Diese Studie wurde unterstützt durch Zuschüsse von Grant der herausragende Wissenschaftler von Shandong (JQ201421) und Zuschuss von NSFC (81371226).

1. Isolation von Mitochondrien
Hinweis: Alle Lösungen und Geräte sollten auf 0 - 4 ° C vorgekühlt und auf Eis gehalten werden.
<ol><li>Samen etwa 3 x 106 SH-SY5Y Zellen pro 10 cm Zelle Kulturschale. Zellkulturen in hoch-Glukose Dulbecco modifizierten Eagle Medium mit 10 % fötalen Rinderserum, Penicillin (100 U/mL)-Streptomycin (100 µg/mL). Bei 37 ° C in einem Inkubator mit 5 % CO2 über Nacht behalten. Hinweis: mindestens 1-2 x 107 SH-SY5Y Zellen für jedes Assay Isolierung erforderlich sind.</li>
	<li>Entfernen Sie das Medium und waschen Sie die Zellen mit etwa 1 mL Eis kaltem PBS in einer Kulturschale 10 cm Zelle 3 Mal zu.</li>
	<li>Fügen Sie neue 1 mL Eis kaltem PBS in 10 cm Zelle Kulturschale und kratzen Sie die adhärenten Zellen in eine neue 1,5 mL Tube. Zentrifuge bei 800 X g für 10 min bei 4 ° C.</li>
	<li>Während der Zentrifugation bereiten Sie 5 mL mitochondriale Isolierung Puffer (250 mM Saccharose, 10 mM KCl, 10 mM HEPES (4-(2-hydroxyethyl)-1-Piperazin-Ethanesulfonic Säure), 1,5 mM MgCl2, 1 mM EDTA) ergänzt mit 50 μL der Protease-Inhibitoren Lager Lösung (100 X Konzentration; 1 EDTA-freie Tablette aufgelöst in 500 µL DdH2O).</li>
	<li>Entfernen des Überstands und Aufschwemmen der Pellet mit 1 mL der mitochondrialen Isolierung Puffer. Inkubieren der 1,5 mL Tube für 30 min auf Eis.</li>
	<li>Lösen Sie die suspendierten Zellen mit einer Glas-Homogenisator (15-25 Schläge) rauf und runter manuell auf Eis. Hinweis: Stellen Sie sicher, dass es gibt keine Luftblasen, wenn Zellen homogenisiert werden. Zählen die intakte Zellen und entblößten Kerne von Mikroskop optisch und bestätigen, dass > 90 % Zelle Bruch aufgetreten.</li>
	<li>Übertragen Sie das Homogenat in einem 1,5 mL-Tube und Zentrifuge bei 1.000 x g für 5 min bei 4 ° C, die Ablagerungen (Kerne und verbleibenden intakten Zellen) zu entfernen.</li>
	<li>Übertragen den überstand in ein neues 1,5 mL-Tube und Zentrifuge bei 1.000 x g für 5 min 4 ° C.</li>
	<li>Den überstand zu übertragen, zu einem neuen Schlauch und Zentrifuge bei 14.000 x g für 15 min 4 ° C.</li>
	<li>Entfernen des Überstands und das Pellet mit Mitochondrien mit 1 mL der mitochondrialen Isolierung Puffer Aufschwemmen.</li>
	<li>Zentrifugieren Sie die Lösung für 15 min bei 14.000 x g 4 ° C um die Mitochondrien auszufällen.</li>
	<li>Das daraus resultierende Mitochondrien Pellet muss ständig auf Eis und Nukleinsäuretablette in 50-100 μl KCl Medien (125 mM KCl, 2 mM K2HPO4, 1 mM MgCl2, 20 mM HEPES, 5 mM Glutamat, 5 mM Malate, und 2 μM Rotenon, pH 7,4) entsprechend dem Pellet-Volumen vor jedem Arsch ay.</li>
