Wir möchten Neej Patel, Thomas Bell, Lyn Mattathil und Mark Mannino für ihre technische Unterstützung zu danken. Diese Arbeit wurde von United States Public Health Service Award R15DK102122 vom National Institute of Diabetes und Magen-Darm und Niere-Krankheiten (NIDDK), Jonathan Fisher unterstützt. Der Manuskript-Inhalt ist ausschließlich in der Verantwortung der Autoren und nicht unbedingt die offizielle Meinung der NIDDK oder die National Institutes of Health.

Hinweis: Siehe Abbildung 1 für einen schematischen Überblick über die wichtigsten Schritte.
1. Reduzierung der WST-1-Assay
<ol><li>Wachsen Sie und differenzieren Sie C2C12 adhärente Zellen mit Standardzellen Kultur Verfahren7 in einer 96-Well-Platte mit Zeilen A-F.<ol>
<li>Verwenden Sie eine Differenzierung Medium aus der Dulbeccos geändert Eagle Medium (DMEM) mit 2 % Pferd Serum, 100 U/mL Penicillin und 0,1 mg/mL Streptomycin ergänzt. Inkubation der Zellen bei 37 ° C mit 5 % CO2.</li>
		<li>Bei der Überwachung von Ascorbat Beteiligung an tPMET ergänzen Sie Differenzierung Medien mit 100 µM Ascorbinsäure. Können Zellen auszubrüten in Differenzierung Medien für ~ 24-48 h.</li>
	</ol>
</li>
	<li>WST-1 und PMS Stammlösungen vorzubereiten.<ol>
<li>Um ein 10 mM Lager von WST-1 machen, lösen 0,033 g WST-1 (Formel Gewicht (FW): 651.34 g/Mol) in 5 mL Phosphat gepufferte Kochsalzlösung (PBS). Shop bei 4 ° C.</li>
		<li>Um einen 5 mM bestand von PMS zu machen, lösen 0,0023 g von PMS (FW: 306.34 g/Mol) in 1,5 mL entionisiertem H2O (DiH2O). Bei-20 ° C lagern und vor Licht schützen.</li>
	</ol>
</li>
	<li>Fügen Sie 0,0108 g Glukose für eine Endkonzentration von 5 mM bis 11,4 mL PBS oder HEPES Kochsalzlösung gepufferten (HBS; 20 mM HEPES Natrium Salz, 140 mM NaCl, KCl, 2,5 mM MgSO4, 5 mM 1 mM CaCl2). Fügen Sie 480 µL 10 mM WST-1 für eine Endkonzentration von 400 µM und 48 µL 5 mM PMS für eine Endkonzentration von 20 µM.</li>
	<li>Bei der Überwachung von Ascorbat Beteiligung an tPMET teilen Sie die Lösung in zwei 6 mL-aliquoten. Eine Aliquote hinzugeben Sie 6 µL DiH2O und die anderen aliquoten fügen Sie 6 µL 2 kU/mL AO für eine Endkonzentration von 2 U/mL hinzu.</li>
	<li>Beim Überwachen der Superoxid-Beteiligung an tPMET teilen Sie die Lösung in zwei 6 mL-aliquoten. Eine Aliquote fügen Sie 55 µL 0.1 M KPO4 Puffer hinzu und fügen Sie der anderen aliquoten 55 µL 6,5 kU/mL SOD für eine Endkonzentration von 60 U/mL hinzu.</li>
	<li>Waschen Sie die Zellen mit PBS. Aspirieren Sie die Medien und fügen Sie 150 µL PBS hinzu. Dann Aspirieren der PBS. Hinweis: Der Test erfolgt mit vergoldeten, angeschlossenen Zellen. So gibt es keine Zentrifugation oder Verwendung von Trypsin im Waschprozess.</li>
	<li>Hinzufügen von 100 µL der WST-1-Lösung (-) SOD oder (-) AO, Spalten 1 bis 6 und 100 µL der WST-1-Lösung (+) SOD oder (+) AO Spalten 7 bis 12 in der 96-Well-Platte hinzufügen. Verwenden Sie Zeilen G und H als Hintergrund-Kontrollen (d. h.Änderung überwachen in Absorption in das Reagenz allein im Brunnen ohne Zellen).</li>
	<li>Messen Sie die Absorption Werte mit dem Spektralphotometer alle 10 min für 1 h bei 438 nm.</li>
	<li>Nach der Lektüre, Aspirieren Sie die Medien und waschen jeweils gut mit 150 µL PBS. Dann Aspirieren der PBS.</li>
	<li>Berechnen Sie die Änderung der Extinktion für jedes gut durch Subtraktion der ersten Extinktion für einen Brunnen aus der Extinktion zu jedem Zeitpunkt für das gut. Korrigieren Sie diese Änderung für die Änderung der Extinktion (falls vorhanden) in den Hintergrund-Brunnen (d.h., Brunnen mit Assay Lösung aber keine Zellen) beobachtet.</li>
