Questo studio è stato sostenuto dal Canadian Institutes of Health Research e Genome Canada. JS detiene premi stipendio sostegno della Alberta Heritage Foundation for Medical Research, Heart and Stroke Foundation of Canada e la Canadian Diabetes Association. Il laboratorio vuole riconoscere l&#39;assistenza tecnica di strumenti Oroboros durante l&#39;acquisizione di saponina permeabilizzate tecnica di fibra.

<ol>
	<li>Saks, V. A., Veksler, V. I., Kuznetsov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilized+cell+and+skinned+fiber+techniques+in+studies+of+mitochondrial+function+in+vivo.">Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo.</a> Mol Cell Biochem. 184, 81-100 (1998).</li><li>Gnaiger, E., Kuznetsov, A. V., Schneeberger, S., Heldmaier, G., Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+in+the+cold.">Mitochondria in the cold.</a> Life in the Cold. , 431-442 (2000).</li><li>Rasmussen, H. N., Rasmussen, U. 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A perspective of mitochondrial physiology.</a> Adv Exp Med Biol. 543, 39-55 (2003).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+oxidative+phosphorylation+in+human+skeletal+muscle:+new+perspectives+of+mitochondrial+physiology.">Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology.</a> Int J Biochem Cell Biol. 41, 1837-1845 .</li><li>Sena, S., Hu, P., Zhang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+signaling+accelerates+cardiac+mitochondrial+dysfunction+after+myocardial+infarction.">Impaired insulin signaling accelerates cardiac mitochondrial dysfunction after myocardial infarction.</a> J Mol Cell Cardiol. 46, 910-918 (2009).</li><li>Boudina, S., Sena, S., O'Neill, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+mitochondrial+oxidative+capacity+and+increased+mitochondrial+uncoupling+impair+myocardial+energetics+in+obesity.">Reduced mitochondrial oxidative capacity and increased mitochondrial uncoupling impair myocardial energetics in obesity.</a> Circulation. 112, 2686-2695 (2005).</li><li>Lenaz, G., Genova, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+organization+of+mitochondrial+respiratory+complexes:+a+new+understanding+of+an+old+subject.">Structure and organization of mitochondrial respiratory complexes: a new understanding of an old subject.</a> Antioxid Redox Signal. 12, 961-1008 .</li><li>Lemieux, H., Hoppel, C. 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I., Kuznetsov, A. V., Sharov, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+parameters+in+cardiac+tissue:+a+novel+method+of+assessment+by+using+saponin-skinned+fibers.">Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers.</a> Biochim Biophys Acta. 892, 191-196 (1987).</li><li>Bangham, A. D., Horne, R. W., Glauert, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+saponin+on+biological+cell+membranes.">Action of saponin on biological cell membranes.</a> Nature. , 196-952 (1962).</li><li>Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids+of+mitochondria.">Lipids of mitochondria.</a> Biochim Biophys Acta. 822, 1-42 (1985).</li><li>Milner, D. 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R., Winkler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+saponin-permeabilized+muscle+fibers+for+the+diagnosis+of+mitochondrial+diseases.">Use of saponin-permeabilized muscle fibers for the diagnosis of mitochondrial diseases.</a> Biofactors. 7, 221-223 (1998).</li><li>Gnaiger, E., Dykens, J., Will, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarographic+oxygen+sensors,+the+oxygraph+and+high-resolution+respirometry+to+assess+mitochondrial+function.">Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function.</a> Drug-Induced Mitochondrial Dysfunction. , 327-352 (2008).</li></ol>