	<li>Verwenden Sie 5 μL der mitochondrialen Lösung zur Messung der mitochondrialen Proteinkonzentration von standard BCA-Test, Messung der OD562 mit einer Platte Leser13.</li>
</ol>2. Bestimmung der mitochondrialen Ca2 + Rückhaltevermögen
<ol><li>Bereiten Sie die KCl-Medien und fügen Sie die Ca2 +-Bindung grünen fluoreszierenden Farbstoff, eine Endkonzentration von 0,5 μM vor dem Experiment.</li>
	<li>1 mL der isolierten Mitochondrien (0,4 mg/mL) in KCl Medien mit 0,5 μM Ca2 +Transfer-grünen fluoreszierenden Farbstoff in jedem 6-Well-Platte auch verbindlich.</li>
	<li>Inkubieren Sie die Mitochondrien und die Ca2 +-Bindung grünen fluoreszierenden Farbstoff in der 6-Well-Platte bei Raumtemperatur geschützt vom Umgebungslicht für 1 min. hinzufügen 4 μL Aliquote eines 20 mm CaCl2 -Lösung (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2 20 mM HEPES, Glutamat 5 mM, 5 mM-Malat, pH 7,4) zu jedem 6-Well-Platte verzichten nun mit Hilfe den automatischen Einstellung 200 Nmol Ca2 +/mg mitochondrialen Proteins einzuführen.</li>
	<li>Verwenden Sie ein Fluoreszenz-Spektrometer, um die Fluoreszenz-Änderungen zu überwachen alle 3 s 2 min mit einer Wellenlänge von Erregung von 506 nm und einer Emissionswellenlänge von 531 nm. Die Platte ist ausgestattet mit schütteln bei 600 u/min für 3 s zwischen den Lesungen von Software (Abbildung 1) gesteuert.</li>
	<li>Fügen Sie eine zusätzliche 4 μL aliquoten 20 mM CaCl2 -Lösung in jede Vertiefung und überwachen den Fluoreszenz Änderungen alle 3 s für 2 min. Wiederholen Sie diesen Schritt, bis die Fluoreszenz mit der Zeit zunimmt. Hinweis: Mitochondrien sind mit der zusätzlichen Ca2 + gekennzeichnet durch die erhöhte Aufnahme Fluoreszenz gefordert. Wenn die mPTP geöffnet wird, steigt die Fluoreszenz mit der Zeit.</li>
	<li>Interpretieren Sie den Gesamtbetrag von Ca2 + hinzugefügt, bis die mPTP als Ca2 + Rückhaltevermögen öffnet.</li>
</ol>3. Ermittlung der Ca 2 +-induzierten mitochondrialen Schwellung
<ol><li>Bereiten Sie die KCl-Medien vor dem Experiment.<ol>
<li>Übertragen Sie 1 mL isolierte Mitochondrien (0,4 mg/mL) in KCl Medien gut in jedes 6-Well-Platte.</li>
	</ol>
</li>
	<li>Fügen Sie 10 μl 20 mm CaCl2 wässrige Lösung (500 Nmol/mg mitochondrialen Proteins) in mitochondrialen Proteins mitochondriale Schwellung auslösen.</li>
	<li>Aufzeichnen den Rückgang der Extinktion bei 540 nm alle 3 s auf einer Mikrotestplatte Reader Software für 10 min (Abbildung 2) gesteuert. Die Fluoreszenz-Änderungen werden durch den Zuzug von gelösten Stoffen durch die innere mitochondriale Membran verursacht. Hinweis: Die verminderte Absorption bei 540 nm entspricht den geschwollenen Mitochondrien. Wenn der Leser keine Abnahme der Absorption beachten, kann eine längere Intervall lesen oder Lesezeit übernommen werden.</li>
</ol>

