Questo studio è stato sostenuto da fondi da Rita Allen sovvenzione della Fondazione per Seena Ajit. Gli autori desiderano ringraziare Erika Balogh e Dr. Soumitra Ghoshroy dall&#39;Università di Centro Electron Microscopy South Carolina per l&#39;uso dello strumento, l&#39;assistenza scientifica e tecnica.

Tutti gli esperimenti usando campioni di sangue da uomo e roditori sono stati eseguiti in conformità a tutte le direttive, i regolamenti e le agenzie di regolamentazione. Soggetti umani sono stati arruolati dopo aver dato il consenso informato come approvato dalla Drexel University College of Medicine Institutional Review Board e di tutte le procedure per gli studi condotti su animali sono stati approvati dalla Institutional Animal Care di Drexel e utilizzo Comitato. 1. Purificazione Exosome dal sangue (~ 5,5 ore) CONSIGLI <ul><li class="jove_content"> Noi Risospendere exosomes da una provetta di sangue (~ 10 ml di sangue umano e ~ 2 ml di sangue del mouse) in 300 ml di RNA tampone di lisi del kit di isolamento miRNA Mirvana. </li><li class="jove_content"> Per purificare exosomes da mezzi di coltura cellulare, il supporto viene centrifugato a 500 xg per 10 minuti per rimuovere le cellule ed anche filtrato attraverso un filtro di 0,22 micron dopo centrifugazione a 12.000 x g. La purificazione exosome da terreni di coltura cellularenon richiede una diluizione in PBS, il lavaggio PBS o il secondo passo ultracentrifugazione. </li><li class="jove_content"> Se vengono utilizzati i exosomes purificati per studi di proteomica, aggiungendo un gradiente di saccarosio fase di centrifugazione dopo il passo ultracentrifugazione diminuirà la quantità di aggregati proteici e contaminanti 15. </li><li class="jove_content"> La morfologia osservata di exosomes può variare a seconda sia della fasi di lavorazione per la TEM sorgente e. Non abbiamo osservato la morfologia a forma di coppa per exosomes che è stato precedentemente attribuite a differenze di trattamento del campione 9,15. Le nostre immagini TEM compreso l&#39;etichettatura immunogold sono simili alle precedenti relazioni provenienti da diverse fonti, tra cui sangue, delle vie biliari e della cultura corticale derivato exosomes 9,16,17. </li></ul><ol><li> Capovolgere tubo rivestito EDTA contenente il sangue 5x e posto in posizione verticale a temperatura ambiente per 10-60 min. </li><li> Centrifugare a 2000 xg per 15 min a 4 ° C. </li&gt; <li> Raccogliere lo strato superiore, il plasma, in 15 ml provette e tenere in ghiaccio. </li><li> Diluire fluido con un volume uguale di PBS. Centrifugare 30 min a 2000 xga 4 ° C. </li><li> Trasferimento di centrifugare provette, aggiungere 1X PBS per un volume totale di 24 ml e centrifugare 45 min a 12.000 xg a 4 ° C. </li><li> Trasferimento a ultracentrifuga tubi e centrifugare 2 ore a 110.000 xg a 4 ° C. </li><li> Risospendere il pellet in PBS e centrifugare 1 ora a 100.000 xg a 4 ° C. </li><li> Risospendere il pellet in tampone appropriato (tampone di lisi RNA). </li><li> Risospendere in PBS o RIPA buffer per la microscopia elettronica e western blot, rispettivamente. </li></ol> 2. RNA Purification Utilizzando un Mirvana Isolation Kit miRNA Modified (~ 1 ora) CONSIGLI <ul><li class="jove_content"> Preparare l&#39;area per l&#39;isolamento di RNA da indossare dispositivi di protezione individuale (DPI), pulendo le superfici e pipette con RNasi Zap, e l&#39;utilizzo di puntali con filtro sterili e tubi. </li><liclass = &quot;jove_content&quot;&gt; Anche se il protocollo dal venditore dà la possibilità di purificare solo miRNA, abbiamo sempre isolare l&#39;RNA totale tra mRNA e miRNA in modo da poter studiare sia dallo stesso campione. </li><li class="jove_content"> Aggiunta di una fase di trattamento DNasi on-column rimuove le tracce di DNA che possono essere presenti. A seconda della sorgente di exosomes, il DNA è stato riportato essere assente in exosomes purificate da alcune fonti tra mouse e linee di mastociti umani 8 ma sequenze di DNA cromosomico è stato isolato exosomes purificati da supporti di cardiomiociti in coltura 18. </li><li class="jove_content"> Abbiamo ridotto il volume della soluzione di lavaggio 1-350 microlitri (Passo 2.4) perché il venditore fornisce abbastanza unica soluzione di lavaggio 1 per un 700 microlitri aggiunta. Se il volume non è ridotta non vi è sufficiente per la soluzione utilizzando l&#39;intero contenuto del kit. Passaggi rimanenti sono state eseguite secondo