Name: Isolamento, caratterizzazione e analisi proteomica di vescicole extracellulari derivate dal plasma per la scoperta di biomarcatori cardiovascolari (Video) | JoVE Journal
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Description: Scopri l'isolamento e la caratterizzazione di vescicole extracellulari dal plasma umano per la ricerca di biomarcatori cardiovascolari. Questo metodo supporta l'integrazione dell'analisi proteomica LC-MS/MS nella pratica clinica di routine.

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<meta charset="utf8"></head><body>Studi sui biomarcatori cardiovascolari utilizzando il proteoma delle vescicole extracellulari plasmatiche

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<li>Nieuwland, R., Siljander, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+beginner%27s+guide+to+study+extracellular+vesicles+in+human+blood+plasma+and+serum%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">A beginner's guide to study extracellular vesicles in human blood plasma and serum.</a> J Extracell Vesicles. 13 (1), e12400(2024).</li>
<li>Lacroix, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+pre-analytical+parameters+on+the+measurement+of+circulating+microparticles%3A+Towards+standardization+of+protocol%5BTitle%5D)+AND+%22J+Thromb+Haemost%22%5BJournal%5D)">Impact of pre-analytical parameters on the measurement of circulating microparticles: Towards standardization of protocol.</a> J Thromb Haemost. 10 (3), 437-446 (2012).</li>
<li>Coumans, F. aW., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodological+guidelines+to+study+extracellular+vesicles%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Methodological guidelines to study extracellular vesicles.</a> Circ Res. 120 (10), 1632-1648 (2017).</li>
<li>Clayton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Considerations+towards+a+roadmap+for+collection%2C+handling+and+storage+of+blood+extracellular+vesicles%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles.</a> J Extracell Vesicles. 8 (1), 1647027(2019).</li>
<li>Lucien, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Miblood-EV%3A+Minimal+information+to+enhance+the+quality+and+reproducibility+of+blood+extracellular+vesicle+research%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Miblood-EV: Minimal information to enhance the quality and reproducibility of blood extracellular vesicle research.</a> J Extracell Vesicles. 12 (12), e12385(2023).</li>
<li>Zhang, X., Takeuchi, T., Takeda, A., Mochizuki, H., Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+serum+and+plasma+as+a+source+of+blood+extracellular+vesicles%3A+Increased+levels+of+platelet-derived+particles+in+serum+extracellular+vesicle+fractions+alter+content+profiles+from+plasma+extracellular+vesicle+fractions%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comparison of serum and plasma as a source of blood extracellular vesicles: Increased levels of platelet-derived particles in serum extracellular vesicle fractions alter content profiles from plasma extracellular vesicle fractions.</a> PLoS One. 17 (6), e0270634(2022).</li>
<li>Gelibter, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+impact+of+storage+on+extracellular+vesicles%3A+A+systematic+study%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">The impact of storage on extracellular vesicles: A systematic study.</a> J Extracell Vesicles. 11 (2), e12162(2022).</li>
<li>Sluijter, J. P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles+in+diagnostics+and+therapy+of+the+ischaemic+heart%3A+Position+paper+from+the+working+group+on+cellular+biology+of+the+heart+of+the+European+Society+of+Cardiology%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Extracellular vesicles in diagnostics and therapy of the ischaemic heart: Position paper from the working group on cellular biology of the heart of the European Society of Cardiology.</a> Cardiovasc Res. 114 (1), 19-34 (2018).</li>
<li>Davidson, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+identification+and+characterization+of+extracellular+vesicles+in+cardiovascular+studies%3A+From+exosomes+to+microvesicles%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: From exosomes to microvesicles.</a> Cardiovasc Res. 119 (1), 45-63 (2023).</li>
<li>Karimi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detailed+analysis+of+the+plasma+extracellular+vesicle+proteome+after+separation+from+lipoproteins%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">Detailed analysis of the plasma extracellular vesicle proteome after separation from lipoproteins.</a> Cell Mol Life Sci. 75 (15), 2873-2886 (2018).</li>
<li>Brennan, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comparison+of+methods+for+the+isolation+and+separation+of+extracellular+vesicles+from+protein+and+lipid+particles+in+human+serum%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum.</a> Sci Rep. 10 (1), 1039(2020).</li>
<li>Palviainen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+vesicles+from+human+plasma+and+serum+are+carriers+of+extravesicular+cargo-implications+for+biomarker+discovery%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-implications for biomarker discovery.</a> PLoS One. 15 (8), e0236439(2020).</li>
<li>Lotvall, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minimal+experimental+requirements+for+definition+of+extracellular+vesicles+and+their+functions%3A+A+position+statement+from+the+international+society+for+extracellular+vesicles%5BTitle%5D)+AND+%22J+Extracell+Vesicles%22%5BJournal%5D)">Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the international society for extracellular vesicles.</a> J Extracell Vesicles. 3, 26913(2014).</li>
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<li>Kreimer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass-spectrometry-based+molecular+characterization+of+extracellular+vesicles%3A+Lipidomics+and+proteomics%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Mass-spectrometry-based molecular characterization of extracellular vesicles: Lipidomics and proteomics.</a> J Proteome Res. 14 (6), 2367-2384 (2015).</li>
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</ol>

