Name: Isolamento di piccole vescicole extracellulari da muscolo scheletrico murino e midollo osseo tramite cromatografia a esclusione di dimensioni (Video) | JoVE Journal
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Description: Scopri l'isolamento di piccole vescicole extracellulari da muscolo scheletrico e midollo osseo murino tramite cromatografia a esclusione di dimensioni. Questo metodo garantisce elevata purezza per analisi molecolari e funzionali.

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<ol>
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<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>Sarstedt</td><td>72.690.300</td><td>General microcentrifuge tube used throughout protocol</td></tr><tr><td>1.5 mL low-protein-binding tube</td><td>Sarstedt</td><td>72.706.600</td><td>Used for EV handling, SEC fraction collection, and storage</td></tr><tr><td>0.2 mL PCR tube</td><td>Fisher Scientific</td><td>14-222-262</td><td>Used for poked-tube bone marrow extraction setup</td></tr><tr><td>16 G needle</td><td>BD</td><td>305197</td><td>Used to puncture the 0.2 mL PCR tube</td></tr><tr><td>30 &micro;m cell strainer</td><td>Miltenyi Biotec&nbsp;</td><td>130-098-458</td><td>Used for skeletal muscle sample filtration</td></tr><tr><td>70% ethanol</td><td>Sigma-Aldrich</td><td>1.11727</td><td>Used to sterilize tweezers</td></tr><tr><td>Biosafety cabinet</td><td>NA</td><td>NA</td><td>Used for sterile tissue handling</td></tr><tr><td>Bone cutting instrument</td><td>Fisher Scientific</td><td>10-001-976</td><td>Used to separate femur and tibia and expose marrow</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>BioShop</td><td>ALB001</td><td>Used for Western blot blocking and antibody dilution</td></tr><tr><td>Calcium chloride dihydrate (CaCl2&middot;2H2O)</td><td>BioShop</td><td>CCL302</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Carbon-coated copper grid, 200-mesh</td><td>Sigma-Aldrich</td><td>930342</td><td>Used for TEM sample preparation</td></tr><tr><td>Centrifugal filter, 100 kDa MWCO</td><td>MilliporeSigma</td><td>UFC810096</td><td>Used for EV concentration</td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td>Used for skeletal muscle enzymatic digestion</td></tr><tr><td>Cryovial</td><td>Sarstedt</td><td>72.690.300</td><td>Used for tissue and bone storage</td></tr><tr><td>Deionized water (ultrapure)</td><td>NA</td><td>NA</td><td>Used for buffer preparation and TEM washing steps</td></tr><tr><td>DNase I</td><td>Roche</td><td>11284932001</td><td>Used for skeletal muscle enzymatic digestion</td></tr><tr><td>Dry ice</td><td>NA</td><td>NA</td><td>Used for sample cooling and transport</td></tr><tr><td>Enhanced chemiluminescence substrate</td><td>Biorad</td><td>1705061</td><td>Used for Western blot detection</td></tr><tr><td>Filter unit, 0.22 &micro;m</td><td>Sarstedt</td><td>83.3941.501</td><td>Used to filter PBS where applicable</td></tr><tr><td>Forceps / tweezers</td><td>MilliporeSigma</td><td>F3892</td><td>Used for tissue handling</td></tr><tr><td>Glutaraldehyde</td><td>NA</td><td>NA</td><td>Used for TEM fixation</td></tr><tr><td>Glycine</td><td>NA</td><td>NA</td><td>Used in TEM grid washing steps</td></tr><tr><td>Hank&rsquo;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14175-095</td><td>Used for DNase I stock preparation</td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.500</td><td>Used for HEPES-NaOH buffer preparation</td></tr><tr><td>Horseradish peroxidase-conjugated streptavidin</td><td>Thermo Fisher</td><td>S911</td><td>Used for Western blot detection</td></tr><tr><td>Imaging system for chemiluminescence</td><td>Biorad</td><td>12003153</td><td>Used to image Western blot membranes</td></tr><tr><td>KCl</td><td>BioShop</td><td>POC308</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>BioShop</td><td>MAG510.500</td><td>Used for HEPES buffer preparation</td></tr><tr><td>2-Methylbutane</td><td>Thermo Fisher</td><td>78-78-4</td><td>Used for skeletal muscle snap-freezing</td></tr><tr><td>Milligram balance</td><td>MilliporeSigma</td><td>Z741359</td><td>Used for tissue and marrow weighing</td></tr><tr><td>NaCl</td><td>BioShop</td><td>SOD004.1</td><td>Used for HEPES buffer preparation</td></tr><tr><td>Nano-particle tracking analysis instrument</td><td>Malvern Panalytical</td><td>NanoSight NS300</td><td>NanoSight NS300 used for particle sizing and counting</td></tr><tr><td>NaOH</td><td>BioShop</td><td>SHY500.500</td><td>Used for pH adjustment</td></tr><tr><td>Nitrocellulose membrane</td><td>BioRad</td><td>1704271</td><td>Used for Western blot transfer</td></tr><tr><td>Nutating mixer</td><td>Labnet</td><td>&nbsp;S0500</td><td>Used during enzymatic digestion</td></tr><tr><td>P1000 pipette</td><td>MilliporeSigma</td><td>FA10006M-1EA</td><td>Used for tissue transfer</td></tr><tr><td>Pipette tip, 1 mL</td><td>Sarstedt</td><td>70.3050.275</td><td>Tip is trimmed to widen opening for tissue fragment transfer</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Wisent</td><td>INC 811.012-FL</td><td>Used throughout protocol; also used for SEC equilibration and elution</td></tr><tr><td>Phosphatase inhibitor</td><td>MilliporeSigma</td><td>4906845001</td><td>Used for Western blot lysate preparation</td></tr><tr><td>Petri dish</td><td>MilliporeSigma</td><td>CLS351146</td><td>Used for bone marrow preparation</td></tr><tr><td>Polyacrylamide gel, 4&ndash;20% gradient stain-free</td><td>BioRad</td><td>4561094</td><td>Used for Western blot separation</td></tr><tr><td>Protease inhibitor</td><td>MilliporeSigma</td><td>11697498001</td><td>Used for Western blot lysate preparation</td></tr><tr><td>RPMI-1640 medium</td><td>Sigma-Aldrich</td><td>R7388-500ML</td><td>Unsupplemented; no FBS or antibiotics</td></tr><tr><td>RIPA buffer, 10&times;</td><td>Merck</td><td>20-188</td><td>Used for protein extraction for Western blotting</td></tr><tr><td>Scalpel, sterile disposable</td><td>Ultident Paragon</td><td>02-90000-10</td><td>Used to mince skeletal muscle tissue</td></tr><tr><td>SEC column, original 70 nm</td><td>IZON</td><td>ICO70P</td><td>Used for skeletal muscle SEC</td></tr><tr><td>SEC column, single 70 nm</td><td>IZON</td><td>ICS70P</td><td>Used for bone marrow SEC</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td>Added to PBS for washing the column</td></tr><tr><td>Sodium cacodylate buffer</td><td>NA</td><td>NA</td><td>Used for TEM fixation buffer</td></tr><tr><td>Luer Slip Single Use Syringe without Needle&nbsp;</td><td>Medline</td><td>SYR101020</td><td>Used during NTA acquisition if applicable</td></tr><tr><td>Transmission electron microscope</td><td>FEI</td><td>NA</td><td>Tecnai G2 Spirit Twin 120 kV Cryo-TEM used for TEM imaging</td></tr><tr><td>Tween-20</td><td>BioShop</td><td>TWN508.1</td><td>Used in PBS-T for Western blotting</td></tr><tr><td>Uranyl acetate</td><td>NA</td><td>NA</td><td>Used for TEM negative staining</td></tr><tr><td>Whatman filter paper</td><td>MilliporeSigma</td><td>WHA1003055</td><td>Used to wick excess TEM stain</td></tr></tbody></table>