le raccomandazioni del fabbricante. </li></ul><ol><li> Aggiungere 1/10 VOLume di miRNA Additivo Omogenato (30 ml) e incubare in ghiaccio per 10 min. </li><li> Estrarre con un volume di acido-fenolo: cloroformio pari al volume iniziale lisato. Centrifugare e recuperare la fase superiore in una nuova provetta. </li><li> Aggiungere 1,25 volumi 100% di etanolo (375 ml). Vortex. Applicare al filtro a cartuccia e centrifugare 15 sec a 10.000 x g. </li><li> Aggiungere la metà del volume di raccomandata miRNA soluzione di lavaggio 1 (350 ml) e centrifugare 15 sec a 10.000 x g. </li><li> Aggiungere 10 microlitri DNasi 1-70 microlitri tampone RDD (Qiagen) per ogni campione. Aggiungere 80 microlitri di colonna e incubare 15 min a temperatura ambiente. </li><li> Aggiungi Soluzione di lavaggio 1 (350 ml) e centrifugare 15 sec a 10.000 x g. </li><li> Aggiungi Soluzione di lavaggio 2/3 (500 microlitri) e centrifugare 15 sec a 10.000 x g. REPEAT. </li><li> Eluire con 40 microlitri 95 ° C nucleasi H 2 O. </li><li> Determinare la concentrazione di RNA e diluire l&#39;RNA a 100 ng / ml. </li></ol> 3. miRNA Profiling di Exosomes da Blood Utilizzando Taqman Low Density Array (TLDA) Cards (~ 6,5 ore) CONSIGLI <ul><li class="jove_content"> Preparare l&#39;area per l&#39;isolamento di RNA da indossare PPE, asciugandosi le superfici e pipette con RNasi Zap, e l&#39;utilizzo di puntali con filtro sterili e tubi. </li><li class="jove_content"> Pre-amplificazione fase (pre-amp) è consigliato per 1-350 ng RNA (350-1,000 campioni ng non richiedono fase di pre-amp dopo sintesi del DNA). E &#39;importante determinare se pre-amp è necessario per il vostro campione, perché è necessario trattare tutti i campioni in condizioni di parità. Fase di pre-amp aggiunge costi e dei valori Ct hanno un basso cut off accettabile (32 invece di 40). </li><li class="jove_content"> È utile preparare la reazione cDNA in tubi striscia invece di una piastra a seconda del numero di carte che possono essere eseguiti in un solo giorno. Questo ridurrà al minimo congelamento-scongelamento dei campioni di cDNA che non saranno trattate il giorno stesso. </li><li class="jove_content"> Exosomes raccolti da diverse fonti hanno diffeaffittare quantità di RNA. Questo protocollo consente un massimo di 3 microlitri stampo di RNA per la reazione di sintesi di cDNA. A seconda della sorgente, la quantità di RNA exosomal che può essere isolata forse limitato. Mentre un tessuto tipico produrrebbe abbastanza RNA che cDNA sarebbe composto da 100 ng di RNA in 3 volumi microlitri, exosomes dal sangue o dal mezzo di coltura di cellule umane spesso contengono meno RNA e cDNA deve essere effettuato da meno materiale di partenza. Exosomes sangue topo presentano livelli di RNA e almeno 100 ng in 3 ml possono essere ottenuti. </li></ul><ol><li> Per preparare cDNA da RNA purificato utilizzando Piscine Megaplex trascrittasi inversa primer (Pool A e Pool B contenere primer per i miRNA su schede di microfluidica array A e B), scongelare i componenti di reazione in ghiaccio. </li><li> Etichetta due provette da 1,5 ml RNasi-free per la reazione RT con piscina A primer o Pool B primer e aggiungere componenti in modo grafico. Aggiungi almeno un volume aggiuntivo di componenti per 2 o più reazioni o fare un master mix come consigliato da tegli venditore. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente reazione RT </td><td> Concentrazione Componente </td><td> Volume per 1 campione (totale V / campione = 4,5 ml) </td></tr><tr><td> Acqua priva di nucleasi </td><td></td><td> 0.20 ml </td></tr><tr><td> Megaplex RT Buffer (10X) </td><td> 1X </td><td> 0,80 ml </td></tr><tr><td> dNTP (100 mm) </td><td> 4.4 mM </td><td> 0.20 ml </td></tr><tr><td> MgCl2 (25 mM) </td><td> 5 mM </td><td> 0,90 ml </td></tr><tr><td> RNase Inhibitor (20U/μl) </td><td> 2 U </td><td> 0,10 ml </td></tr><tr><td> Megaplex RT Primer A o B (10 volte) </td><td> 1X </td><td> 0,80 ml </td></tr><tr><td> MultiScribe trascrittasi inversa </td><td> 75 U </td><td> 1,50 ml </td></tr></tbody></table><ol start="3"><li> Invertire i tubi 6 volte e centrifuge brevemente. Poi, pipetta 4,5 microlitri mix di reazione RT in MicroAmp strisce da 8 provette o una piastra di reazione ottica a 96 pozzetti MicroAmp. </li><li> Aggiungere 100 ng di RNA e di regolare il volume a 3 ml con acqua trattata DEPC o aggiungere 3 RNA totale microlitri, mescolate con la punta della pipetta, e sigillare le provette o la piastra. Spin