Name: Isolatie, karakterisering en proteomische analyse van plasma-afgeleide extracellulaire blaasjes voor de ontdekking van cardiovasculaire biomarkers (Video) | JoVE Journal
Uploaded: 2025-01-31T12:30:00.000+00:00
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Description: Leer de isolatie en karakterisering van plasma-afgeleide extracellulaire blaasjes voor cardiovasculaire biomarkerontdekking. Deze methode combineert ultracentrifugatie met LC-MS/MS-proteoomanalyse voor klinische toepassingen.

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<meta charset="utf8"></head><body>Cardiovasculaire biomarkerstudies met behulp van plasma extracellulair blaasje-proteoom

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<table><tbody><tr><td>4x Laemmli sample bufffer</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>26.3 mL Polycarbonate Bottle with Cap Assembly for Ultracentrifugation</td><td>Beckman Coulter</td><td>355654</td><td></td></tr><tr><td>5810R Benchtop centrifuge</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>Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>43816-10ML</td><td></td></tr><tr><td>Dulbecco's Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Formic Acid 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>Mass Spectrometer Orbitrap Q Exactive HF</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>MOPS SDS running buffer</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;%, Bis-Tris protein gels</td><td>Invitrogen</td><td>NP0323</td><td></td></tr><tr><td>Optima XPN-80 floor-standing ultracentrifuge</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>Protease/Phosphatase Inhibitor Cocktail (100X)</td><td>Cell Signaling Technology</td><td>5872S</td><td></td></tr><tr><td>Protein marker</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 Hybrid Quadrupole-Orbitrap Mass Spectrometer</td><td>Thermo Fisher Scientific</td><td>IQLAAEGAAPFALGMBDK</td><td></td></tr><tr><td>Quick stain coomasssie</td><td>Serva</td><td>35081.01</td><td></td></tr><tr><td>ReproSil-Pur 120 C18-AQ, 1.9 &micro;m 1 g</td><td>Dr. Maisch</td><td>r119.aq.0001</td><td></td></tr><tr><td>SDS-PAGE commercial gel</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>Speed vac concentrator</td><td>Savant</td><td></td><td></td></tr><tr><td>Swiss-Prot (Uniprot) Homo sapiens (UP000005640, June 2020) protein database</td><td>UniProt</td><td>https://www.uniprot.org/proteomes/UP000005640</td><td></td></tr><tr><td>Triethylammonium bicarbonate buffer (TEAB)</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid (TFA) for HPLC >99%, 100 mL</td><td>Sigma-Aldrich</td><td>302031-100ML</td><td></td></tr><tr><td>Trypsin MS-Grade</td><td>Thermo Fisher</td><td>90058</td><td></td></tr><tr><td>Type 50.2 Ti Fixed-Angle Rotor</td><td>Beckman Coulter</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>

isolatie, karakterisering en proteomische analyse van plasma-afgeleide extracellulaire blaasjes voor de ontdekking van cardiovasculaire biomarkers

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

Isolatie, karakterisering en proteomische analyse van plasma-afgeleide extracellulaire blaasjes voor de ontdekking van cardiovasculaire biomarkers

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

Research

JoVE Journal

Medicine

Geneeskunde

Extracellulaire vesicules in bloed zijn voorgesteld als nieuwe biomarkers voor verschillende ziekten. Hier presenteren we een protocol voor de isolatie van grote plasmamembraan-afgeleide microvesicles uit perifere bloedmonsters en hun daaropvolgende analyse door conventionele flowcytometrie en Western Blotting.

Isolatie en karakterisering van microvesicles uit perifeer bloed

Hier presenteren we een protocol om te zuiveren van exosomes van plasma en serum met verminderde CO zuivering van niet-exosomal bloedeiwitten. Het geoptimaliseerde protocol bevat ultrafiltratie, protease behandeling en grootte uitsluiting chromatografie. Verbeterde zuivering van exosomes voordelen downstream analyses, met inbegrip van nauwkeuriger kwantificering van blaasjes en proteomic karakterisering.