<ol>
	<li>Rossi, C. S., Lehninger, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stoichiometry+of+respiratory+stimulation,+accumulation+of+Ca+++and+phosphate,+and+oxidative+phosphorylation+in+rat+liver+mitochondria.">Stoichiometry of respiratory stimulation, accumulation of Ca++ and phosphate, and oxidative phosphorylation in rat liver mitochondria.</a> J Biol Chem. 239 (11), 3971-3980 (1964).</li><li>Gunter, T. E., Buntinas, L., Sparagna, G., Eliseev, R., Gunter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+transport:+mechanisms+and+functions.">Mitochondrial calcium transport: mechanisms and functions.</a> Cell Calcium. 28 (5-6), 285-296 (2000).</li><li>Crofts, A. R., Chappell, J. 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Reversal of Calcium Ion-Induced Swelling.</a> Biochem J. 95, 387-392 (1965).</li><li>Wojda, U., Salinska, E., Kuznicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+ions+in+neuronal+degeneration.">Calcium ions in neuronal degeneration.</a> IUBMB Life. 60 (9), 575-590 (2008).</li><li>Celsi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria,+calcium+and+cell+death:+a+deadly+triad+in+neurodegeneration.">Mitochondria, calcium and cell death: a deadly triad in neurodegeneration.</a> Biochim Biophys Acta. 1787 (5), 335-344 (2009).</li><li>Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E., Fiskum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+potentiates+the+maximal+calcium+uptake+capacity+of+neural+cell+mitochondria.">Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.</a> Proc Natl Acad Sci U S A. 93 (18), 9893-9898 (1996).</li><li>Rajdev, S., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+green-5N,+a+novel+fluorescent+probe+for+monitoring+high+intracellular+free+Ca2++concentrations+associated+with+glutamate+excitotoxicity+in+cultured+rat+brain+neurons.">Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons.</a> Neurosci Lett. 162 (1-2), 149-152 (1993).</li><li>Stout, A. K., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+calcium+indicators+underestimate+increases+in+intracellular+calcium+concentrations+associated+with+excitotoxic+glutamate+stimulations.">High-affinity calcium indicators underestimate increases in intracellular calcium concentrations associated with excitotoxic glutamate stimulations.</a> Neuroscience. 89 (1), 91-100 (1999).</li><li>Bambrick, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin+A+increases+mitochondrial+calcium+uptake+capacity+in+cortical+astrocytes+but+not+cerebellar+granule+neurons.">Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.</a> J Bioenerg Biomembr. 38 (1), 43-47 (2006).</li><li>Zhao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-permeable+peptide+antioxidants+targeted+to+inner+mitochondrial+membrane+inhibit+mitochondrial+swelling,+oxidative+cell+death,+and+reperfusion+injury.">Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury.</a> J Biol Chem. 279 (33), 34682-34690 (2004).</li><li>Muriel, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+free+calcium+levels+(Rhod-2+fluorescence)+and+ultrastructural+alterations+in+neuronally+differentiated+PC12+cells+during+ceramide-dependent+cell+death.">Mitochondrial free calcium levels (Rhod-2 fluorescence) and ultrastructural alterations in neuronally differentiated PC12 cells during ceramide-dependent cell death.</a> J Comp Neurol. 426 (2), 297-315 (2000).</li><li>Allman, R., Hann, A. C., Phillips, A. P., Martin, K. L., Lloyd, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Azotobacter+vinelandii+with+correlation+of+Coulter+cell+size,+flow+cytometric+parameters,+and+ultrastructure.">Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.</a> Cytometry. 11 (7), 822-831 (1990).</li><li>Wiechelman, K. J., Braun, R. D., Fitzpatrick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+bicinchoninic+acid+protein+assay:+identification+of+the+groups+responsible+for+color+formation.">Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation.</a> Anal Biochem. 175 (1), 231-237 (1988).</li><li>Petronilli, V., Nicolli, A., Costantini, P., Colonna, R., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+permeability+transition+pore,+a+voltage-dependent+mitochondrial+channel+inhibited+by+cyclosporin+A.">Regulation of the permeability transition pore, a voltage-dependent mitochondrial channel inhibited by cyclosporin A.</a> Biochim Biophys Acta. 1187 (2), 255-259 (1994).</li><li>Pastorino, J. 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X. Sonne überwacht die Experimente. Wei Li durchgeführt die Experimente. Chen Zhang und X. Sun schrieb die Zeitung.