brevemente e incubare in ghiaccio per 5 min. </li><li> Caricare la reazione nella macchina di PCR e correre con le seguenti condizioni (come raccomandato dal Biosystems protocollo Piscine Megaplex Applicata): </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Tappa </td><td> Temp </td><td> Volta </td></tr><tr><td> Ciclo (40x) </td><td> 16 ° C </td><td> 2 min </td></tr><tr><td></td><td> 42 ° C </td><td> 1 min </td></tr><tr><td></td><td> 50 ° C </td><td> 1 sec </td></tr><tr><td> Tenere </td><td> 85 ° C </td><td> 5 min </td></tr><tr><td> Tenere </td><td> 4 ° C </td><td> ∞ </td></tr></tbody></table><ol start="6"><li> Conservare cDNA a -20 ° C (buono per almeno una settimana) o passare alla fase di pre-Amp, che possono essere conservati per una settimana. </li><li> Scongelare primer preamplificazione per la Pool A e Pool B sul ghiaccio e miscelare. Swirl Taqman Pre-Amp Master Mix e tenere in ghiaccio anche. </li><li> Seguire la tabella aggiungendo almeno un volume aggiuntivo di componenti per 2 o più reazioni o fare un master mix come raccomandato dal produttore. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente Reazione Pre-Amp </td><td> Volume per 1 campione </td></tr><tr><td> Acqua priva di nucleasi </td><td> 7,5 microlitri </td></tr><tr><td> TaqMan PreAmp Master Mix (2X) </td><td> 12,5 ml </td></tr><tr><td> Megaplex Primer PreAmp A o B (10 volte) </td><td> 2,5 microlitri </td></tr></tbody></table><ol start="9"><li> Pipettare 2,5 ml RT produttorict in MicroAmp Strips 8-tubo o una piastra di reazione ottica a 96 pozzetti MicroAmp ed erogare 22,5 ml mix di reazione PreAmp in ciascun pozzetto. </li><li> Sigillare la piastra, incubare in ghiaccio 5 min e quindi caricare il campione nella macchina PCR. Eseguire le seguenti condizioni. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Tappa </td><td> Temp </td><td> Volta </td></tr><tr><td> Tenere </td><td> 95 ° C </td><td> 10 min </td></tr><tr><td> Tenere </td><td> 55 ° C </td><td> 2 min </td></tr><tr><td> Tenere </td><td> 72 ° C </td><td> 2 min </td></tr><tr><td> Ciclo (12x) </td><td> 95 ° C </td><td> 15 sec </td></tr><tr><td></td><td> 60 ° C </td><td> 4 min </td></tr><tr><td> Tenere </td><td> 99,9 ° C </td><td> 10 min </td></tr><tr><td> Tenere </td><td> 4 ° C </td><td> ∞ </td></tr></tbody></table><ol start="11"><li> Dopo la reazione preamplificatore è finito, centrifugare brevemente le provette o la piastra e poi aggiungere 75 ml 0,1 X. TE pH 8.0 per ciascun bene o un tubo. </li><li> Sigillare e mescolare e centrifugare brevemente. Conservare il prodotto diluito per una settimana a -20 ° C o proseguire per caricare la piastra. </li><li> Unire la segue in un RNase-free 1,5 ml tubo in ghiaccio. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente </td><td> Volume per 1 scheda </td></tr><tr><td> TaqMan Universal PCR Master Mix, No AmpErase UNG (2X) </td><td> 450 microlitri </td></tr><tr><td> PREAMP prodotto diluito </td><td> 9 ml </td></tr><tr><td> Acqua priva di nucleasi </td><td> 441 microlitri </td></tr></tbody></table><ol start="14"><li> Capovolgere la provetta per mescolare e centrifugare brevemente. Dispensare 100 ul miscela di reazione PCR in ogni porta della scheda Array MicroRNA Taqman. </li><li> Centrifugare la scheda 2 x 1.000 xg per 1 minuto. Sigillare la carta ed eseguirlo utilizzando il modello fornito con i primer nel manuale Applied Biosystems e le Taqman impostazioni Array 384 e bassa densità. Usiamo Applied Biosystems 7900HT veloce Real-Time PCR. </li></ol> 4. mRNA Analisi per Taqman (~ 1,5 ore) <ol><li> Per preparare reazione di qPCR, miscelare i componenti di reazione nell&#39;ordine nel grafico. Tutti i campioni saranno analizzati con sonde di primer per il gene di interesse e 18S rRNA come il gene normalizzatore. </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td> Componente Reazione Taqman </td><td> Volume per 1 campione </td></tr><tr><td> Acqua priva di nucleasi </td><td> 7 ml (regolare sulla base del campione individuale) </td></tr><tr><td> Taqman Veloce Master Mix (2X) </td><td> 10 microlitri </td></tr><tr><td> Sonde TaqMan Primer (20x) </td><td> 1 microlitri </td></tr><tr><td> cDNA da 100 ng di RNA </td><td> 2 &amp; mu, L (regolare sulla base del campione individuale) </td></tr></tbody></table><ol start="2"><li> Esegui il pozzo piastra 96 ​​tramite il blocco a 96 pozzetti veloce come consigliato dal produttore (Biosystems 7900HT veloce Real-Time PCR System Applicata). </li></ol>

