Questo studio è stato sostenuto da sovvenzioni da parte di grant della eccezionale scienziato dello Shandong (JQ201421) e grant da NSFC (81371226).

1. isolamento dei mitocondri
Nota: Tutte le soluzioni e apparecchiature dovrebbe essere preraffreddate a 0 - 4 ° C e tenute su ghiaccio.
<ol><li>Seme circa 3 x 106 SH-SY5Y cellule per piastra di coltura delle cellule di 10 cm. Cellule di cultura in alto-glucosio Dulbecco s modified medium di Eagle con 10% siero bovino fetale, penicillina (100 U/mL)-streptomicina (100 µ g/mL). Mantenere a 37 ° C in un incubatore contenente 5% CO2 durante la notte. Nota: almeno 1-2 x 107 SH-SY5Y cellule sono necessari per ciascun test di isolamento.</li>
	<li>Rimuovere il supporto e lavare le cellule con circa 1 mL di PBS freddo di ghiaccio in una piastra di coltura cellulare di 10cm 3 volte.</li>
	<li>Aggiungere nuovo 1 mL di PBS freddo ghiaccio nella piastra di coltura cellulare di 10cm e raschiare le celle aderenti in una nuova provetta da 1,5 mL. Centrifuga a 800 x g per 10 min a 4 ° C.</li>
	<li>Durante la centrifugazione, preparare il tampone di isolamento mitocondriale di 5 mL (250 mM saccarosio, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazina ethanesulfonic acido), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA) completato con 50 µ l di brodo di inibitori di proteasi soluzione (una concentrazione di 100 x; 1 compressa EDTA-libero dissolto in 500 µ l di ddH2O).</li>
	<li>Rimuovere il supernatante e risospendere il pellet con 1 mL di tampone di isolamento mitocondriale. Incubare la provetta da 1,5 mL in ghiaccio per 30 min.</li>
	<li>Lisare le cellule sospese con un omogeneizzatore di vetro (15-25 colpi) su e giù manualmente sul ghiaccio. Nota: Assicurarsi che non ci sono bolle quando le cellule sono omogeneizzate. Contare le cellule intatte e nuclei scoperti da microscopio visivamente e confermare che > rottura cellulare 90% si è verificato.</li>
	<li>Trasferire l'omogeneizzato in una provetta da 1,5 mL e centrifugare a 1.000 x g per 5 min a 4 ° C per rimuovere i detriti (nuclei e restanti cellule intatte).</li>
	<li>Trasferire il surnatante in una nuova provetta da 1,5 mL e centrifugare a 1.000 x g per 5 min 4 ° C.</li>
	<li>Trasferire il surnatante in una nuova provetta e centrifugare a 14.000 x g per 15 min 4 ° C.</li>
	<li>Rimuovere il supernatante e risospendere il pellet contenente i mitocondri con 1 mL di tampone di isolamento mitocondriale.</li>
	<li>Centrifugare la soluzione per 15 min a 4 ° C di 14.000 x g per precipitare i mitocondri.</li>
	<li>Il pellet di mitocondri risultante deve essere tenuto sul ghiaccio e risospesi in 50-100 media KCl μL (125 mM KCl, 2 mM K2HPO4, 1mm MgCl2, 20mm HEPES, 5mm glutammato, malate di 5 mM e 2 rotenone μM, pH 7.4) secondo il volume di pellet prima qualsiasi culo Ay.</li>
	<li>Utilizzare 5 μL di soluzione di mitocondriale per misurare la concentrazione di proteina mitocondriale di test standard di BCA, OD562 di misura utilizzando un lettore di piastra13.</li>
</ol>2. la determinazione della capacità di ritenzione di mitocondriale di Ca2 +
<ol><li>Preparare il supporto di KCl e aggiungere il Ca2 +-associazione tintura fluorescente verde a una concentrazione finale di 0,5 μM prima dell'esperimento.</li>
	<li>Trasferire 1 mL di mitocondri isolati (0,4 mg/mL) in media di KCl che contengono 0,5 μM Ca2 +-associazione tintura fluorescente verde in ogni piastra a 6 pozzetti bene.</li>
	<li>Incubare i mitocondri e il Ca2 +-associazione tintura fluorescente verde nel piatto 6 pozzetti a temperatura ambiente al riparo dalla luce ambiente per 1 min. aggiungere 4 aliquote del μL di un soluzione di CaCl2 (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2 da 20 mM 20 mM HEPES, 5mm glutammato, malate di 5 mM, pH 7.4) per ogni piastra a 6 pozzetti pozzetto usando l'automatico dispensa impostazione di introdurre 200 nmol proteina mitocondriale di Ca2 +/mg.</li>
	<li>Utilizzare uno spettrometro di fluorescenza per monitorare i cambiamenti di fluorescenza ogni 3 s per 2 min con una lunghezza d'onda di eccitazione di 506 nm e una lunghezza d'onda di emissione di 531 nm. La piastra è dotata di agitazione a 600 rpm per 3 s tra letture controllato dal software (Figura 1).</li>
	<li>Aggiungere un'aliquota di ulteriori 4 μL di soluzione di 20 mM CaCl2 in ciascun pozzetto e monitorare i cambiamenti di fluorescenza ogni 3 s per 2 min. Ripetere questo passaggio finché la fluorescenza aumenta con il tempo. Nota: I mitocondri sono sfidati con l'aggiunto Ca2 + indicato dalla registrazione maggiore fluorescenza. Quando si apre il mPTP, la fluorescenza aumenta con il tempo.</li>
	<li>Interpretare la quantità totale di Ca2 + aggiunte fino a quando il mPTP apre come la capacità di ritenzione di Ca2 + .</li>
</ol>3. determinazione del Ca 2 +-indotto gonfiamento mitocondriale
<ol><li>Preparare il supporto di KCl prima dell'esperimento.<ol>
<li>Trasferire 1 mL di mitocondri isolati (0,4 mg/mL) in media di KCl in ogni piastra a 6 pozzetti bene.</li>
	</ol>
</li>
	<li>Aggiungere 10 μL di 20 mM CaCl2 soluzione acquosa (500 nmol/mg di proteina mitocondriale) nella proteina mitocondriale di innescare gonfiamento mitocondriale.</li>
	<li>Registrare la diminuzione di assorbanza a 540 nm ogni 3 s su un lettore di micropiastre controllato dal software per 10 min (Figura 2). Le modifiche di fluorescenza sono causate da un afflusso di soluti attraverso la membrana mitocondriale interna. Nota: La diminuzione assorbanza a 540 nm corrisponde ai mitocondri gonfiati. Se il lettore non ha rispettato una diminuzione di assorbanza, un più lungo intervallo di lettura o di tempo di lettura può essere adottato.</li>
</ol>