<table><tbody><tr><td>4x Bufffer per campioni Laemmli</td><td>Bio-rad</td><td>1610747</td><td></td></tr><tr><td>10x RIPA-Buffer</td><td>Abcam</td><td>ab156034</td><td></td></tr><tr><td>Flacone in policarbonato da 26,3 mL con tappo per ultracentrifugazione</td><td>Beckman Coulter</td><td>355654</td><td></td></tr><tr><td>5810R Centrifuga da banco</td><td>Eppendorf</td><td>5811000015</td><td></td></tr><tr><td>Acetonitrile (ACN)</td><td>Biosolve</td><td>0001204101BS</td><td></td></tr><tr><td>Ditiotreitolo (DTT)</td><td>Sigma-Aldrich</td><td>43816-10ML</td><td></td></tr><tr><td>Soluzione salina tamponata con fosfato di Dulbecco (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Acido formico 99% ULC/MS 100</td><td>Biosolve</td><td>0006914143BS</td><td></td></tr><tr><td>Histopaque - 1077</td><td>Sigma-Aldrich</td><td>10771</td><td></td></tr><tr><td>Iodoacetamide (IAA)</td><td>Sigma-Aldrich</td><td>I6125-5G</td><td></td></tr><tr><td>Spettrometro di massa Orbitrap Q Exactive HF</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>MOPS SDS tampone di corsa</td><td>Thermo Fisher</td><td>B0001</td><td></td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical&nbsp;</td><td>NS300</td><td></td></tr><tr><td>Nanosight NTA 3.2 Software</td><td>Malvern Panalytical&nbsp;</td><td></td><td></td></tr><tr><td>NuPAGE 4&nbsp; bis 12&nbsp;%, gel proteici Bis-Tris</td><td>Invitrogen</td><td>NP0323</td><td></td></tr><tr><td>Optima XPN-80 ultracentrifuga da pavimento</td><td>Beckman Coulter</td><td>521-4180</td><td></td></tr><tr><td>PageRuler Plus Prestained Protein Ladder</td><td>Thermo Fisher Scientific</td><td>26619</td><td></td></tr><tr><td>Cocktail di inibitori della proteasi/fosfatasi (100X)</td><td>Cell Signaling Technology</td><td>5872S</td><td></td></tr><tr><td>Marcatore proteico</td><td>Thermo Fisher</td><td>26616</td><td></td></tr><tr><td>Proteome Discoverer 2.5</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>Q Exactive Plus Spettrometro di massa ibrido quadrupolo-orbitrap</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Colorazione rapida Serva</td><td></td><td>35081.01</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 1.9 &; m 1 g</td><td>Dr. Maisch</td><td>r119.aq.0001</td><td></td></tr><tr><td>SDS-PAGE gel commerciale</td><td>Thermo Fisher</td><td>NW00100BOX</td><td></td></tr><tr><td>S-Monovette&nbsp; Citrat 9NC 0,106 mol/l 3,2%</td><td>Sarstedt</td><td>02.1067.001</td><td></td></tr><tr><td>Concentratore Speed vac</td><td>Savant</td><td></td><td></td></tr><tr><td>Swiss-Prot (Uniprot) Homo sapiens (UP000005640, giugno 2020) database di proteine</td><td>UniProt</td><td>https://www.uniprot.org/proteomes/UP000005640</td><td></td></tr><tr><td>Tampone bicarbonato di trietilammonio (TEAB)</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Acido trifluoroacetico (TFA) per HPLC >99%, 100 mL</td><td>Sigma-Aldrich</td><td>302031-100ML</td><td></td></tr><tr><td>Tripsina MS-Grade</td><td>Thermo Fisher</td><td>90058</td><td></td></tr><tr><td>Tipo 50.2 Ti Rotore ad angolo fisso</td><td>Coltro Beckman</td><td>337901</td><td></td></tr><tr><td>UHPLC Dionex Ultimate 3000</td><td>Thermo Fisher Scientific</td><td>ULTIM3000RSLCNANO</td><td></td></tr><tr><td>Water</td><td>Biosolve</td><td>0023214102BS</td><td></td></tr></tbody></table>