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	<li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Mendell, J. T., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+stress+signaling+and+human+disease.">MicroRNAs in stress signaling and human disease.</a> Cell. 148, 1172-1187 (2012).</li><li>Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-coding+RNAs+in+human+disease.">Non-coding RNAs in human disease.</a> Nature reviews. Genetics. 12, 861-874 (2011).</li><li>Mitchell, P. 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+as+stable+blood-based+markers+for+cancer+detection.">Circulating microRNAs as stable blood-based markers for cancer detection.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 10513-10518 (2008).</li><li>Chen, X., 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=Characterization+of+microRNAs+in+serum:+a+novel+class+of+biomarkers+for+diagnosis+of+cancer+and+other+diseases.">Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.</a> Cell research. 18, 997-1006 (2008).</li><li>Gilad, 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=Serum+microRNAs+are+promising+novel+biomarkers.">Serum microRNAs are promising novel biomarkers.</a> PloS one. 3, e3148 (2008).</li><li>Mittelbrunn, M., 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=Unidirectional+transfer+of+microRNA-loaded+exosomes+from+T+cells+to+antigen-presenting+cells.">Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells.</a> Nature. 2, 282 (2011).</li><li>Valadi, H., 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=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nat Cell Biol. 9, 654-659 (2007).</li><li>Gyorgy, B., 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=Membrane+vesicles,+current+state-of-the-art:+emerging+role+of+extracellular+vesicles.">Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.</a> Cell Mol Life Sci. 68, 2667-2688 (2011).</li><li>Mathivanan, S., Ji, H., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+extracellular+organelles+important+in+intercellular+communication.">Exosomes: extracellular organelles important in intercellular communication.</a> J. Proteomics. 73, 1907-1920 (2010).</li><li>Ramachandran, S., Palanisamy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+transfer+of+RNAs:+exosomes+as+mediators+of+intercellular+communication.">Horizontal transfer of RNAs: exosomes as mediators of intercellular communication.</a> Wiley Interdiscip Rev. RNA. , (2011).</li><li>Record, M., Subra, C., Silvente-Poirot, S., Poirot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+as+intercellular+signalosomes+and+pharmacological+effectors.">Exosomes as intercellular signalosomes and pharmacological effectors.</a> Biochem Pharmacol. 81, 1171-1182 (2011).</li><li>Thery, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat Rev Immunol. 9, 581-593 (2009).</li><li>Ludwig, A. K., Giebel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+small+vesicles+participating+in+intercellular+communication.">Exosomes: small vesicles participating in intercellular communication.</a> The international journal of biochemistry &amp; cell biology. 44, 11-15 (2012).</li><li>Thery, 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> Curr. Protoc. Cell Biol. Chapter 3, Unit 3 22 (2006).</li><li>Masyuk, A. I., 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=Biliary+exosomes+influence+cholangiocyte+regulatory+mechanisms+and+proliferation+through+interaction+with+primary+cilia.">Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia.</a> American journal of physiology. Gastrointestinal and liver physiology. 299, G990-G999 (2010).</li><li>Faur&#233;, J., 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+are+released+by+cultured+cortical+neurones.">Exosomes are released by cultured cortical neurones.</a> Molecular and Cellular Neuroscience. 31, 642-648 (2006).</li><li>Waldenstrom, A., Genneback, N., Hellman, U., Ronquist, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+microvesicles+contain+DNA/RNA+and+convey+biological+messages+to+target+cells.">Cardiomyocyte microvesicles contain DNA/RNA and convey biological messages to target cells.</a> PloS one. 7, e34653 (2012).</li><li>Simpson, R. J., Lim, J. W., Moritz, R. L., Mathivanan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+proteomic+insights+and+diagnostic+potential.">Exosomes: proteomic insights and diagnostic potential.</a> Expert Rev Proteomics. 6, 267-283 (2009).</li><li>Turchinovich, A., Weiz, L., Langheinz, A., Burwinkel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+extracellular+circulating+microRNA.">Characterization of extracellular circulating microRNA.</a> Nucleic acids research. 39, 7223-7233 (2011).</li><li>El-Andaloussi, 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=Exosome-mediated+delivery+of+siRNA+in+vitro+and+in+vivo.">Exosome-mediated delivery of siRNA in vitro and in vivo.</a> Nature. 7, 2112-2126 (2012).</li><li>Singh, P. P., Smith, V. L., Karakousis, P. C., Schorey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+isolated+from+mycobacteria-infected+mice+or+cultured+macrophages+can+recruit+and+activate+immune+cells+in+vitro+and+in+vivo.">Exosomes isolated from mycobacteria-infected mice or cultured macrophages can recruit and activate immune cells in vitro and in vivo.</a> J. Immunol. 189, 777-785 (2012).</li><li>Etheridge, A., Lee, I., Hood, L., Galas, D., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNA:+A+new+source+of+biomarkers.">Extracellular microRNA: A new source of biomarkers.</a> Mutat. Res. , (2011).</li><li>Stenvang, J., Silahtaroglu, A. N., Lindow, M., Elmen, J., Kauppinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+LNA+in+microRNA-based+cancer+diagnostics+and+therapeutics.">The utility of LNA in microRNA-based cancer diagnostics and therapeutics.</a> Seminars in cancer biology. 18, 89-102 (2008).</li><li>Mathivanan, S., Fahner, C. J., Reid, G. E., Simpson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExoCarta+2012:+database+of+exosomal+proteins,+RNA+and+lipids.">ExoCarta 2012: database of exosomal proteins, RNA and lipids.</a> Nucleic acids research. 40, D1241-D1244 (2012).</li></ol>