Wir beschrieben hier, ein einfaches und effektives Protokoll für die mitochondriale Ca2 + Retention Kapazität Assay und Ca2 +-mitochondriale Schwellung Assay ausgelöst.
Stellen Sie für die mitochondriale Isolierung sicher, dass alle Materialien und Rohre auf Eis, besonders wenn die Zellen zu homogenisieren. 0,25 % Trypsin-EDTA kann auch verwendet werden, um Zellen aus der Schale für 5 min bei 37 ° c zu lösen Nach 15-25 Schlägen ist es notwendig, die intakte Zellme...

Mitochondrien sind die zellulären Hauptorgane, fast 95 % der verwendeten in den Säugetier-Zellen durch Oxidative Phosphorylierung ATP zu produzieren. Es wird berichtet, dass die abgesonderten mikromolaren Konzentration von Ca2 + von Mitochondrien, die Anwesenheit von ADP und anorganischem Phosphat ADP zu ATP-Synthese1phosphorylieren verwendet werden können. Wenn die Konzentration der cytosolischen Ca2 + einen Schwellenwert überschreitet, Mitochondrien können Aufnahme Ca2 + schnell und Ausfluss es langsam. So können die funktionierenden Mitochondrien Zustrom der erhöhten cytosolischen Ca2 +. Irrelevant für die Teilnahme an der Oxidative Phosphorylierung, die mitochondriale Ca2 + auch an der cytosolischen kalziumsignale teilnehmen und aktivieren den mitochondrialen Apoptose-Mechanismus durch Induktion die Eröffnung der mPTP in der inneren mitochondrialen Membran-2. Es wurde allgemein anerkannt, dass abnorme Erhöhung der intrazellulären Ca2 + mitochondriale massive Schwellung hervorrufen kann, durch die Eröffnung der mPTP-3. So, dieses Protokoll zielt darauf ab, die Funktion der Mitochondrien zu bewerten, indem Sie die Quantifizierung der mitochondrialen Ca2 + Rückhaltevermögen und Ca2 +-trigged mitochondriale Schwellung.
Ca2 + Rückhaltevermögen ist das Maß für die Fähigkeit der Mitochondrien zur Aufnahme zytosolischen Kalzium. Mitochondrien können Puffer freie zytosolischen Kalzium und Kalzium-abhängigen zellulären Prozesse zu regulieren, indem Calciumaufnahme in Form von inaktiven Ausscheidungen. Beeinträchtigt Ca2 + Rückhaltevermögen tritt in Stress-Erscheinungen, die mit Energie-Begrenzung und auch Neurodegenerative Krankheiten4,5. Isolierte Mitochondrien oder Digitonin-permeabilized Zellen eingesetzt werden, um die Ca2 + Rückhaltevermögen zu identifizieren, und die erhöhte Fähigkeit zu akkumulieren Ca2 + durch isolierte Mitochondrien mit dem Additonal Glutamat und Malat erfolgt nicht durch die Mitochondriale Isolierung Verfahren6. Eine betitelten Menge an Digitonin sollte verwendet werden, um die Plasmamembranen von verschiedenen Zelltypen permeabilize. Das Hexapotassium Salz der Ca2 +-Bindung grünen fluoreszierenden Farbstoff, Ca2 +-sensible Zelle-impermeant Licht-erregbare Sichtanzeige, wurde weit verbreiteten7. Ca2 +-Bindung grün fluoreszierenden Farbstoff ist ein Low-Affinität-Indikator und verwendet, um anzuzeigen, dass intrazelluläre freie Ca2 + Konzentrationen im steigen weiter (5 min) Stimulationen8verlängert. Diese Methode war weniger empfindlich als die radioaktive Filtertechnik aber wesentlich vereinfacht. Das zusätzliche Ca2 + Kalzium grüne Fluoreszenz erhebt und die Ca2 + Aufnahme von Mitochondrien kehrt die Fluoreszenz Baseline. Fortlaufende Ergänzungen von Ca2 + wurden vorgenommen, bis die Mitochondrien zur Aufnahme Extramitochondrial Ca2 + 9nicht. Ein Fluoreszenz Mikrotestplatte Leser lässt sich regelmäßig Kalzium grüne Fluoreszenz Berichten.
Nach Ca2 + Akkumulation Mitochondrien depolarisiert, Ca2 + in das Medium abgegeben und begann anzuschwellen. Ca2 +-Mitochondrien Schwellung verwendet wird, um die mPTP Öffnung zeigen ausgelöst. Elektronenmikroskopie und die Abnahme der leichten Extinktion bei 540 nm kann verwendet werden, um die Ca2 +Messen-mitochondriale Schwellung10,11ausgelöst. Mitochondrien Volumen kann direkt durch nach vorne Winkel Lichtstreuung12, ermittelt werden, wo sinkt in die Extinktion passive Schwellung der mitochondrialen Matrix widerspiegeln.
Hier zeigen wir die Methodik zur Prüfung der mitochondrialen Ca2 + Rückhaltevermögen und Ca2 +-mitochondriale Schwellung in isolierten Mitochondrien aus SH-SY5Y Zellen ausgelöst.