	<li>Für die Analyse normalisieren die Extinktion Daten der Kontrollmessung 60 min oder waschen mit PBS oder HBS und führen Sie dann einen Bicinchoninic Säuren (BCA) Protein Assay.</li>
	<li>Hinzufügen von 2 mg/mL Rinderserumalbumin (BSA) Standard (Bereich von 0,5-2 µL für die Standardkurve, je nach dem Grad der Zusammenfluss der Zellen), die leeren Brunnen (Zeilen G und H), fügen Sie das BCA-Reagenz in alle Vertiefungen.</li>
	<li>Die Daten über Nmol WST-1 Reduktion pro µg Protein zu quantifizieren. Verwenden Sie 37 mM-1 cm-1 22 als vom Aussterben bedroht-Koeffizient für reduzierte WST-1 438 nm.</li>
</ol>2. ‑Aufklärung-Reduktion-Assay
<ol><li>Wachsen und C2C12 adhärente Zellen mit dem gleichen Verfahren wie Schritt 1.1 zu unterscheiden.</li>
	<li>Lager 10 mM ‑Aufklärung Lösung wie folgt vorbereiten. Lösen Sie 0,029 g ‑Aufklärung (FW: 290.08 g/Mol) in 10 mL DiH2O. bestätigen die Konzentration des ‑Aufklärung durch Messung der Extinktion bei 600 nm mit einem Spektrophotometer. Verwenden Sie 1 mM-1 cm-1 23 als vom Aussterben bedroht-Koeffizient für reduzierte ‑Aufklärung bei 600 nm. Shop bei 4 ° C.</li>
	<li>11,880 mL PBS für eine Endkonzentration von 5 mM 0,0108 g Glukose hinzufügen. Fügen Sie 120 µL 10 mM ‑Aufklärung für eine Endkonzentration von 100 µM.</li>
	<li>Bei der Überwachung von Ascorbat Beteiligung an tPMET teilen Sie die Lösung in zwei 6 mL-aliquoten. Eine Aliquote hinzugeben Sie 6 µL DiH2O und die anderen aliquoten fügen Sie 6 µL 2 kU/mL AO für eine Endkonzentration von 2 U/mL hinzu.</li>
	<li>Beim Überwachen der Superoxid-Beteiligung an tPMET teilen Sie die Lösung in zwei 6 mL-aliquoten. Eine Aliquote fügen Sie 55 µL 0.1 M KPO4 Puffer hinzu und fügen Sie der anderen aliquoten 55 µL 6,5 kU/mL SOD für eine Endkonzentration von 60 U/mL hinzu.</li>
	<li>Aspirieren Sie die Medien und waschen Sie Zellen in 150 µL PBS. Aspirieren der PBS und 100 µL der ‑Aufklärung Lösung (-) hinzufügen SOD oder (-) AO, Spalten 1 bis 6 und 100 µL der ‑Aufklärung Lösung (+) SOD oder (+) AO Spalten 7 bis 12 in der 96-Well-Platte hinzufügen. Verwenden Sie Zeilen G und H wird als Hintergrund-Kontrollen (d. h.Änderung überwachen in Absorption in das Reagenz allein im Brunnen ohne Zellen).</li>
	<li>Messung der Extinktion bei 600 nm mit Spektralphotometer alle 10 min für 1 h quantifizieren die Änderung der Extinktion relativ zum Steuerelement bei 60 min Schritten 1.9 1.13 ähnlich.</li>
</ol>3. Ermittlung der ob reduzierte Elektronen Akzeptoren Substrate für AO oder SOD sind
<ol><li>5,436 mL PBS oder HBS fügen Sie 240 µL 10 mM WST-1 für eine Endkonzentration von 400 µM und 24 µL 5 mM PMS für eine Endkonzentration von 20 µM hinzu.<ol>
<li>Fügen Sie für ‑Aufklärung 60 µL 10 mM ‑Aufklärung, für eine Endkonzentration von 100 µM zu 5,940 mL PBS oder HBS hinzu.</li>
	</ol>
</li>
	<li>Hinzufügen von 100 µL Lösung jeweils gut in eine flach-Boden-96-Well-Platte in der Abwesenheit von Zellen und Messen Sie die Absorption in einem Spektrophotometer bei 438 nm für WST-1 oder 600 nm für ‑Aufklärung.</li>
	<li>1 µL 10 mM Ascorbat hinzu kommt die Hälfte der Brunnen für eine Endkonzentration von 100 μM. Überwachen Sie die Extinktion, bis es stabilisiert.</li>
	<li>Nach Stabilisierung 1 µL 200 U/mL AO für eine Endkonzentration von 2 U/mL in jede Vertiefung oder fügen Sie 1 µL 6 kU/mL SOD für eine Endkonzentration von 60 U/mL für jeden gut und überwachen die Extinktion für 1 h hinzu.</li>
</ol>