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In questo protocollo, mostriamo la quantificazione dei miRNA e mRNA da exosomes purificati mediante centrifugazione differenziale di sangue e di cultura dei media. Exosomes hanno diverse componenti che dipendono dalla loro origine e sono coinvolti in una serie di funzioni biologiche, compresa la risposta immunitaria, presentazione dell&#39;antigene, comunicazione intracellulare, e il trasferimento di RNA e proteine ​​9,11,12,19,20. Mentre la dimensione e la forma è un determinante di purezza exosome, una...

Brevi non codificanti miRNA modulano l&#39;espressione genica legandosi al mRNA bersaglio. Seme sequenza di complementarità ~ 7 coppie di basi miRNA consente di legarsi al mRNA bersaglio conseguente inibizione della traduzione o nella riduzione della stabilità del mRNA, entrambi i quali possono portare a diminuzione dell&#39;espressione della proteina bersaglio 1. La ricerca negli ultimi dieci anni ha inequivocabilmente dimostrato un ruolo fondamentale per il miRNA nel mediare le funzioni cellulari. C&#39;è stato anche un notevole sforzo diretto verso la dissezione miRNA cambiamenti molecolari alla base di diverse malattie mediate 2,3. Inoltre, la recente identificazione di miRNA stabili in fluidi corporei 4-6 aperto la strada per il loro uso come nuovi biomarcatori suscettibili di diagnosi clinica. Un modo di trasporto miRNA nei fluidi corporei è via exosomes, piccole vescicole che trasportano mRNA, proteine, mediatori lipidici, e miRNA alle cellule riceventi tramite cir arteriosa sistemicaione 7-14. Ciò si traduce in modulazione dell&#39;espressione genica in cellule riceventi e rappresenta un nuovo meccanismo di comunicazione cellulare. Per esempio, le cellule possono modulare processi immuno-regolatori secernendo e / o exosomes assorbenti contenenti biomolecole coinvolte nella infiammazione, come interleuchina-1β (IL1β), fattore di necrosi tumorale-α (TNFa), fattore di crescita trasformante-β5 (TGFβ5), e i miRNA che regolano questi geni 13. Come espressione di miRNA aberrante è una caratteristica comune in una varietà di malattie umane, queste molecole offrono nuove interessanti opportunità per la scoperta e la validazione di nuovi bersagli terapeutici 2. MicroRNA circolanti sono presenti in tutti i fluidi corporei ed è noto che la composizione del exosomes è diverso in base alle celle di origine da cui sono stati rilasciati. Così essi offrono una via per studiare lo stato fisiologico delle cellule e come le cellule cambiano a segnalazione anchets in risposta a stress comprese malattie. Studiare le alterazioni nella composizione exosome può fornire indicazioni in trasduzione del segnale e studiare il loro potenziale utilità come biomarcatori o percorsi di intervento terapeutico. Qui dimostreremo la purificazione exosomes provenienti da fonti multiple basate su protocolli pubblicati. Questi exosomes saranno utilizzati per l&#39;isolamento dell&#39;RNA seguito da qPCR per identificare e misurare i livelli di miRNA presenti nelle exosomes.