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La saponina permeabilizzate tecnica fibra cardiaca offre un compromesso tra le uniche in vitro e in vivo, la valutazione del consumo di ossigeno mitocondriale OXPHOS. I vantaggi di questa tecnica includono una maggiore rilevanza fisiologica rispetto ai mitocondri isolati come architettura cellulare è conservata. Mentre la membrana plasmatica è degradata, strutture a membrana intracellulari tra cui i mitocondri 12, 14, reticolo sarcoplasmatico 14, miofilamenti e il citoscheletro ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100% Ethanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>HC600</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>HC-1000</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate monopotassium salt dihydrate (ADP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5285</td>
<td>&nbsp;</td>
<tr>
<td>Albumin from bovine serum essentially fatty acid&#x2013;free</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-6003</td>
<td>&nbsp;</td>
<tr>
<td>Antimycin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8674</td>
<td>&nbsp;</td>
<tr>
<td>Ascobic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4403</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-triphosphate disodium salt hydrate (ATP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2383</td>
<td>&nbsp;</td>
<tr>
<td>Atractyloside</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6882</td>
<td>&nbsp;</td>
<tr>
<td>Calcium carbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4830</td>
<td>&nbsp;</td>
<tr>
<td>Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Cytochrome c</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7752</td>
<td>&nbsp;</td>
<tr>
<td>Digitonin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D141</td>
<td>&nbsp;</td>
<tr>
<td>Dithiothreitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D9779</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis-(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E4378</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>27647</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>Imidazole</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I5513</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Pfizer</td>
<td>&nbsp;</td>
<td>Ketaset</td>
</tr>
<tr>
<td>Lactobionic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>153516</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride hexahydrate (MgCl2&#x2219;6H2O)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9272</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
<td>&nbsp;</td>
<tr>
<td>N,N,N&#x2019;,N&#x2019;-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T3134</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Phosphocreatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7936</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Hydroxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5958</td>
<td>&nbsp;</td>
<tr>
<td>Potassium cyanide</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>60178</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Rotenone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R8875</td>
<td>&nbsp;</td>
<tr>
<td>Saponin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>47036</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pentobarbital</td>
<td>&nbsp;</td>
<td>Ceva Sante Animale</td>
<td>1715 138</td>
<td>Conc. 54.7 mg/ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2256</td>
<td>&nbsp;</td>
<tr>
<td>Succinic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3674</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S7903</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8691</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Bayer</td>
<td>&nbsp;</td>
<td>Rompun</td>
</tr>
<tr>
<td>ddH2O</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ice</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oroboros Oxygraph-2k</td>
<td>&nbsp;</td>
<td>Oroboros Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>21905-026</td>
<td>&nbsp;</td>
<tr>
<td>15ml polypropylene centrifuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89004-368</td>
<td>&nbsp;</td>
<tr>
<td>50ml polypropylene centrifuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89004-364</td>
<td>&nbsp;</td>
<tr>
<td>Straight Jewelers Forceps</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>160-50B</td>
<td>&nbsp;</td>
<tr>
<td>Curved Jewelers Forceps</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>160-57B</td>
<td>&nbsp;</td>
<tr>
<td>Straight Surgery Scissors</td>
<td>&nbsp;</td>
<td>George Tiemann &amp; Co.</td>
<td>105-402</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Surgical Blade</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BD371610</td>
<td>&nbsp;</td>
<tr>
<td>0.45-&mu;m Syringe filters</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA28145-485</td>
<td>&nbsp;</td>
<tr>
<td>pH meter</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA11388-308</td>
<td>&nbsp;</td>
<tr>
<td>Glass Petri dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89000-300</td>
<td>&nbsp;</td>
<tr>
<td>12-well Polystyrene Tissue Culture Plates</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82050-926</td>
<td>&nbsp;</td>
<tr>
<td>Plate Stirrer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>97042-594</td>
<td>&nbsp;</td>
<tr>
<td>Fisherbrand Microbars</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-511-67</td>
<td>&nbsp;</td>
<tr>
<td>Weigh Scale</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CA11278-162</td>
<td>&nbsp;</td>
<tr>
<td>10&mu;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-815-1</td>
<td>&nbsp;</td>
<tr>
<td>25&#x03BC;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-824-7</td>
<td>&nbsp;</td>
<tr>
<td>50&mu;l Hamilton Micro Syringe</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>14-824-5</td>
<td>&nbsp;</td>
<tr>
<td>Nalgene Squeeze Bottles</td>
<td>&nbsp;</td>
<td>Wilkem Scientific</td>
<td>LNA2407-1000</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene Weighing Dishes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>89106-750</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

respirometric oxidative phosphorylation assessment in saponin-permeabilized cardiac fibers

Indagine della funzione mitocondriale rappresenta un importante parametro di fisiologia cardiaca come i mitocondri sono coinvolti nel metabolismo energetico, stress ossidativo, apoptosi, invecchiamento, encefalomiopatie mitocondriali e la tossicità dei farmaci. Detto questo, le tecnologie per misurare la funzione mitocondriale cardiaco sono molto richiesti. Una tecnica che si avvale di un approccio integrato per misurare la funzione mitocondriale è respirometrico fosforilazione ossidativa (OXPHOS) analisi. Il principio di valutazione respirometrico OXPHOS è incentrata la misurazione della concentrazione di ossigeno utilizzando un elettrodo di Clark. Mentre il fascio di fibre permeabilizzate consuma ossigeno, concentrazione di ossigeno nel declina camera chiusa. Utilizzando selezionati substrato-inibitore-disaccoppiatore protocolli di titolazione, gli elettroni sono forniti a siti specifici della catena di trasporto degli elettroni, che consentono la valutazione della funzione mitocondriale. Tecniche di preparazione prima dell&#39;analisi respirometrico della funzione mitocondriale, meccanici e chimici sono utilizzati per permeabilize il sarcolemma delle fibre muscolari. Permeabilizzazione chimica impiega saponina per perforare modo selettivo la membrana cellulare, pur mantenendo l&#39;architettura cellulare. Questo documento descrive accuratamente le fasi del processo di preparazione di fibre cardiache saponina di pelle per le misure di consumo di ossigeno per valutare OXPHOS mitocondriale. Inoltre, la risoluzione dei problemi indicazioni, nonché a specifici substrati, inibitori e uncouplers che possono essere utilizzati per determinare la funzione dei mitocondri in siti specifici della catena di trasporto degli elettroni sono forniti. È importante sottolineare che il protocollo descritto può essere facilmente applicata al tessuto cardiaco e scheletrico di vari modelli animali e campioni umani.

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Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Saponina permeabilizzate preparazione fibra in collaborazione con respirometrico analisi fosforilazione ossidativa fornisce una valutazione integrativa della funzione mitocondriale. Respirazione mitocondriale in stati fisiologici e patologici possono riflettere varie influenze normative ivi comprese le interazioni mitocondriale, morfologia e biochimica.

Respirometriche valutazione fosforilazione ossidativa in fibre cardiache saponina permeabilizzate

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

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24.5K Views.  University of Calgary. Saponina permeabilizzate preparazione fibra in collaborazione con respirometrico analisi fosforilazione ossidativa fornisce una valutazione integrativa della funzione mitocondriale. Respirazione mitocondriale in stati fisiologici e patologici possono riflettere varie influenze normative ivi comprese le interazioni mitocondriale, morfologia e biochimica.