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G., Babcock, G. T., Dye, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+5-methylphenazinium+methyl+sulfate.+Reaction+of+the+oxidized+form+with+NADH+and+of+the+reduced+form+with+oxygen.">Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.</a> J Biol Chem. 257 (3), 1458-1461 (1982).</li><li>Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+derived+from+the+family+of+NOX+NADPH+oxidases.">Detection of reactive oxygen species derived from the family of NOX NADPH oxidases.</a> Free Radic Biol Med. 53 (10), 1903-1918 (2012).</li></ol>

Die Autoren haben nichts preisgeben.

Wir haben zwei Methoden zur Nutzung von extrazellulären Elektronen Akzeptoren, WST-1 und ‑Aufklärung, in spektralphotometrische Untersuchungen zur Überwachung von tPMET in C2C12 Myotubes vorgestellt. Mit dem Wachstum von Zelllinien im standard Kultur Verfahren und ein Spektralphotometer Platte Leser ist es möglich, tPMET mit dieser Elektronen-Akzeptoren in einem einfachen Mikrotestplatte Assay zu überwachen. WST-1 Reduzierung ist reproduzierbar aus Brunnen, Brunnen in einen Test, aber es gibt tägliche Variabilit?...

Die Fähigkeit des gereinigten Plasmamembranen, Elektronen-Akzeptoren reduzieren führte zu der Ansicht, dass der Plasmamembran ein Redox-inhärente Kapazität1hat. Zuvor in Pilzen, Pflanzen und Tiere gesehen, ist tPMET ein Prozess üblich, mehrere Organismen2,3,4,5. Insbesondere wurde dabei in Saccharomyces Cerevisiae, Karotte Zellen, Erythrozyten, Lymphozyten, Osteosarkom, Melanom, Makrophagen, Skelettmuskel und Neutrophilen2,3, nachgewiesen 4 , 5 , 6 , 7. in einem Prozess, der Elektronen über die Plasmamembran extrazelluläre Oxidantien reduzieren transportiert, tPMET engagiert sich in vielen Zellfunktionen einschließlich: Zelle Wachstum5,8, Zelle überleben9, Eisen Stoffwechsel10, Zelle11,12,13, und Schutz vor reaktiven Sauerstoff-Spezies12,14,15. Aufgrund des tPMET Engagements in vielen Zellfunktionen, Hypothese aufgestellt die ein Ungleichgewicht von tPMET als Beitrag zu der Entwicklung von einigen schweren gesundheitlichen Bedingungen, einschließlich Krebs16, Herz-Kreislauf-Krankheit17, und Stoffwechselerkrankungen Syndrom18.
Es gibt mehrere Möglichkeiten, um die Übertragung von Elektronen in der Plasmamembran zu überwachen, aber die am häufigsten verwendete Technik ist die Reduzierung der extrazellulären Elektronen Akzeptoren durch farbmetrische Assays zu beurteilen. Häufig verwendete extrazelluläre Elektronen Akzeptoren sind tetrazoliumsalz Salze, ‑Aufklärung, FeCN und Ferricytochrome C19,20. Das am häufigsten verwendete Tetrazolium-Salz ist als WST-119eine zweite Generation Salz bekannt. Diese Verbindung ist einfacher zu nutzen in farbmetrische Untersuchungen im Vergleich zur ersten Generation