<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (10 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>366643</td>
 <td>For exosome purification from human blood</td>
 </tr>
 <tr>
 <td>EDTA coated vacutainer (2 ml tubes)</td>
 <td>BD Diagnostics</td>
 <td>367841</td>
 <td>For exosome purification from mouse blood</td>
 </tr>
 <tr>
 <td>PAXgene Blood RNA Tube</td>
 <td>BD Diagnostics</td>
 <td>762165</td>
 <td>For miRNA isolation from total blood</td>
 </tr>
 <tr>
 <td>miRVana microRNA isolation kit</td>
 <td>Ambion</td>
 <td>AM1561</td>
 <td></td>
 </tr>
 <tr>
 <td>Acid-Phenol: CHCl3</td>
 <td>Ambion</td>
 <td>9721G</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase 1</td>
 <td>Qiagen</td>
 <td>79254</td>
 <td></td>
 </tr>
 <tr>
 <td>TaqMan Universal PCR Master Mix, No AmpErase UNG</td>
 <td>Applied Biosystems</td>
 <td>4326614</td>
 <td>For TLDA cards</td>
 </tr>
 <tr>
 <td>Megaplex RT rodent Pool Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444746</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex preamp rodent Pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444747</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Rodent MicroRNA A+B set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444909</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex RT Human Pool set V3.0</td>
 <td>Applied Biosystems</td>
 <td>4444745</td>
 <td></td>
 </tr>
 <tr>
 <td>Megaplex Preamp human pool set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444748</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman Array Human MicroRNA A+B Cards Set v3.0</td>
 <td>Applied Biosystems</td>
 <td>4444913</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman MicroRNA RT kit</td>
 <td>Applied Biosystems</td>
 <td>4366596</td>
 <td></td>
 </tr>
 <tr>
 <td>Taqman fast universal PCR master mix </td>
 <td>Applied Biosystems</td>
 <td>4366072</td>
 <td>For mRNA qRT-PCR</td>
 </tr>
 <tr>
 <td>Tumor necrosis factor (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs01113624_g1 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vascular endothelial growth factor A (primer probe)</td>
 <td>Applied Biosystems</td>
 <td>Hs00900055_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>Maxima First Strand cDNA Synthesis Kit for RT-qPCR</td>
 <td>Thermo Scientific</td>
 <td>K1642</td>
 <td>For mRNA</td>
 </tr>
 <tr>
 <td>HSP70 antibody</td>
 <td>Abcam</td>
 <td>ab94368 </td>
 <td>For western blot</td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG-Gold</td>
 <td>Sigma</td>
 <td>G7402</td>
 <td>For electron microscopy</td>
 </tr>
 <tr>
 <td>Rabbit-anti CD81</td>
 <td>Sigma</td>
 <td>SAB3500454</td>
 <td></td>
 </tr>
 <tr>
 <td>Nickel 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Ni</td>
 <td></td>
 </tr>
 <tr>
 <td>Copper 300 mesh carbon formvar grids</td>
 <td>Electron Microscopy Sciences</td>
 <td>FCF300-Cu</td>
 <td></td>
 </tr>
 <tr>
<td></td>
<td></td>
<td></td>
<td>Table 1. Table of specific reagents.</td>
</tr>
 </tbody>
 </table>