isolamento, caratterizzazione e analisi proteomica di vescicole extracellulari derivate dal plasma per la scoperta di biomarcatori cardiovascolari

Isolation, Characterization, and Proteomic Analysis of Plasma-Derived Extracellular Vesicles for Cardiovascular Biomarker Discovery

Le EV sono state isolate da campioni di plasma (n = 3) di pazienti senza malattia cardiovascolare conclamata, nonché da pazienti con infarto del miocardio senza sopraslivellamento del tratto ST (NSTEMI; n = 2), utilizzando il protocollo di ultracentrifugazione differenziale stabilito. Un'adeguata separazione delle EV plasmatiche è stata confermata dall'immunoblotting delle proteine arricchite di EV TSG-101, annessina 5 (Anx5) e CD9 negli isolati di EV rispetto al plasma depleto di EV degli stessi pazienti (<strong class=...

Isolamento, caratterizzazione e analisi proteomica di vescicole extracellulari derivate dal plasma per la scoperta di biomarcatori cardiovascolari

Extracellular vesicles (EV) are cell-derived, lipid bilayer-enclosed, non-replicable nanoparticles. EV currently gain attention in cardiovascular research ...

isolation-characterization-proteomic-analysis-plasma-derived

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JoVE Journal

Medicine

Medicina

Le vescicole extracellulari presenti nel sangue sono state suggerite come nuovi biomarcatori per varie malattie. Qui, presentiamo un protocollo per l'isolamento di microvescicole derivate da grandi membrane plasmatiche da campioni di sangue periferico e la loro successiva analisi mediante citometria a flusso convenzionale e Western Blotting.

Isolamento e caratterizzazione di microvescicole da sangue periferico

Qui, presentiamo un protocollo per purificare gli esosomi da siero con ridotta co-purificazione non-exosomal di proteine del sangue e del plasma. Il protocollo ottimizzato include ultrafiltrazione, trattamento con proteasi e cromatografia di esclusione di formato. Una maggiore purificazione di esosomi analisi a valle di vantaggi, tra cui più accurata quantificazione delle vescicole e caratterizzazione proteomica.