tetrazoliumsalz Salze durch zwei Sulfonate Gruppen, die die Wasser Löslichkeit21zu erhöhen. WST-1, wird in Verbindung mit der mittleren Elektron Akzeptor 1-Methoxy-phenazinderivat Methosulfate (Komponenten), in zwei Einzel-Elektronentransfer Ereignisse reduziert. Diese Reduktion ändert die schwach gefärbte oxidierte Form von WST-1 zu einer intensiveren, gelbe Formazan20,22. WST-1 hat eine hohe molare Aussterben Koeffizient von 37 x 103 M-1cm-1, führt zu einer hohen Assay Empfindlichkeit21,22. ‑Aufklärung wird auch als eine extrazelluläre Elektron Akzeptor tPMET Überwachung genutzt. Es hat sich gezeigt, dass ‑Aufklärung tPMET ohne die Hilfe eines zwischengeschalteten Elektronen Akzeptoren23,24extrazellulär gesenkt werden kann. Aufgrund des Fehlens der Mittelstufe Elektronen-Akzeptoren ‑Aufklärung können direkt Pick-up Elektronen aus der Plasmamembran, im Gegensatz zu WST-124. Ähnlich wie bei ‑Aufklärung, FeCN nachweislich extrazellulär tPMET ohne die Hilfe eines zwischengeschalteten Elektronen Akzeptoren19,24, ferrocyanid gesenkt werden. Im Gegensatz zu WST-1 und ‑Aufklärung hat FeCN eine niedrige molare Aussterben Koeffizient führt zu einem geringeren Assay Empfindlichkeit9. Eine andere häufig verwendete extrazelluläre Elektron Akzeptor, tPMET zu überwachen ist Ferricytochrome c. WST-1, Ferricytochrome C Reduktion steigt mit dem Einsatz von mittleren Elektron Akzeptor, Komponenten22ähnlich. Im Gegensatz zu WST-1 ist jedoch die Ferricytochrome C Methode aufgrund hoher Hintergrund und eine niedrige molare Aussterben Koeffizient22weniger empfindlich.
Hier präsentieren wir eine Methode zur Echtzeit-Analyse des tPMET durch photometrische Tests. Die Methode genutzt die extrazelluläre Elektronen-Akzeptoren WST-1 und ‑Aufklärung, da sie beide einen hohen molaren Aussterben Koeffizienten haben, während weniger teuer im Vergleich zu den anderen häufig extrazelluläre Elektronen-Akzeptoren wie Ferricytochrome c eingesetzt. Wir nutzten phenazinderivat Methosulfate (PMS) anstelle von Komponenten haben sie eine ähnliche chemische Zusammensetzung und PMS ist weit weniger teuer. Komponenten ist photochemisch stabilen ein wichtiges Merkmal für eine kommerzielle Kit, die eine lange Haltbarkeit. Jedoch machen wir PMS frisch für jeden Test, damit Stabilität kein Problem sein sollte. Wir präsentieren auch eine Methode zur Evaluierung möglicher enzymatischer Interaktionen (siehe Abbildung 1) zwischen der extrazellulären Elektron Akzeptor und Enzyme, die genutzt werden können, um den Prozess der tPMET weiter zu charakterisieren. Insbesondere ermitteln die Enzyme, die AO und SOD verwendet werden kann, welcher Teil des tPMET ist durch Ascorbat Transport oder extrazellulären Superoxid Version, zwei gängige Methoden für Elektronen über die Plasmamembran transportiert werden.