Vertering van eiwitten, ultrafiltratie en Size Exclusion Chromatography voor het optimaliseren van het isolement van Exosomes uit menselijk bloed Plasma en Serum

Het protocol wordt een reproduceerbare methode ontworpen voor gebruik met cel cultuur supernatant voor het opsporen van oppervlakte epitopes op kleine extracellulaire blaasjes (EV) beschreven. Het maakt gebruik van specifieke EV immunoprecipitation met parels in combinatie met de antilichamen die surface antigeen CD9 herkent, CD63, en CD81. De methode is geoptimaliseerd voor downstream stroom cytometry analyse.

Cytometrische analyse van extracellulaire blaasjes van cel-geconditioneerd Media stromen

Het onderhavige rapport belicht chronologische eisen voor extracellulaire vesicle (EV) isolatie van microglia of bloedmacrofagen. Microglia-afgeleide EV's werden geëvalueerd als regelgevers van de neurite uitgroei, terwijl bloed macrofaag-afgeleide EV's werden bestudeerd in de controle van C6 glioom cel invasie in in vitro testen. Het doel is om deze EV-functies als immuunbemiddelaars in specifieke micro-omgevingen beter te begrijpen.

Karakterisering van immuuncel-afgeleide extracellulaire vesicles en het bestuderen van functionele impact op celmilieu

Het huidige protocol beschrijft een methode om extracellulaire blaasjes uit het perifere bloed en vaste weefsels te extraheren met daaropvolgende profilering van oppervlakteantigenen en eiwitladingen.

Isolatie en analyse van traceerbare en gefunctionaliseerde extracellulaire blaasjes uit het plasma en vaste weefsels

De nieuwste generatie EV-karakteriseringstools is in staat om één EV-analyse over meerdere parameters tegelijk uit te voeren. Nano-flow cytometrie meet alle biologische deeltjes groter dan 45 nm zonder labeling en identificeert specifieke kenmerken van subpopulaties door een verscheidenheid aan fluorescerende etiketteringstechnieken.

Single Extracellular Vesicle Transmembrane Protein Characterization door Nano-Flow Cytometry

Dit protocol biedt gedetailleerde beschrijvingen voor de efficiënte isolatie van extracellulaire blaasjes in de urine met behulp van gefunctionaliseerde magnetische kralen. Bovendien omvat het latere analyses, waaronder western blotting, proteomics en phosphoproteomics.

Chemische affiniteit-gebaseerde isolatie van extracellulaire blaasjes uit biovloeistoffen voor proteomics en fosfoproteomics-analyse

Er wordt een gedetailleerd protocol opgesteld voor het reinigen en hergebruiken van ultracentrifugebuisjes van polycarbonaat om extracellulaire blaasjesisolatie uit te voeren die geschikt is voor proteomics-experimenten.

Hergebruik van polycarbonaat ultracentrifugebuis in proteomische analyses van extracellulaire blaasjes

Hier demonstreren we de karakteriseringsmethode voor extracellulaire blaasjes (EV's) verzameld uit biologische vloeistoffen, zoals tranen en speeksel, van menselijke proefpersonen. De scanner die bij deze methode wordt gebruikt, is in staat om het fenotype, de grootte en het totale aantal deeltjes van EV's van 1 μL van het monster te detecteren.

Karakterisering van extracellulaire blaasjes uit biologische vloeistoffen

Deze video toont een effectieve techniek voor het differentiëren en het ontleden van de verschillende semi-transparante structuren van het menselijk glasachtig lichaam in post mortem ogen.

Dissectie van de Mens glasvocht Elementen voor proteoomanalyse

Leer de isolatie en karakterisering van plasma-afgeleide extracellulaire blaasjes voor cardiovasculaire biomarkerontdekking. Deze methode combineert ultracentrifugatie met LC-MS/MS-proteoomanalyse voor klinische toepassingen.

Isolatie, karakterisering en proteomische analyse van plasma-afgeleide extracellulaire blaasjes voor de ontdekking van cardiovasculaire biomarkers (Video) | JoVE Journal

Isolatie, karakterisering en proteomische analyse van plasma-afgeleide extracellulaire blaasjes voor de ontdekking van cardiovasculaire biomarkers

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