Name: Karakterisering van door adipocyten afgeleide extracellulaire vesikelsecretie met behulp van een CD63-GFP Reporter Mouse Model in vivo en in vitro (Video) | JoVE Journal
Uploaded: 2025-12-05T13:20:00.000+00:00
Duration: 10 min 40 s
Description: Leer hoe adipocyt-afgeleide extracellulaire vesikels worden gekarakteriseerd met behulp van een CD63-GFP reporter muismodel. Deze methode visualiseert en kwantificeert EV-secretie in vivo en in vitro.

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</ol>

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Fisher</td><td>AM9260G</td><td>500x verdunning</td></tr><tr><td>10x PBS</td><td>Fisher Scientific</td><td>70-013-032</td><td>1000x verdunning met water</td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td>1000x verdunning</td></tr><tr><td>Adipose Tissue Progenitor Isolation Kit, mouse</td><td>Miltenyi biotec</td><td>30-106-639</td><td>Klaar voor gebruik</td></tr><tr><td>Amicon Ultra Centrifugal Filter, 100 kDa MWCO</td><td>Sigma</td><td>UFC9100</td><td>15 mL monstervolume</td></tr><tr><td>Amicon Ultra Centrifugal Filter, 100 kDa MWCO</td><td>Sigma</td><td>UFC9100</td><td>4 mL monstervolume</td></tr><tr><td>BSA</td><td>Sigma</td><td>A6003</td><td>Klaar voor gebruik</td></tr><tr><td>Calnexin antibody</td><td>calnexin</td><td>ab22595</td><td>1000x verdunning</td></tr><tr><td>CD63 antibody</td><td>abcam</td><td>ab217345</td><td>500x verdunning</td></tr><tr><td>CD81</td><td>cell signaling Technology</td><td>10037s</td><td>500x verdunning</td></tr><tr><td>Corning&nbsp;BioCoat&nbsp;Collagen I Culture Slide</td><td>Sigma</td><td>CLS354630</td><td>Klaar voor gebruik</td></tr><tr><td>Corning&nbsp;cell strainer</td><td>Sigma</td><td>CLS431750</td><td>40 μm poriegrootte</td></tr><tr><td>Corning&nbsp;cell strainer</td><td>Sigma</td><td>CLS431752</td><td>100 μm poriegrootte</td></tr><tr><td>COX IV antibody</td><td>Cell Signaling Technology</td><td>4844</td><td>1000x verdunning</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td>Klaar voor gebruik</td></tr><tr><td>DMEM/F-12, GlutaMAX supplement</td><td>Thermofisher</td><td>10565018</td><td>Klaar voor gebruik</td></tr><tr><td>Dnase 1</td><td>Sigma</td><td>D4513</td><td>Stock 5 KU/mL bij -20 °C</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>100x verdunning</td></tr><tr><td>Halt&trade; Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermofisher</td><td>78444</td><td>Klaar voor gebruik</td></tr><tr><td>HSP70 antibody</td><td>abcam</td><td>ab181606</td><td>1000x verdunning</td></tr><tr><td>Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species)</td><td>Fisher Scientific</td><td>50-112-9743</td><td>10x verdunning in water</td></tr><tr><td>Izon Science Usa Ltd QEVORIGINAL 70NMGEN2 5/PK SDP</td><td>Fisher Scientific</td><td>NC2042225</td><td>Klaar voor gebruik</td></tr><tr><td>LEICA STELLARIS 8 confocal microscope</td><td>Leica Microscopes</td><td>N/A</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Sigma</td><td>LIBTM-RO</td><td>Stock 5 mg/mL bij -20 °C</td></tr><tr><td>Millex MCE syringe filter</td><td>Sigma</td><td>SLAA025</td><td>0.8 μm poriegrootte</td></tr><tr><td>NanoSight Pro</td><td>Malvern Panalytical</td><td>N/A</td><td></td></tr><tr><td>Novex Tris-Glycine Mini Protein Gels, 4&ndash;12%,</td><td>Thermofisher</td><td>XP04120BOX</td><td>Klaar voor gebruik</td></tr><tr><td>Novex Tris-Glycine SDS Sample Buffer (2x)</td><td>Thermofisher</td><td>LC2676</td><td>Klaar voor gebruik</td></tr><tr><td>Pacific Blue anti-mouse Ly-6A/E (Sca-1) Antibody</td><td>BioLegend</td><td>108119</td><td>25x verdunning</td></tr><tr><td>Paraformaldehyde solution (16%)</td><td>Fisher Scientific</td><td>50-980-487</td><td>Verdun tot 4% met PBS</td></tr><tr><td>pluriStrainer S / 30 &micro;m (Cell Strainer)</td><td>Fisher Scientific</td><td>NC0922459</td><td>30 μm poriegrootte</td></tr><tr><td>Ponceau S solution</td><td>Sigma</td><td>P7170-1L</td><td>Klaar voor gebruik</td></tr><tr><td>Primocin</td><td>Invivogen</td><td>NC9141851</td><td>500x verdunning</td></tr><tr><td>ProLong Gold Antifade Reagent with DAPI</td><td>Cell Signaling Technology</td><td>8961S</td><td>Klaar voor gebruik</td></tr><tr><td>Purified anti-mouse CD16/32 Antibody</td><td>BioLegend</td><td>101301</td><td>100x verdunning</td></tr><tr><td>RIPA buffer&nbsp;</td><td>Fisher Scientific</td><td>PI89900</td><td>10x verdunning in water</td></tr><tr><td>Spectral flow cytometer (Aurora 3)</td><td>Cytek Biosciences</td><td>N/A</td><td></td></tr><tr><td>Syntaxin 4 antibody</td><td>Synaptic Systems</td><td>110042</td><td>5000x verdunning</td></tr><tr><td>Tecnai 20 (Transmission Electron Microscope)</td><td>FEI</td><td>N/A</td><td></td></tr><tr><td>Tergeo-EM</td><td>PIE Scientific LLC</td><td>N/A</td><td></td></tr><tr><td>Vinculin antibody</td><td>abcam</td><td>ab18508</td><td>5000x verdunning</td></tr></tbody></table>

