelle magnetico anti-umano, esosomi vengono prima precipitati da fluidi biologici. Dopo l&#39;estrazione, a bassa tensione onde elettriche quadre ciclici (CSW) vengono applicati per distruggere la membrana vescicolare e causare carico scarico. Il contenuto del exosome viene ibridato al primer DNA o anticorpi immobilizzati su una superficie dell&#39;elettrodo per quantification contenuto molecolare. Il metodo Efirm è vantaggioso per l&#39;estrazione di esosomi e scarico merci per analisi senza tampone di lisi. Questo metodo è in grado di eseguire il rilevamento preciso dei due bersagli di RNA e proteine ​​biomarcatori nella exosome. Efirm estrae esosomi specificamente in base alle loro marcatori di superficie al contrario di tecniche basate dimensioni. La microscopia elettronica a trasmissione (TEM) e test dimostrano la funzionalità del metodo per exosome acquisizione e l&#39;analisi. Il metodo è stato applicato a Efirm exosomal analisi di 9 topi iniettati con le cellule H640 cancro del polmone umano (una linea cellulare trasfettata per esprimere il marcatore exosome umana CD63-GFP), al fine di testare il loro profilo exosome contro 11 topi che ricevono controlli saline. Elevati livelli di biomarkers exosomal (riferimento gene GAPDH e proteine ​​marcatori superficie umana CD63-GFP) sono stati trovati per l&#39;H640 iniettato topi in entrambi i campioni di siero e saliva. Inoltre, saliva e campioni di siero sono stati dimostrato di avere linearità (R = 0,79). Questi risultati sono suggestivi per la vitalità di biomarcatori exosome salivari per la rilevazione di malattie distali.

Validazione di Exosome Cattura di Beads Uso TEM Isolamento di esosomi da saliva utilizzando sfere magnetiche CD63 anti-umani è stato convalidato dopo protocollo di estrazione mediante microscopia elettronica a trasmissione (TEM) immagini. TEM mostra sfere magnetiche con granuli 70-100 nm immediatamente adiacenti (vedi Figura 3A, 3B e), in coerenza con il profilo noto di esosomi. Nessun 70-100 granuli nm sono stati osservati per le sfere magnetiche di saliva...

Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Rilevazione di Exosomal Biomarker da campo elettrico indotto rilascio e di misura (Efirm)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

detection-exosomal-biomarker-electric-field-induced-release

Research

JoVE Journal

Bioengineering

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

18.3K Views.  University of California, Los Angeles. Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves. 

Video: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Sviluppo di ovaio di criceto cinese-dosaggi delle proteine ​​della cellula ospite con la tecnologia Acoustic microparticelle di membrana

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Ricerca

Bioingegneria

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

Misura della coesione aggregata da parte del tessuto superficiale Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

Misura della tensione di uscita durante Laser Induced Axon Lesione di valutare Adesione assonale al substrato a piconewton e Millisecond Risoluzione

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Rilevamento ottico di<em&gt; E. coli</em&gt; Batteri di mesoporosi Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Misurazione della massima isometrica forza generata dal permeabilizzate scheletrici fibre muscolari

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Rilevamento dell'endotossina in Nano-formulazioni utilizzando dosaggi di Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Differenziazione delle cellule precursori neurali indotte dal campo elettrico nei dispositivi microfluidici

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Modellazione agli elementi finiti di un microambiente elettrico cellulare

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivi elettrici e a campo magnetico per la stimolazione dei tessuti biologici

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.