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	<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. 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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. sole sotto la supervisione degli esperimenti. Wei Li eseguito gli esperimenti. Chen Zhang e X. Sun ha scritto il libro.

Qui, abbiamo descritto un protocollo semplice ed efficace per il mitocondriale Ca2 + ritenzione capacità dosaggio e Ca2 +-attivato dosaggio gonfiamento mitocondriale.
Per l'isolamento mitocondriale, assicurarsi che tutti i materiali e i tubi sono sul ghiaccio, soprattutto quando le cellule di omogeneizzazione. 0,25% tripsina-EDTA può essere utilizzato anche per staccare le cellule dal piatto per 5 min a 37 ° C. Dopo 15-25 colpi, è necessario osservare la membrana dell...

I mitocondri sono i principali organi cellulari per produrre quasi il 95% del trifosfato di adenosina usato nelle cellule dei mammiferi di fosforilazione ossidativa. È stato riferito che la concentrazione micromolar sequestrata di Ca2 + dai mitocondri, la presenza di ADP e fosfato inorganico può essere utilizzato per fosforilare ADP a sintesi ATP1. Quando la concentrazione di Ca citosolico2 + supera una soglia, i mitocondri possono l'assorbimento di Ca2 + rapidamente e di efflusso e lentamente. Così, i mitocondri di funzionamento possono afflusso l'aumento citosolico Ca2 +. Irrilevante per la partecipazione nella fosforilazione ossidativa, il mitocondriale Ca2 + anche partecipare i segnali di calcio citosolico e attivare il meccanismo apoptotico mitocondriale inducendo l'apertura della mPTP nella parte interna mitocondriale membrana2. È stato ampiamente riconosciuto che l'elevazione anormale del intracellulare di Ca2 + può indurre mitocondriale gonfiamento voluminoso aprendo il mPTP3. Così, questo protocollo ha lo scopo di valutare la funzione mitocondriale di quantificare la capacità di ritenzione mitocondriale Ca2 + e Ca2 +-innescato gonfiamento mitocondriale.
Capacità di ritenzione di CA2 + è la misura della capacità dei mitocondri di calcio citosolico di assorbimento. I mitocondri possono buffer il calcio libero citosolico e regolano i processi cellulari di calcio-dipendente dall'assorbimento di calcio sotto forma di precipitati inattivi. Capacità di ritenzione alterata Ca2 + si verifica nei fenomeni di stress associati a limitazione di energia e anche neurodegenerative malattie4,5. Mitocondri isolati o cellule della digitonina-permeabilized possono essere utilizzate per identificare la capacità di ritenzione di Ca2 + , e l'elevata capacità di accumulare Ca2 + di mitocondri isolati con il glutammato additonal e malate non avviene mediante il isolamento mitocondriale procedura6. Una quantità di Estratto titolata di digitonina deve essere utilizzata per permeabilize le membrane del plasma di diversi tipi di cellule. Il sale di hexapotassium di Ca2 +-associazione tintura fluorescente verde, Ca2 +-indicatore luce-eccitabile visibile cellula-impermeant sensibile, è stato ampiamente usato7. CA2 +-associazione verde colorante fluorescente è un indicatore di bassa affinità e utilizzati per dimostrare che intracellulare Ca2 + le concentrazioni libere di continuare ad aumentare durante prolungato di stimolazioni (5 min)8. Questo metodo era meno sensibile che la tecnica di filtro radioattivo, ma è stato notevolmente semplificato. Ulteriori Ca2 + eleva fluorescenza verde di calcio e l'assorbimento di Ca2 + dai mitocondri ritorna la fluorescenza ai valori basali. Fino a quando i mitocondri non è riuscito a assorbimento extramitochondrial Ca2 + 9, sono state fatte aggiunte sequenziale di Ca2 + . Un lettore di micropiastre di fluorescenza può essere utilizzato per segnalare continuamente la fluorescenza verde di calcio.
Dopo Ca2 + accumulo, mitocondri depolarizzarono, scaricata Ca2 + nel terreno e ha cominciato a gonfiarsi. CA2 +-innescato mitocondri gonfiore viene utilizzato per indicare l'apertura di mPTP. La microscopia elettronica e la diminuzione di luce assorbanza a 540 nm può essere usato per misurare il Ca2 +-innescato mitocondriale gonfiore10,11. Volume di mitocondri può essere determinata direttamente da angolo in avanti dispersione della luce12, dove diminuisce di assorbanza riflettono passiva gonfiore della matrice mitocondriale.
Qui, vi illustriamo la metodologia per esaminare la capacità di ritenzione mitocondriale Ca2 + e Ca2 +-innescato gonfiamento mitocondriale in mitocondri isolati da cellule SH-SY5Y.