<table><tbody><tr><td>SH-SY5Y cell line</td><td>ATCC (The Global Bioresource Center)</td><td>ATCC Number: CRL-2266</td><td></td></tr><tr><td>Calcium green-5N</td><td>Life Technologies</td><td>c-3737</td><td>Ca&lt;sup&gt;2+&lt;/sup&gt;-binding green fluorescent dye</td></tr><tr><td>complete protease inhibitors</td><td>Roche Molecular Biochemicals</td><td>4693116001</td><td></td></tr><tr><td>glass homogenizer</td><td>Kimble Chase</td><td>9885303002</td><td></td></tr><tr><td>glutamate</td><td>Sigma-Aldrich</td><td>RES5063G-A7</td><td></td></tr><tr><td>malate</td><td>Sigma-Aldrich</td><td>46940-U</td><td></td></tr><tr><td>rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>00457</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900050</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V900266</td><td></td></tr><tr><td>Varioskan flash instruments</td><td>Thermo Scientific</td><td>IC100E</td><td></td></tr><tr><td>Bongkrekate</td><td>Biovision</td><td>1820-100</td><td></td></tr><tr><td>atractyloside</td><td>Sigma-Aldrich</td><td>C4992</td><td></td></tr><tr><td>high-glucose Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Hyclone</td><td>SH30022</td><td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td></tr><tr><td>fetal bovine serum</td><td>Hyclone</td><td>SV30087</td><td></td></tr><tr><td>penicillin</td><td>Sigma-Aldrich</td><td>P3032</td><td></td></tr><tr><td>streptomycin</td><td>Invitrogen</td><td>11860-038</td><td></td></tr><tr><td>BCA assay kit</td><td>Thermo Scientific</td><td>NCI3225CH</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>10 cm cell culture dish</td><td>NEST</td><td>704001</td><td></td></tr></tbody></table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

Die Produktion von ATP durch Oxidative Phosphorylierung ist die primäre Funktion der Mitochondrien. Mitochondrien in den höheren Eukaryotes auch an cytosolischen Ca2 + Pufferung und die ATP-Produktion in Mitochondrien kann durch intramitochondrial frei Ca2 + Konzentration vermittelt werden. Ca2 + Rückhaltevermögen kann als die Fähigkeit der Mitochondrien, Kalzium in der mitochondrialen Matrix zu behalten. Kumulierte intrazellulären Ca2 + führt zu der Durchlässigkeit der inneren mitochondrialen Membran bezeichnet die Öffnung des mitochondrialen Permeabilität Übergang Pore (mPTP), was dazu führt, das Austreten von Molekülen mit einer molekularen Gewicht weniger als 1,5 kDa. Ca2 +-Mitochondrien Schwellung verwendet wird, um die mPTP Öffnung zeigen ausgelöst. Hier beschreiben wir zwei Tests untersuchen die Ca2 + Rückhaltevermögen und Ca2 +-mitochondriale Schwellung in isolierten Mitochondrien ausgelöst. Nach bestimmten Mengen von Ca2 + werden hinzugefügt, können alle Schritte innerhalb eines Tages abgeschlossen und von einer Mikrotestplatte Leser aufgenommen. Somit können diese zwei einfachen und effektiven Assays ergriffen werden, um die Ca2 +bewerten-mitochondriale Funktionen.

