Name: Beoordeling van mitochondriale splijting/fusiedynamiek in proximale tubulaire cellen van de nier (Video) | JoVE Journal
Uploaded: 2025-11-14T14:40:00.000+00:00
Duration: 6 min 14 s
Description: Leer mitochondriale splijting/fusiedynamiek beoordelen in proximale tubulaire cellen van de nier met confocale microscopie en Dendra2-fluorescentie. Deze methode helpt onderzoekers bij het volgen van getransplanteerde mitochondriën.

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</ol>

```html
<table><tbody><tr><td>Celcultuur&nbsp;</td><td></td><td></td><td></td></tr><tr><td>DMEM/F12&nbsp;</td><td>Basismedium&nbsp;</td><td>(Gibco/Cat. Nr.11320033 )&nbsp;</td><td>Proximale tubulaire celcultuur&nbsp;</td></tr><tr><td>Foetaal Runderserum (FBS)&nbsp;</td><td>5-10% suppletie&nbsp;</td><td>(Biowest/Cat. Nr.S181B)&nbsp;</td><td>Verrijking van cultuurmedium&nbsp;</td></tr><tr><td>Penicilline-Streptomycine&nbsp;</td><td>100X&nbsp;</td><td>(Gibco/Cat. Nr.15140122)&nbsp;</td><td>Antibioticabescherming&nbsp;</td></tr><tr><td>Collagene IV&nbsp;</td><td>coating&nbsp;</td><td>( Sigma-C5533-5M)&nbsp;</td><td>Ibidi plaatcoating&nbsp;</td></tr><tr><td>Trypsine-EDTA&nbsp;</td><td>0,25% oplossing&nbsp;</td><td>(Gibco/Cat. Nr.25200056)&nbsp;</td><td>Celdetachering&nbsp;</td></tr><tr><td>Fosfaat-Gebufferd Saline (PBS)&nbsp;</td><td></td><td>(Gibco/Cat. Nr.10010023 )&nbsp;</td><td>Celwassing&nbsp;</td></tr><tr><td>Behandelingen&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Cisplatin&nbsp;</td><td>2 &micro;M werkconcentratie&nbsp;</td><td>(Selleck Chemicals/Cat. Nr.S1166)&nbsp;</td><td>Stressinductie&nbsp;&nbsp;</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)&nbsp;</td><td>voertuigcontrole&nbsp;</td><td>(Fisher Scientific/Cat. Nr.D1391)&nbsp;</td><td>Cisplatin solvent controle&nbsp;</td></tr><tr><td>Beeldvorming&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Fenol Rood-Vrij Medium&nbsp;</td><td>Live beeldvorming&nbsp;</td><td>(Gibco/Cat. Nr.21041025)&nbsp;</td><td>Verminderde achtergrondfluorescentie&nbsp;</td></tr><tr><td>Moleculair Biologie&nbsp;</td><td></td><td></td><td></td></tr><tr><td>TRIzol&reg;&nbsp;</td><td>RNA extractie&nbsp;</td><td>(Gibco/Cat. Nr.15596018)&nbsp;</td><td>Totale RNA isolatie&nbsp;</td></tr><tr><td>SYBR Green Master Mix&nbsp;</td><td>qPCR chemie&nbsp;</td><td>(Thermo Scientific/Cat. Nr.K0221)&nbsp;</td><td>Mitochondriale dynamic genexpressie (DNM1L, FIS1, MFN1, MFN2, OPA1)&nbsp;</td></tr><tr><td>Fluorescente Hulpmiddelen&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Dendra2&nbsp;</td><td>Mitochondriale matrix tag&nbsp;</td><td>Jacson Laboratory(PhAMverwijderd)&nbsp;</td><td>Foto-wisselaar reporter (405nm omzetting, 488/561nm beeldvorming)&nbsp;</td></tr><tr><td>Microscopen&nbsp;</td><td></td><td></td><td></td></tr><tr><td>Confocale Microscoop&nbsp;</td><td>Leica DMi8&nbsp;</td><td>Live-cel beeldvorming en FRAP&nbsp;</td><td>405nm/488nm/561nm lasers&nbsp;</td></tr><tr><td>Image J&nbsp;</td><td></td><td>https://imagej.net/software/fiji/downloads&nbsp;</td><td></td></tr><tr><td>RGB Profiler Plugin&nbsp;</td><td></td><td>https://imagej.net/ij/plugins/rgb-profiler.html.&nbsp;</td><td>Fusiegebeurtenis analyse (rood/groen signaaloverlappend)&nbsp;</td></tr></tbody></table>
```

beoordeling van mitochondriale splijting/fusiedynamiek in proximale tubulaire cellen van de nier

Assessment of Mitochondrial Fission/Fusion Dynamics in Kidney Proximal Tubular Cells

Beoordeling van mitochondriale splijting/fusiedynamiek in proximale tubulaire cellen van de nier

Mitochondria are best recognized for their role in ATP synthesis and serve as key regulators of cellular metabolism. Mitochondrial dynamics comprehend the ...

assessment-mitochondrial-fissionfusion-dynamics-kidney-proximal

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Mitochondriale fusie werd gemeten door het bijhouden van de gelijkschakeling van de photoconverted matrix-gerichte GFP over het mitochondriale netwerk in de tijd. Tot dusver kon slechts een cel worden onderworpen aan een uur lang kinetische analyse tegelijk. Wij presenteren een methode die meet gelijktijdig meerdere cellen, waardoor versnelling van het proces van gegevensverzameling.