<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

La produzione di ATP da fosforilazione ossidativa è la funzione primaria dei mitocondri. I mitocondri negli eucarioti superiori partecipano anche Ca citosolico2 + buffer, e la produzione di ATP in mitocondriale può essere mediata da intramitochondrial Ca2 + concentrazione libera. Capacità di ritenzione di CA2 + può essere considerata come la capacità dei mitocondri di trattenere calcio nella matrice mitocondriale. Accumulato intracellulare Ca2 + conduce alla permeabilità della membrana mitocondriale interna, definito l'apertura del poro di transizione di permeabilità mitocondriale (mPTP), che conduce alla perdita di molecole con un molecolare peso meno di 1,5 kDa. CA2 +-innescato mitocondri gonfiore viene utilizzato per indicare l'apertura di mPTP. Qui, descriviamo due saggi per esaminare la capacità di ritenzione di Ca2 + e Ca2 +-innescato gonfiamento mitocondriale in mitocondri isolati. Dopo determinate quantità di Ca2 + vengono aggiunti, tutti i passaggi possono essere completati in un giorno e registrati da un lettore di micropiastre. Così, queste due analisi semplice ed efficace possono essere adottate per valutare il Ca2 +-relative funzioni mitocondriali.

Rappresentante risultati di Ca2 + capacità di ritenzione: Risultati sono stati espressi come valori di fluorescenza. Con gli ulteriori impulsi del Ca2 + (200 proteina mitocondriale nmols/mg), la fluorescenza aumentata del doppio di sopra della linea di base con l'apertura di mPTP. 5 μM Bongkrekate (BKA), un inibitore di Ca2 +-indotto mPTP apertura, o 1 μM atractyloside (ATR), un attivatore di Ca2 +-indotto mPTP apertura, sono stat...

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

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

Mitocondriale di Ca2 + ritenzione capacità Assay e Ca2 +-innescato gonfiamento mitocondriale Assay

Questo protocollo ha lo scopo di descrivere un metodo per esaminare la capacità di ritenzione di Ca2 + e Ca2 +- innescato mitocondriale gonfiore di mitocondri isolati di SH-SY5Y cellule passo-passo.

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

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Neuroscience

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

11.1K Views.  Qilu Hospital of Shandong University. Questo protocollo ha lo scopo di descrivere un metodo per esaminare la capacità di ritenzione di Ca2 + e Ca2 +- innescato mitocondriale gonfiore di mitocondri isolati di SH-SY5Y cellule passo-passo.

Video: 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.

Visualizzazione della replicazione del DNA mitocondriale in singole celle da amplificazione del segnale EdU

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

Ricerca

Neuroscienze

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.

Neuromodulazione e mitocondriale di trasporto: Imaging Live in neuroni dell&#39;ippocampo oltre durate a lungo

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.

Determinazione della mitocondriale Oxygen Species potenziale di membrana e reattiva in neuroni corticali di ratto dal vivo

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.

Analisi di afflusso del calcio mitocondriale in mitocondri isolati e cellule coltivate

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

Biochimica

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.

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

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

Biologia

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-parametro di misura della apertura dei pori di transizione di permeabilità nel Cuore mouse mitocondri isolati

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.

Misurazione simultanea di mitocondriale calcio e mitocondriale potenziale di membrana in cellule vive di Microscopia fluorescente

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.

Saggio dell'afflusso di calcio per misurare l'assorbimento mitocondriale di calcio nelle cellule in coltura

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

Saggio di captazione del calcio mitocondriale: un metodo basato su lettore di piastre per misurare l'assorbimento di calcio nei mitocondri isolati

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)
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Mitocondriale di Ca^{2 +} ritenzione capacità Assay e Ca^{2 +}-innescato gonfiamento mitocondriale Assay

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