<table><tbody><tr><td>C2C12 myoblasts</td><td>American Type Culture Collection&amp;nbsp;</td><td>CRL-1772</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle&#039;s medium - low glucose</td><td>Sigma</td><td>D6046</td><td></td></tr><tr><td>Fetal Plex animal serum complex</td><td>Gemini Bio-Products&amp;nbsp;</td><td>100-602</td><td></td></tr><tr><td>penicillin-streptomycin</td><td>Sigma</td><td>516106</td><td></td></tr><tr><td>horse serum</td><td>Gibco Technologies</td><td>16050-130</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>15 cm culture dishes</td><td>TPP</td><td>93150</td><td></td></tr><tr><td>96 well culture plates</td><td>TPP</td><td>92096</td><td></td></tr><tr><td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td><td>Accela ChemBio&amp;nbsp; Inc</td><td>SY016315</td><td></td></tr><tr><td>phenazine methosulfate&amp;nbsp;</td><td>Sigma</td><td>P9625</td><td></td></tr><tr><td>L-ascorbic acid</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>ascorbate oxidase&amp;nbsp;</td><td>Sigma</td><td>A0157</td><td></td></tr><tr><td>superoxide dismutase&amp;nbsp;</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>2,6-dichloroindophenol sodium salt&amp;nbsp;</td><td>ICN Biomedicals</td><td>215011825</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Sigma</td><td>G7528</td><td></td></tr><tr><td>HEPES sodium salt</td><td>Sigma</td><td>H3784</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>potassium chloride</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366</td><td></td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Sigma</td><td>M5921</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>potassium phosphate</td><td>Fisher Scientific&amp;nbsp;</td><td>BP363</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Powerwave X-I spectrophotometer</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr><tr><td>Spectronic Genesys 5 Spectrophotometer</td><td>Thermo Scientific</td><td>336001</td><td></td></tr><tr><td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td><td>MidSci</td><td>781602</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma</td><td>220299</td><td></td></tr><tr><td>Iron (II) sulfate heptahydrate</td><td>Sigma</td><td>215422</td><td></td></tr><tr><td>hypoxanthine</td><td>Sigma</td><td>H9636</td><td></td></tr><tr><td>xanthine oxidase</td><td>Sigma</td><td>X4500</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>R Studio</td><td>Rstudio</td><td></td><td>https://www.rstudio.com/products/rstudio/</td></tr><tr><td>KC4</td><td>Biotek Insturments</td><td>discontinued&amp;nbsp;</td><td></td></tr></tbody></table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