isolamento di piccole vescicole extracellulari da muscolo scheletrico murino e midollo osseo tramite cromatografia a esclusione di dimensioni

Isolation of Small Extracellular Vesicles from Murine Skeletal Muscle and Bone Marrow by Size-Exclusion Chromatography

Isolamento di piccole vescicole extracellulari da muscolo scheletrico murino e midollo osseo tramite cromatografia a esclusione di dimensioni

Small extracellular vesicles (sEVs) are nanosized, lipid-bound particles that mediate intercellular communication through the transfer of proteins, ...

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Fonte: Watson, D. C., et al. Isolamento e purificazione scalabili di vescicole extracellulari da Escherichia coli e altri batteri. J. Vis. Exp. (2021)Questo video mostra l'uso della cromatografia ad esclusione dimensionale con perle di resina porosa per isolare le vescicole extracellulari batteriche (EV) da un filtrato di coltura batterica. Le vescicole extracellulari più grandi eluiscono presto bypassando i pori della resina, mentre le proteine più piccole entrano nei pori ed eluiscono più...

Isolamento di vescicole extracellulari batteriche mediante cromatografia ad esclusione dimensionale 

In questo video, dimostriamo una tecnica di cromatografia ad esclusione dimensionale per separare una popolazione eterogenea di vescicole della membrana esterna o OMV derivate da batteri. Queste vescicole sono strutture sferiche di dimensioni nanometriche e vengono separate in sottopopolazioni grandi e piccole in base alle loro dimensioni attraverso la cromatografia ad esclusione dimensionale.