purification and microrna profiling of exosomes derived from blood and culture media

MiRNA stabili sono presenti in tutti i fluidi corporei e di alcuni microRNA circolanti sono protetti dalla degradazione di sequestro in piccole vescicole chiamate exosomes. Exosomes possono fondersi con la membrana plasmatica con conseguente trasferimento di RNA e proteine ​​per la cellula bersaglio. Le loro funzioni biologiche includono la risposta immunitaria, presentazione dell&#39;antigene, e la comunicazione intracellulare. Consegna dei miRNA che possono regolare l&#39;espressione genica nelle cellule riceventi attraverso il sangue ha aperto nuove strade per l&#39;intervento di destinazione. Oltre ad offrire una strategia per la consegna di farmaci o RNA agenti terapeutici, contenuti exosomal possono servire come biomarcatori che possono aiutare nella diagnosi, determinare le opzioni di trattamento e la prognosi. Qui descriveremo la procedura per analizzare quantitativamente miRNA e RNA messaggero (mRNA) da exosomes secreti nel sangue e nei mezzi di coltura cellulare. Exosomes purificati saranno caratterizzati mediante analisi Western Blot per exosmarcatori Omal e PCR per mRNA di interesse. Microscopia elettronica a trasmissione (TEM) e l&#39;etichettatura immunogold verranno utilizzati per convalidare la morfologia e l&#39;integrità exosomal. L&#39;RNA totale saranno purificati da questi exosomes per assicurare che siamo in grado di studiare sia l&#39;mRNA e miRNA dallo stesso campione. Dopo la convalida integrità dell&#39;RNA da Bioanalyzer, eseguiremo una produttività quantitativa real time PCR media (qPCR) per identificare il miRNA exosomal utilizzando Taqman Low Density Array (TLDA) carte e studi di espressione genica per le trascrizioni di interesse. Questi protocolli possono essere usati per quantificare variazioni miRNAs exosomal in pazienti, modelli di roditori e terreni di coltura cellulare prima e dopo l&#39;intervento farmacologico. Contenuto Exosomal variabili per la fonte di origine e le condizioni fisiologiche di cellule che secernono exosomes. Queste variazioni possono fornire informazioni su come le cellule e sistemi di far fronte allo stress o perturbazioni fisiologiche. I nostri dati rappresentativi mostrano variations in miRNA presenti in exosomes purificate dal sangue del mouse, sangue umano e mezzi di coltura di cellule umane. Qui descriveremo la procedura per analizzare quantitativamente miRNA e RNA messaggero (mRNA) da exosomes secreti nel sangue e nei mezzi di coltura cellulare. Exosomes purificati saranno caratterizzati mediante analisi western blot per i marcatori exosomal e PCR per mRNA di interesse. Microscopia elettronica a trasmissione (TEM) e l&#39;etichettatura immunogold verranno utilizzati per convalidare la morfologia e l&#39;integrità exosomal. L&#39;RNA totale saranno purificati da questi exosomes per assicurare che siamo in grado di studiare sia l&#39;mRNA e miRNA dallo stesso campione. Dopo la convalida integrità dell&#39;RNA da Bioanalyzer, eseguiremo una produttività quantitativa real time PCR media (qPCR) per identificare il miRNA exosomal utilizzando Taqman Low Density Array (TLDA) carte e studi di espressione genica per le trascrizioni di interesse. Questi protocolli possono essere usati per quantificare variazioni miRNA exosomal in pazienti, modelli di roditori e terreni di coltura delle cellule prima e dopo l&#39;intervento farmacologico. Contenuto Exosomal variabili per la fonte di origine e le condizioni fisiologiche di cellule che secernono exosomes. Queste variazioni possono fornire informazioni su come le cellule e sistemi di far fronte allo stress o perturbazioni fisiologiche. I nostri dati rappresentativi mostrano variazioni di miRNA presenti in exosomes purificate dal sangue del mouse, sangue umano e mezzi di coltura di cellule umane