Trans-Plasma Membran Elektronentransport (tPMET) spielt eine Rolle im Schutz der Zellen vor intrazellulären reduktiven Stress sowie Schutz vor Schäden durch extrazelluläre Oxidantien. Dieser Prozess für den Transport von Elektronen von intrazellulären Reduktionsmitteln extrazelluläre Oxidantien ist nicht gut definiert. Hier präsentieren wir Ihnen spektralphotometrische Proben von C2C12 Myotubes tPMET unter Verwendung der extrazellulären Elektronen-Akzeptoren zu überwachen: wasserlösliche tetrazoliumsalz Salz-1 (WST-1) und 2,6-Dichlorophenolindophenol (‑Aufklärung oder DCIP). Durch die Reduzierung der diese Elektronen-Akzeptoren sind wir in der Lage, diesen Prozess in eine Echtzeit-Analyse zu überwachen. Mit dem Zusatz von Enzymen wie Ascorbat Oxidase (AO) und Superoxid-Dismutase (SOD) für die Tests können wir bestimmen, welcher Teil des tPMET durch Ascorbat Export oder Superoxid Produktion, bzw. ist. Während WST-1 zu stabilen Ergebnissen mit niedrigen Hintergrund gezeigt wurde, konnte ‑Aufklärung nach der Zugabe von AO und SOD, die photometrische Analyse zeigte wieder oxidiert werden. Diese Methode zeigt einen in Echtzeit, Multi-gut, schnelle photometrische Assay mit Vorteilen gegenüber anderen Methoden verwendet, um tPMET, wie ferricyanid (FeCN) und Ferricytochrome C Reduktion zu überwachen.

Statistiken wurden mit ANOVA mit wiederholten Messungen mit RStudio Statistiksoftware25durchgeführt. Stichprobengrößen entnehmen Sie bitte der Abbildung Legenden.
Um tPMET zu überwachen, wurden C2C12 Myotubes zusammen mit extrazellulären Elektronen Akzeptoren, WST-1 und ‑Aufklärung eingesetzt. AO wurde verwendet, um festzustellen, welcher Teil der WST-1 und ‑Aufklärung Reduktion wurde durch Asc...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Messung der Trans-Plasma Membran Elektronentransport durch C2C12 Myotubes

Das Ziel dieses Protokolls ist, spektralphotometrisch Trans-Plasma Membran Elektronentransport Nutzung extrazelluläre Elektronen Akzeptoren überwachen und enzymatische Interaktionen zu analysieren, die mit dieser extrazellulären Elektronen-Akzeptoren auftreten können.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

measuring-trans-plasma-membrane-electron-transport-by-c2c12-myotubes

Research

JoVE Journal

Biochemistry

6.9K Aufrufe.  Saint Louis University. Das Ziel dieses Protokolls ist, spektralphotometrisch Trans-Plasma Membran Elektronentransport Nutzung extrazelluläre Elektronen Akzeptoren überwachen und enzymatische Interaktionen zu analysieren, die mit dieser extrazellulären Elektronen-Akzeptoren auftreten können.

Serie: Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Verwendung von isolierten Mitochondrien aus minimalen Mengen an Maus-Skelettmuskel für die Hochdurchsatz-Mikroplatten-Atemmessungen

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Forschung

Entwicklungsbiologie

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Einzelne Liposomen Messungen für das Studium der Protonen-Pumpen Membran Enzyme mit Elektrochemie und Fluoreszenz-Mikroskopie

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Biochemie

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Molecular Imaging Transplantierte Muscle Progenitor Zielzellen

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Medizin

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Innere Empfindlichkeit der mitochondrialen Membran gegenüber Na+ zeigt teilweise segmentierte funktionelle CoQ-Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Vorbereitung und Respirometrische Bewertung der Mitochondrien isoliert aus dem Skelettmuskelgewebe durch perkutane Nadelbiopsie Erhalten

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Bioenergetische Profil Experiment mit C2C12 Myoblasten Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Biologie

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

Modellierung Muskeldystrophie 1 in C2C12 Myoblastenzellen

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluoreszenz-basierte Messung von Calcium-Shop-Eintrag in lebenden Zellen: aus kultivierten Krebszelle zu Skelettmuskelfaser

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-Parameter-Messung der Durchlässigkeit Transition Pore Opening in isolierten Maus-Mitochondrien

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...