Name: Robuuste mitochondriale isolatie van knaagdierweefsel (Video) | JoVE Journal
Uploaded: 2024-08-23T14:10:00.000+00:00
Duration: 7 min 3 s
Description: Leer een robuuste methode voor mitochondriale isolatie uit knaagdierweefsel met hoge resolutie-respirometrie. Deze techniek levert hoogwaardige mitochondriën voor onderzoek naar ziekteprocessen en celbiologie.

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<meta charset="utf8"></head><body>Mitochondriale isolatie van knaagdieren voor hoogwaardige bio-energetische studies

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<li>Tomar, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Substrate-dependent+differential+regulation+of+mitochondrial+bioenergetics+in+the+heart+and+kidney+cortex+and+outer+medulla%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Bioenerg%22%5BJournal%5D)">Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla.</a> Biochim Biophys Acta Bioenerg. 1863 (2), 148518(2022).</li>
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<li>Strubbe-Rivera, J. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mitochondrial+permeability+transition+phenomenon+elucidated+by+cryo-em+reveals+the+genuine+impact+of+calcium+overload+on+mitochondrial+structure+and+function%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">The mitochondrial permeability transition phenomenon elucidated by cryo-em reveals the genuine impact of calcium overload on mitochondrial structure and function.</a> Sci Rep. 11 (1), 1037(2021).</li>
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<li>Will, Y., Hynes, J., Ogurtsov, V. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+mitochondrial+function+using+phosphorescent+oxygen-sensitive+probes%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes.</a> Nat Protoc. 1 (6), 2563-2572 (2006).</li>
<li>Huerta, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sea+lamprey+cardiac+mitochondrial+bioenergetics+after+exposure+to+TFM+and+its+metabolites%5BTitle%5D)+AND+%22Aquat+Toxicol%22%5BJournal%5D)">Sea lamprey cardiac mitochondrial bioenergetics after exposure to TFM and its metabolites.</a> Aquat Toxicol. 219, 105380(2020).</li>
<li>Malyala, S., Zhang, Y., Strubbe, J. O., Bazil, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+phosphate+precipitation+inhibits+mitochondrial+energy+metabolism%5BTitle%5D)+AND+%22PLoS+Comput+Biol%22%5BJournal%5D)">Calcium phosphate precipitation inhibits mitochondrial energy metabolism.</a> PLoS Comput Biol. 15 (1), e1006719(2019).</li>
</ol>

<table><tbody><tr><td>1.7 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>10 mL glazen beker</td><td></td><td></td><td>Voor orgaan dissectie en fijnhakken</td></tr><tr><td>50 mL centrifugebuizen</td><td></td><td></td><td>Centrifugatie</td></tr><tr><td>Adenosine 5'-difosfaat monokaliumzout dihydraat</td><td>Sigma&nbsp;</td><td>A5285</td><td>Respirometrie-assays</td></tr><tr><td>BSA-eiwitasey-kit</td><td>Thermo Scentific</td><td>PI23225</td><td>Mitochondriale eiwitkwantificering</td></tr><tr><td>Dextrose</td><td>Sigma&nbsp;</td><td>DX0145</td><td>Voor buffer (CB)</td></tr><tr><td>Ethyleenglycol-bis(2-amino-ethylether)-N,N,N',N'-tetraazijnzuur&nbsp;</td><td>Sigma&nbsp;</td><td>E4378</td><td>Voor buffers (CB en IB) en respirometrie-assays</td></tr><tr><td>Glazen canule</td><td>Radnoti</td><td></td><td>Perfusie van hart van cavia's en ratten</td></tr><tr><td>Heparine natrium varkensslijmvlies</td><td>Sigma&nbsp;</td><td>SRE0027-250KU</td><td>Dierlijke IP-injectie</td></tr><tr><td>Hoge-resolutie respirometer</td><td></td><td></td><td>Clark-type elektrode; oxygraaf met 2 mL kamers</td></tr><tr><td>Inductiekamer</td><td></td><td></td><td></td></tr><tr><td>Isofluraan</td><td>Sigma&nbsp;</td><td>792632</td><td>Anesthesie</td></tr><tr><td>L-appelzuur</td><td>Sigma&nbsp;</td><td>02288-50G</td><td>Respirometrie-assays</td></tr><tr><td>Magnesiumchloride hexahydraat</td><td>Sigma&nbsp;</td><td>M9272</td><td>Voor buffer (RB)</td></tr><tr><td>Mannitol</td><td>Sigma&nbsp;</td><td>MX0214</td><td>Voor buffer (IB)</td></tr><tr><td>Microliter spuiten</td><td></td><td></td><td>Groottes variërend van 5&ndash;50 &micro;L</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>Moet in staat zijn om te incuberen bij 37 &deg;C&nbsp;</td></tr><tr><td>MOPS</td><td>Sigma&nbsp;</td><td>475898</td><td>Voor alle buffers</td></tr><tr><td>O2 tank</td><td></td><td></td><td></td></tr><tr><td>OMNI THQ Homogenisator</td><td>OMNI International&nbsp;</td><td>12-500</td><td>Vergelijkbare rotor stator homogenisatoren zullen werken</td></tr><tr><td>pipetten</td><td></td><td></td><td>Volumes van&nbsp; 2&ndash;20 &micro;L; 20&ndash;200 &micro;L; 200&ndash;1000 &micro;L</td></tr><tr><td>Kaliumchloride</td><td>Sigma&nbsp;</td><td>P3911</td><td>Voor buffers (RB en CB)</td></tr><tr><td>Kaliumfosfaat dibasisch</td><td>Sigma&nbsp;</td><td>795496</td><td>Voor buffers (IB en RB)</td></tr><tr><td>Protease van Bacillus licheniformis</td><td>Sigma&nbsp;</td><td>P5459</td><td></td></tr><tr><td>Natriumchloride</td><td>Sigma&nbsp;</td><td>S9888</td><td>Voor buffer (CB)</td></tr><tr><td>Natriumpyruvaat</td><td>Fisher bioreagents</td><td>BP356-100</td><td>Respirometrie-assays</td></tr><tr><td>Sucrose</td><td>Sigma&nbsp;</td><td>8510-OP</td><td>Voor buffer (IB)</td></tr><tr><td>Chirurgisch dissectiekit</td><td></td><td></td><td>Afhankelijk van dier en weefselbron</td></tr><tr><td>Centrifuge op tafel</td><td></td><td></td><td>Moet afkoelen tot 4 &deg;C&nbsp;</td></tr></tbody></table>