Dopo aver isolato exosomes da sangue o terreni di coltura cellulare, la purezza dei exosomes può essere testata mediante microscopia elettronica (EM) e western blot (Figure 1A e 1B). Abbiamo confermato la nostra preparazione exosome da varie fonti con EM e western blot utilizzando anticorpi multipli. Figura 1A mostra EM immagini confermando che exosomes sono intatte con un diametro di ~ 30 -100 nm e contengono CD81 mediante l&#39;etichettatura immunogold. Marcatori exo...

Watch this Scientific Journal Video about Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media at JoVE.com

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

La presenza di microRNA stabili (miRNA) in exosomes ha generato enorme interesse come un romanzo modalità di comunicazione intercellulare, per la loro potenziale utilità come biomarcatori e come via per l&#39;intervento terapeutico. Qui mostriamo purificazione exosome dal sangue e cultura mediatica seguita da PCR quantitativa per identificare miRNA trasportati.

Purificazione e microRNA profiling di Exosomes derivati ​​dal sangue e cultura dei media

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

purification-microrna-profiling-exosomes-derived-from-blood-culture

Research

JoVE Journal

Biology

31.7K Views.  Drexel University College of Medicine. La presenza di microRNA stabili (miRNA) in exosomes ha generato enorme interesse come un romanzo modalità di comunicazione intercellulare, per la loro potenziale utilità come biomarcatori e come via per l'intervento terapeutico. Qui mostriamo purificazione exosome dal sangue e cultura mediatica seguita da PCR quantitativa per identificare miRNA trasportati.

Video: Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

La colorazione delle proteine ​​in gel con Coomassie G-250, senza solventi organici acido acetico e

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Ricerca

Biologia

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Isolamento e la coltura di cellule endoteliali polmonari da topi neonati

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Characterization of RNA-Containing Exosomes

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

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

Isolamento e caratterizzazione di RNA contenenti esosomi

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

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e purificazione di chinesina dal Drosophila Embrioni

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Caratterizzazione fenotipica e funzionale delle cellule endoteliali Colony Forming derivato dal sangue del cordone ombelicale umano

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

Purificazione di Trascrizioni e metaboliti<em&gt; Drosophila</em&gt; Teste

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

MicroRNA Expression Profili di cellule iPS umane, dell&#39;epitelio pigmentato retinico derivate da iPS, e fetale dell&#39;epitelio pigmentato retinico

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Generazione e Cultura Sangue escrescenza endoteliali cellule da sangue periferico umano

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Purification of Platelets from Mouse Blood

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), and plasma proteins. We describe here purification of platelets from mouse blood using three-fold more iohexol gradient medium relative to blood sample volume and centrifugation in a swinging bucket rotor at 400 x g for 20 min at 20 &#176;C. The recovery/yield of the purified platelets were 18.2-38.5%, and the purified platelets were in a resting state, which did not contain any significant number of RBC and WBC. The purified platelets treated with thrombin showed up to 93% activation, indicating their viability. We confirmed that the purified platelets are sufficiently pure using flow cytometric and microscopic evaluation. These platelets can be used for gene expression, activation, granule release, aggregation, and adhesion assays. This method can be used for purification of platelets from the blood of other species as well.

Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy.

Purificazione delle piastrine dal sangue del topo

Platelets are purified from whole blood to study their functional properties, which should be free from red blood cells (RBC), white blood cells (WBC), ...

Purificazione e microRNA profiling di Exosomes derivati ​​dal sangue e cultura dei media

Purificazione e microRNA profiling di Exosomes derivati dal sangue e cultura dei media