Separazione basata sulla cromatografia ad esclusione dimensionale di vescicole extracellulari batteriche: una tecnica per separare la popolazione eterogenea di vescicole batteriche Gram-negative della membrana esterna in base alle loro dimensioni

Questo video mostra la tecnica di cromatografia ad esclusione dimensionale (SEC) per purificare le vescicole extracellulari batteriche (EV) dal filtrato di coltura di Mycobacterium tuberculosis (Mtb). La tecnica utilizza una colonna porosa impaccata di resina per ottenere frazioni purificate di EV. Le particelle porose consentono selettivamente alle proteine più piccole di entrare nei pori, con conseguente eluizione più lenta. Allo stesso tempo, le vescicole extracellulari sono escluse...

Una tecnica di cromatografia ad esclusione dimensionale per purificare le vescicole extracellulari micobatteriche

Questo protocollo illustra 1) l'isolamento e la coltura di fibroblasti primari dal muscolo gastrocnemio di topo adulto e 2) la purificazione e la caratterizzazione degli esosomi utilizzando un metodo di ultracentrifugazione differenziale combinato con gradienti di densità del saccarosio seguito da analisi western blot.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

Le vescicole batteriche svolgono un ruolo importante nella patogenesi e hanno promettenti applicazioni biotecnologiche. L'eterogeneità delle vescicole complica l'analisi e l'uso; pertanto, è necessario un metodo semplice e riproducibile per separare le varie dimensioni delle vescicole. Qui, dimostriamo l'uso della cromatografia di esclusione dimensionale per separare vescicole eterogenee prodotte da Aggregatibacter actinomycetemcomitans.

Cromatografia di esclusione dimensionale per analizzare l'eterogeneità delle vescicole della membrana esterna batterica

Il protocollo qui dimostra che le vescicole extracellulari possono essere adeguatamente separate dai terreni di coltura cellulare condizionati utilizzando la cromatografia ad esclusione dimensionale.

Cromatografia ad esclusione dimensionale per separare le vescicole extracellulari dai terreni di coltura cellulare condizionati

Questo protocollo dimostra come isolare le piccole vescicole extracellulari derivate dal cordone ombelicale umano (hUC-MSC-sEV) con una semplice impostazione da banco su scala di laboratorio. La distribuzione dimensionale, la concentrazione proteica, i marcatori sEVs e la morfologia degli hUC-MSC-sEV isolati sono caratterizzati rispettivamente dall'analisi del tracciamento delle nanoparticelle, dal saggio della proteina BCA, dal western blot e dal microscopio elettronico a trasmissione.

Un semplice metodo di filtrazione da banco per isolare piccole vescicole extracellulari da cellule staminali mesenchimali umane

Questo protocollo fornisce un metodo di isolamento rapido e specifico per le dimensioni delle piccole vescicole extracellulari ottimizzando le dimensioni dell'ugello di nebulizzazione dell'aria, la pressione del fluido della guaina, la pressione del flusso del campione, la tensione, il guadagno e i parametri di soglia di attivazione.

Ottimizzazione dei parametri di selezione citometrica a flusso per l'isolamento e la purificazione ad alta produttività di piccole vescicole extracellulari

Le vescicole extracellulari sono estremamente promettenti per le applicazioni biomediche, ma gli attuali metodi di isolamento richiedono molto tempo e non sono pratici per l'uso clinico. In questo studio, presentiamo un dispositivo microfluidico che consente l'isolamento diretto di vescicole extracellulari da grandi volumi di biofluidi in modo continuo con passaggi minimi.

Isolamento in un'unica fase di vescicole extracellulari da campioni di grande volume con un dispositivo microfluidico A4F biforcato

Questo protocollo mira a isolare e purificare le vescicole extracellulari interstiziali del muscolo scheletrico (SkM-EVs) dai tessuti muscolari dei roditori attraverso il distacco meccanico, la dissociazione enzimatica, la filtrazione e l'ultracentrifugazione differenziale. Dimostra coerenza e affidabilità. Le SkM-EV isolate forniscono informazioni sull'omeostasi muscolare e sulle malattie, con potenziali applicazioni come biomarcatori diagnostici o veicoli terapeutici.

Isolamento enzimatico delle vescicole extracellulari interstiziali del muscolo scheletrico

Scopri l'isolamento di piccole vescicole extracellulari da muscolo scheletrico e midollo osseo murino tramite cromatografia a esclusione di dimensioni. Questo metodo garantisce elevata purezza per analisi molecolari e funzionali.

Isolamento di piccole vescicole extracellulari da muscolo scheletrico murino e midollo osseo tramite cromatografia a esclusione di dimensioni (Video) | JoVE Journal

Isolamento di piccole vescicole extracellulari da muscolo scheletrico murino e midollo osseo tramite cromatografia a esclusione di dimensioni

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