Ca2 + Rückhaltevermögen Vertreter folgendes Ergebnis: Die Ergebnisse wurden als Fluoreszenz-Werte. Mit der zusätzlichen Impulsen von Ca2 + (200 Nmols/mg mitochondrialen Proteins), die Fluoreszenz durch doppelte über dem Ausgangswert mit der Eröffnung von mPTP erhöht. 5 μM Bongkrekate (BKA), ein Inhibitor von Ca2 +-induzierte mPTP Öffnung oder 1 μM Atractyloside (ATR), Aktivator von Ca2 +-induzierte mPTP Öffnung, wurden in d...

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Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Mitochondriale Ca2 + Retention Kapazität Assay und Ca2 +-mitochondriale Schwellung Assay ausgelöst

Dieses Protokoll zielt darauf ab, beschreiben eine Methode zur Prüfung der Ca2 + Rückhaltevermögen und Ca2 +- ausgelöst mitochondriale Schwellung des isolierten Mitochondrien von SH-SY5Y Zellen Schritt für Schritt.

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

mitochondrial-ca2-retention-capacity-assay-ca2-triggered

Research

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

11.1K Aufrufe.  Qilu Hospital of Shandong University. Dieses Protokoll zielt darauf ab, beschreiben eine Methode zur Prüfung der Ca2 + Rückhaltevermögen und Ca2 +- ausgelöst mitochondriale Schwellung des isolierten Mitochondrien von SH-SY5Y Zellen Schritt für Schritt.

Serie: Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy cells1-3. One approach for monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication4. We developed a sensitive technique to label newly synthesized mtDNA in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of 5-ethynyl-2'-deoxyuridine (EdU)5-7 with a tyramide signal amplification (TSA)8 protocol to visualize mtDNA replication within subcellular compartments of neurons. EdU is superior to other thymidine analogs, such as 5-bromo-2-deoxyuridine (BrdU), because the initial click reaction to label EdU5-7 does not require the harsh acid treatments or enzyme digests that are required for exposing the BrdU epitope. The milder labeling of EdU allows for direct comparison of its incorporation with other cellular markers9-10. The ability to visualize and quantify mtDNA biogenesis provides an essential tool for investigating the mechanisms used to regulate mitochondrial biogenesis and would provide insight into the pathogenesis associated with drug toxicity, aging, cancer and neurodegenerative diseases. Our technique is applicable to sensory neurons as well as other cell types. The use of this technique to measure mtDNA biogenesis has significant implications in furthering the understanding of both normal cellular physiology as well as impaired disease states.

We developed a sensitive technique to label newly synthesized mitochondrial DNA (mtDNA) in individual cells in order to study mtDNA biogenesis. The technique combines the incorporation of EdU together with a tyramide signal amplification (TSA) protocol to visualize mtDNA replication within subcellular compartments of neurons.

Visualisierung der mitochondrialen DNA-Replikation in einzelnen Zellen von Edu Signalverstärkung

Mitochondria are key regulators of cellular energy and mitochondrial biogenesis is an essential component of regulating mitochondria numbers in healthy ...

Forschung

Neurowissenschaften

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulation und Mitochondriale Transport: Live Imaging in Hippocampus-Neuronen über lange Zeiträume

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

Bestimmung des mitochondrialen Membranpotentials und Reactive Oxygen Species in Live Rat kortikalen Neuronen

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Analysen der mitochondrialen Calcium Zustrom in isolierten Mitochondrien und kultivierten Zellen

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Biochemie

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Überwachung dynamischen Veränderungen in der mitochondrialen Kalzium während der Apoptose mit einem genetisch kodierten Kalzium-Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Biologie

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 ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Simultane Messung von Mitochondriale Calcium und mitochondrialen Membranpotentials in lebenden Zellen durch Fluoreszenzmikroskopie

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

Calcium-Influx-Assay zur Messung der mitochondrialen Calciumaufnahme in kultivierten Zellen

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Mitochondrial Calcium Uptake Assay: Eine auf Plattenlesern basierende Methode zur Messung der Kalziumaufnahme in isolierten Mitochondrien

Biochemical assays

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
This video ...

Mitochondriale Ca^{2 +} Retention Kapazität Assay und Ca^{2 +}-mitochondriale Schwellung Assay ausgelöst

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