Een snellere, met hoge resolutie, MTPA-GFP-gebaseerde Mitochondriale Fusion Assay verwerven Kinetische gegevens van meerdere cellen parallel geschakeld Met behulp van confocale microscopie

De effecten van activering van eiwit kinase C (PKC) isozymen op mitochondriële functies geassocieerd met ademhaling en oxidatieve fosforylering en cellevensvatbaarheid beschreven. De aanpak past adenovirale techniek om selectief PKC overexpressie isozymen in primaire celkweek en verschillende assays voor mitochondriële functies en energie status van de cel te bepalen.

Beoordeling van Mitochondriale functies en levensvatbaarheid van de cellen in de nier overexpressie Proteïne kinase C-isozymen

Hier schetsen we hoe we de mitochondriale lokalisatie van een (celcyclus) kinase kunnen bestuderen, en hoe we de sub-mitochondriale locatie evenals potentiële mitochondriale substraten/doelen kunnen bepalen. Gedwongen expressie van eiwitten in de mitochondriën biedt een nuttige tool voor het bestuderen van de functionele consequenties van de mitochondriale lokalisatie van een eiwit van belang.

Experimentele benaderingen om te studeren mitochondriale lokalisatie en functie van een nucleair celcyclus Kinase, Cdk1

Dit protocol biedt een rendabele aanpak om te isoleren en karakteriseren van muis primaire renale tubulaire cellen die vervolgens sub kunnen worden gekweekt om te beoordelen renale biologische functies- ex vivo, met inbegrip van de mitochondriale Bioenergetica.

Isolatie, karakterisering en High Throughput extracellulaire Flux-analyse van muis primaire renale tubulaire epitheliale cellen

Hier presenteren we een protocol om veranderingen in mitochondriale dichtheid en longitudinale distributie te analyseren door middel van beeldvorming van levende skeletspieren met behulp van confocale microscopie voor het scannen van mitochondriale netwerken.

Analyse van de mitochondriale dichtheid en longitudinale verdeling in levende skeletspiervezels van ratten door confocale microscopie

In deze studie beschrijven we een stapsgewijs protocol en benadrukken we de belangrijkste details voor het bepalen van morfologische kenmerken van mitochondriën in levende cellen, inclusief monstervoorbereiding, beeldacquisitie en gegevensanalyse. Deze methode wordt vaak gebruikt om de mitochondriale morfologie te onderzoeken voor het bestuderen van verschillende aandoeningen.

Bepaling van mitochondriale morfologie in levende cellen met behulp van confocale microscopie

Hier beschrijven we de mitochondriale gebeurtenislokalisator (MEL), een ImageJ-plug-in die nuttig is bij het kwantificeren van de 3-dimensionale veranderingen in mitochondriale splijting en fusieactiviteit in de loop van de tijd. We beschrijven ook een beeldverwerkingspijplijn die nuttig is voor het opschonen van microfoto's voorafgaand aan analyse in ImageJ.

Inzicht in de veranderingen in mitochondriale morfologie door middel van dynamische en driedimensionale fluorescentiemicrofoto's

Hier beschrijven we een in vitro protocol om hypoxie/reoxygenatieschade te modelleren in de sterk metabole proximale tububulaire epitheelcellen. Dit model is ontworpen om schade veroorzaakt door metabole stress te induceren, die kan worden gemeten door de expressie van proximale tubuli-schademarkers en de beoordeling van de mitochondriale ademhalingsfunctie.

Modellering van hypoxie/reoxygenatieschade in proximale tubulära epitheelcellen

Lokale injecties van een mitochondriale kleurstof, labelen terminale Schwann-celmitochondriën in vivo voor confocale microscopie. Deze methode maakt gebruik van proof of concept aangetoond in skeletspierweefsel van muizen met gezonde of zieke spieren om een hoogresolutie mitochondriale visualisatie te bieden.

Methoden voor in situ kwantificering van mitochondriale morfologie in spier- en terminale Schwann-cellen van muizen

Stress korrels (SG) zijn cytoplasmatische RNA korrels die geïnstalleerd ribonucleoproteïnepartikels (RNP), en belangrijk in cellulaire reactie op diverse spanningen. Dynamiek van SG's kunnen in levende cellen worden gevolgd door het visualiseren van de lokalisatie van een gelabeld onderdeel van SG in getransfecteerde primaire cellen na stress.

Visualisatie van G3BP Stress Korrels Dynamics Live primaire cellen

Leer mitochondriale splijting/fusiedynamiek beoordelen in proximale tubulaire cellen van de nier met confocale microscopie en Dendra2-fluorescentie. Deze methode helpt onderzoekers bij het volgen van getransplanteerde mitochondriën.

Beoordeling van mitochondriale splijting/fusiedynamiek in proximale tubulaire cellen van de nier

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