karakterisering van door adipocyten afgeleide extracellulaire vesikelsecretie met behulp van een cd63-gfp reporter mouse model in vivo en in vitro

Characterization of Adipocyte-Derived Extracellular Vesicle Secretion Using a CD63-GFP Reporter Mouse Model In Vivo and In Vitro

Met dit protocol van AdEV-detectie via APC-opname met confocale microscopie isoleerden we APC's uit het inguinale vetweefsel van AdipCD63-GFP en Stopfl/fl/CD63-GFP-muizen en kleurden ze met DAPI voor visualisatie van de kernen. Bij onderzoek met confocale microscopie toonden APC's van Stopfl/fl/CD63-GFP-muizen geen detecteerbare GFP puncta (Figuur 1A). Daarentegen vertoonden APC's geïsoleerd van AdipCD63-GFP-muizen talrijke onderscheidende GFP-puncta door het hele cytop...

Karakterisering van door adipocyten afgeleide extracellulaire vesikelsecretie met behulp van een CD63-GFP Reporter Mouse Model in vivo en in vitro

Adipocytes are increasingly recognized as active endocrine cells that secrete substantial quantities of EVs enriched in proteins, lipids, and nucleic ...

characterization-adipocyte-derived-extracellular-vesicle-secretion

Research

JoVE Journal

Biology

Biologie

In this video, we demonstrate an assay to visualize and quantify the cellular uptake of extracellular vesicles or EVs. This assay utilizes 3D fluorescent imaging using a confocal microscope and allows for distinguishing internalized EVs from ones adhered to the cell surface.

Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

De endotheliale glycocalyx / endotheliale oppervlaktelaag is ideaal bestudeerd met behulp van intravitale microscopie. Intravitale microscopie is technisch uitdagend in een bewegend orgaan zoals de longen. We zien hoe gelijktijdige helderveld en fluorescentiemicroscopie kan worden gebruikt om endotheeloppervlak laagdikte schatten in een vrij...

In vivo meting van de muis Pulmonary Endothelial oppervlaktelaag

Dit protocol beschrijft methoden voor het zuiveren, kwantificeren en karakteriseren van extracellulaire vesicles (EVs) / exosomen van niet-aanhechtende / mesenchymale mammary epitheelcellen en voor het gebruik ervan om het vermogen van de borstkanker te overdragen naar luminale mammary epitheelcellen. EV's / exosomen afgeleid van stamachtige mammale epitheelcellen kunnen deze celeigenschap overbrengen aan cellen die de EV's / exosomen innemen.

Overdracht van Mammary Gland-vormende Vermogen Tussen Mammary Basal Epithelial Cellen en Mammary Luminale Cellen via Extracellulaire Vesicles / Exosomen

In dit werk beschrijven we protocollen voor het onderzoek naar de rol van extracellulaire blaasjes (EVs) uitgebracht door Plasmodium falciparum geïnfecteerd erytrocyten. In het bijzonder richten we ons op de interacties van EVW met endotheliale cellen.

Beoordeling van extracellulaire vesikel functie tijdens Malaria infectie

In dit protocol beschrijven we de complete workflow voor snelle isolatie van extracellulaire blaasjes uit menselijk bloed en karakterisering van specifieke markers door fluorescentie-gebaseerde analyse van het nanoparticle-tracking. De gepresenteerde resultaten geven een hoge graad van reproduceerbaarheid en kunnen worden aangepast aan de cel cultuur supernatant.

Snelle fluorescentie gebaseerde karakterisering van één extracellulaire blaasjes in menselijk bloed met Nanoparticle-tracking analyse

We beschrijven een 3D menselijke extracellulaire matrix-adipocyte in vitro cultuur systeem dat het mogelijk maakt dissectie van de rollen van de matrix en adipocytes in bij te dragen tot vetweefsel metabole fenotype.

Een menselijke 3D-extracellulaire matrix-Adipocyte cultuur model voor het bestuderen van matrix-cel metabole Overspraak

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 beschrijft alle procedures, van het kweken van humane, van vet afgeleide mesenchymale stamcellen (ADSC's) en het verzamelen van supernatans tot het extra-extracellulaire blaasjes (EV's) met behulp van ultracentrifugatie.

Zuivering en karakterisering van extracellulaire blaasjes uit humane van vet afgeleide mesenchymale stamcellen

Hier beschrijven we een protocol voor het isoleren, kweken en fenotyperen van microvasculaire endotheelcellen uit menselijk onderhuids vetweefsel (hSATMVEC's). Daarnaast beschrijven we een experimenteel model van hSATMVEC-adipocyte overspraak.

Bestudering van overspraak van vet-endotheelcellen en adipocyten in menselijk onderhuids vetweefsel

Met een ex vivo explant-model worden kleine stukjes van het rostrale deel van visceraal wit vetweefsel van magere muizen 7–10 dagen gekweekt, waardoor ontsteking in vetweefsel tijdens vetrijke voeding kan worden geïnduceerd en bestudeerd.

Een ex vivo muismodel voor vetrijke vetachtige vetweefselontsteking

Leer hoe adipocyt-afgeleide extracellulaire vesikels worden gekarakteriseerd met behulp van een CD63-GFP reporter muismodel. Deze methode visualiseert en kwantificeert EV-secretie in vivo en in vitro.

Karakterisering van door adipocyten afgeleide extracellulaire vesikelsecretie met behulp van een CD63-GFP Reporter Mouse Model in vivo en in vitro (Video) | JoVE Journal

Karakterisering van door adipocyten afgeleide extracellulaire vesikelsecretie met behulp van een CD63-GFP Reporter Mouse Model in vivo en in vitro

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