robuuste mitochondriale isolatie van knaagdierweefsel 

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Robuuste mitochondriale isolatie van knaagdierweefsel 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

robust-mitochondrial-isolation-from-rodent-cardiac-tissue

Research

JoVE Journal

Biochemistry

Biochemie

We beschrijven а protocol voor isolatie van pure, sterk gekoppeld rat hart mitochondriën voor functionele of structurele studies van cellulaire bio-energetica, biofysische metingen, proteomics of mitochondriale DNA en lipiden-analyse.

Bereiding van zeer Gekoppeld Rat Hart Mitochondriën

Dit protocol beschrijft een procedure om de ademhaling van mitochondriën geïsoleerd van skeletspieren studie. Deze methode werd aangepast van...

Mitochondriale Isolatie van skeletspieren

Een spectrofluorimetrische protocol voor het meten van de mitochondriale permeabiliteit transitie porie opening in geïsoleerde muizenhart mitochondria wordt hier gepresenteerd. De bepaling omvat de gelijktijdige meting van Ca...

Multi-parameter meting van de doorlatendheid Transitie Pore Openen in Geïsoleerde Muis Heart Mitochondria

Werkwijze voor snelle isolatie van mitochondria uit zoogdierweefsel biopsies beschreven. Rat lever of skeletspieren voorbereidingen werden gehomogeniseerd met een commerciële weefsel Dissociator en mitochondriën werden door differentiële filtratie geïsoleerd door nylon mesh filters. Mitochondriale isolatie is &lt;30 min ten opzichte van 60-100 min met behulp van alternatieve methoden.

Rapid isolatie en zuivering van Mitochondriën voor transplantatie Door Tissue Dissociatie en differentiële Filtratie

Vergelijking van het mitochondriale membraanpotentieel tussen monsters levert waardevolle informatie op over de cellulaire status. Gedetailleerde stappen voor het isoleren van mitochondriën en het beoordelen van de respons op remmers en ontkoppelaars met behulp van fluorescentie worden beschreven. De methode en het nut van dit protocol worden geïllustreerd door het gebruik van een celcultuur en een diermodel van cellulaire stress.

Isolatie en functionele analyse van Mitochondriën uit gekweekte cellen en muis Tissue

Hier presenteren we een aanpassing van een eerder gerapporteerde methode die de isolatie van hoogwaardige en gezuiverde mitochondriën mogelijk maakt uit kleinere hoeveelheden muisskeletspier. Deze procedure resulteert in sterk gekoppelde mitochondriën die ademen met hoge functionaliteit tijdens microplate-gebaseerde respirometrische analyses.

Isolatie van Mitochondriën uit Minimale hoeveelheden muis skeletspieren voor Metingen High Throughput Microplate Respiratory

Hier wordt een quadricepsspiermonster genomen van een geanesthetiseerd varken en mitochondriën worden geïsoleerd door differentiële centrifugatie. Vervolgens worden de ademhalingssnelheden van mitochondriale ademhalingsketencomplexen I, II en IV bepaald met behulp van hoogwaardige respirometrie.

Isolatie van Intact Mitochondriën zijn van skeletspieren door differentiële centrifugatie voor High-resolution respirometrietest Metingen

Hier presenteren we twee protocollen voor het meten van mitochondriale Ca2 + toestroom in geïsoleerde mitochondriën en gekweekte cellen. Voor geïsoleerde mitochondriën, detailleren wij een plaat lezer gebaseerde Ca2 + import assay met behulp van de Ca2 + gevoelige kleurstof calcium groen-5N. Voor gekweekte cellen beschrijven we een methode van de confocal microscopie met behulp van de Ca2 + kleurstof Rhod-2/AM.

Analyses van mitochondriale Calcium toestroom in geïsoleerde Mitochondria en gekweekte cellen

Hier beschrijven we een gedetailleerde methode voor mitochondriale isolatie van de skeletspieren van muizen en de daaropvolgende analyse van ademhaling door oxygen consumption rate (OCR) met behulp van op microplaten gebaseerde respirometrische assays. Deze pijplijn kan worden toegepast om de effecten van meerdere omgevings- of genetische interventies op het mitochondriale metabolisme te bestuderen.

Isolatie van mitochondriën uit de skeletspieren van muizen voor respirometrische assays

Dit protocol beschrijft een techniek voor de analyse van respiratoire supercomplexen wanneer slechts kleine hoeveelheden monsters beschikbaar zijn.

Isolatie van mitochondriën voor mitochondriale supercomplexanalyse uit kleine weefsel- en celcultuurmonsters 

Leer een robuuste methode voor mitochondriale isolatie uit knaagdierweefsel met hoge resolutie-respirometrie. Deze techniek levert hoogwaardige mitochondriën voor onderzoek naar ziekteprocessen en celbiologie.

Robuuste mitochondriale isolatie van knaagdierweefsel

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