Digestione delle proteine, ultrafiltrazione e cromatografia di esclusione di formato per ottimizzare l'isolamento degli esosomi da siero e Plasma sanguigno umano

Il protocollo descrive un metodo riproducibile progettato per l'utilizzo con i surnatanti di coltura cellulare per rilevare superfici epitopi sulle piccole vescicole extracellulari (EV). Utilizza specifici EV immunoprecipitazione usando i branelli accoppiati con gli anticorpi che riconoscono l'antigene di superficie CD9, CD63 e CD81. Il metodo è ottimizzato per l'analisi di citometria a flusso a valle.

Analisi citofluorimetrica delle vescicole extracellulari dal supporto di cella-condizionato

La presente relazione evidenzia i requisiti cronologici per l'isolamento delle vesciche extracellulari (EV) da microglia o macrofagi di sangue. Gli EV derivati dalla microglia sono stati valutati come regolatori dell'escrescenza di neurite, mentre gli EV derivati dai macrofaci di sangue sono stati studiati nel controllo dell'invasione delle cellule glioma C6 nei saggi in vitro. L'obiettivo è comprendere meglio queste funzioni EV come mediatori immunitari in microambienti specifici.

Caratterizzazione delle vescicoli extracellulari derivati dalle cellule immunitarie e studio dell'impatto funzionale sull'ambiente cellulare

Il presente protocollo descrive un metodo per estrarre vescicole extracellulari dal sangue periferico e dai tessuti solidi con successiva profilazione degli antigeni di superficie e dei carichi proteici.

Isolamento e analisi di vescicole extracellulari tracciabili e funzionalizzate dal plasma e dai tessuti solidi

L'ultima generazione di strumenti di caratterizzazione dei veicoli elettrici è in grado di eseguire l'analisi di singoli veicoli elettrici su più parametri contemporaneamente. La citometria a nano-flusso misura tutte le particelle biologiche più grandi di 45 nm senza etichettatura e identifica le caratteristiche specifiche delle sottopopolazioni mediante una varietà di tecniche di etichettatura fluorescente.

Caratterizzazione di proteine transmembrana di vescicole extracellulari singole mediante citometria a nano-flusso

Il presente protocollo fornisce descrizioni dettagliate per l'isolamento efficiente delle vescicole extracellulari urinarie utilizzando microsfere magnetiche funzionalizzate. Inoltre, comprende analisi successive, tra cui western blotting, proteomica e fosfoproteomica.

Isolamento chimico basato sull'affinità di vescicole extracellulari da biofluidi per analisi proteomiche e fosfoproteomiche

Viene fornito un protocollo dettagliato per la pulizia e il riutilizzo delle provette per ultracentrifuga in policarbonato per eseguire l'isolamento delle vescicole extracellulari adatto per esperimenti di proteomica.

Riutilizzo della provetta per ultracentrifuga in policarbonato nelle analisi proteomiche delle vescicole extracellulari

Qui, dimostriamo il metodo di caratterizzazione delle vescicole extracellulari (EV) raccolte da fluidi biologici, come lacrime e saliva, di soggetti umani. Lo scanner utilizzato in questo metodo è in grado di rilevare il fenotipo, le dimensioni e il conteggio totale delle particelle delle vescicole extracellulari a partire da 1 μL del campione.

Caratterizzazione delle vescicole extracellulari da fluidi biologici

Questo video mostra una tecnica efficace per differenziare e sezionare le varie semitrasparente strutture del corpo vitreo in post mortem occhi.

Dissezione di Human Elements corpo vitreo per l&#39;analisi proteomica

Scopri l'isolamento e la caratterizzazione di vescicole extracellulari dal plasma umano per la ricerca di biomarcatori cardiovascolari. Questo metodo supporta l'integrazione dell'analisi proteomica LC-MS/MS nella pratica clinica di routine.

Isolamento, caratterizzazione e analisi proteomica di vescicole extracellulari derivate dal plasma per la scoperta di biomarcatori cardiovascolari (Video) | JoVE Journal

Isolamento, caratterizzazione e analisi proteomica di vescicole extracellulari derivate dal plasma per la scoperta di biomarcatori cardiovascolari

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