Ringraziamo il Centro Tufts for Neuroscience Research, P30 NS047243 (Jackson), i mitocondri Affinity Collaborative Research (mtARC) supportate dal Centro Interdisciplinare Evans per la Ricerca Biomedica presso la Boston University Medical Campus, Medicina Link Corporation, e Zeiss per sostenere questo lavoro.

1. Immagine Piastra Preparazione <ol><li> Cultura INS1 cellule RPMI supporti contenenti standard del 10% siero fetale bovino, 1% di penicillina-streptomicina, 2 mM L-glutammina, 50 pM 2-mercaptoetanolo, 5 mM NaHCO 3, 2 mM di HEPES, 2 mM acido piruvico, e 11 mM di glucosio al 80% di confluenza. </li><li> Tripsinizzare le colture cellulari INS1 con 0,05% tripsina e li piastra su poli-D-lisina coprioggetto rivestiti con fondo fosfori confluenza (30-40%). </li><li> Lasciare per piatti di raggiungere confluenza del 60-80% (circa 2 giorni) e aggiungere matrice mitocondriale mirati (COXVIII) adenovirale PA-GFP per 24 ore (MOI = 5). Media Exchange e consentono alle cellule di crescere per altri 2 giorni prima di imaging. </li><li> Il giorno di immagini, aggiungere TMRE 7-15 nM per le piastre di imaging, ed equilibrare per almeno 45 min. </li><li> Durante questo tempo sul giro l&#39;incubatore (fase superiore incubatore è stato usato qui) e permettono il microscopio equilibrare a 37 ° C per circa 1 ora. Accendere il 5% di CO 2. </li&gt; </ol> 2. Parametri di imaging per la Zeiss LSM 710 microscopio confocale <ol><li> Dopo che il microscopio ha equilibrato, nella scheda di acquisizione, fare clic su &quot;Strumenti manuale mostrano&quot;, aprire l&#39;imaging istituito pannello e scegliere &quot;la modalità del canale&quot; e passare rintracciare ogni &quot;frame&quot;. </li><li> Nel pannello percorso della luce, scegliete LSM e la modalità del canale. Lavorare da l&#39;icona del campione verso l&#39;alto, selezionare &quot;posteriore&quot;, MBS 690 + e MBS 488. Nella parte inferiore del pannello, scegliere &quot;T-PMT&quot; per consentire la visualizzazione del canale in campo chiaro o DIC. </li><li> Terminare la regolazione dei parametri di Light Path impostando l&#39;intervallo per il colorante GFP a 490-540 nm e 580-700 nm per il colorante TMRE. </li><li> Nella modalità di acquisizione, utilizzare un 512 × 512 pixel campo di scansione, 4x media, e scegliere un fattore di zoom (che si consiglia di mantenere coerente per scopi di taratura). </li></ol> 3. Ottimizzazione dei parametri di imaging <ol><li> Utilizzando il piano apocromatico 100x (1,4 NA) lente dell&#39;obiettivo, focnoi le vostre cellule con la luce alogena non, luce fluorescente, per proteggere i mitocondri da fototossicità. </li><li> Schermo per PA-GFP cellule che esprimono con un foro di massima apertura e la scansione per individuare le cellule che sono più luminosi verdi. </li><li> Trovare la minima potenza del 2-fotone laser necessaria per attivare il PA-GFP e ottimizzare i parametri di imaging garantire che il segnale non saturare il rivelatore. Controllare che il PA-GFP colocalizza segnale con il segnale TMRE per pochi z-serie. Perdita del segnale TMRE indica depolarizzazione mitocondriale o fototossicità, e cellule che presenta queste caratteristiche non devono essere utilizzati per l&#39;analisi. </li><li> Trova un PA-GFP cellula che esprime e specificare un fattore di zoom. </li><li> Impostare la serie Z gamma di raccogliere 6 fette (questo intervallo sarà necessario per soddisfare tutte le 10 celle a meno che una specifica z-messa a fuoco è impostata a ciascuna posizione). </li><li> Salvare il metodo di imaging per ogni cella (cella 1, cella 2 ecc.) </li></ol> 4. Regolare il AutomatePorzione d del programma e designare i 10 celle si seguirà per il test 1 Fusion Hour mitocondriale <ol><li> Sul pannello di sinistra della finestra Multitime, selezionare il pannello &quot;risparmio&quot;, e specificare il percorso dove i file verranno salvati. </li><li> Nel pannello &quot;Acquisizione&quot;, caricare il metodo di acquisizione salvato per cella 1 nella configurazione di scansione e selezionare la casella pila z. Scegli anche &quot;segnato Z-middle dello stack z&quot;. </li><li> Nel pannello &quot;blocchi&quot;, selezionare &quot;blocco unico in ogni sede&quot;. Fare clic su &quot;Aggiungi&quot; per blocchi di ogni intervallo da misurare. </li><li> Nel pannello &quot;timing&quot;, selezionare &quot;attendere l&#39;intervallo&quot; e tipo &quot;0&quot; in &quot;intervallo di attesa prima del blocco alla prima posizione solo&quot;. Blocchi 2-4 avrà &quot;15 minuti&quot; in questa sezione. </li><li> Nel pannello posizione, scegliere &quot;spostare lo stato attivo per caricare posizione tra percorsi&quot; e &quot;carico scansione config quando &#39;passare alla loc&#39; o &#39;Avanti loc&#39; cliccato. Sotto&quot; modifica posizioni &quot;scegliere&quot; cancella tutto &quot;e poi selezionare&quot; multiple posizioni motorizzata stage &quot;. </li><li> Under il pannello di &quot;Bleach&quot;, selezionare la casella &quot;candeggina&quot;, e designa il file di configurazione in &quot;config&quot; menu a discesa, come indicato nella finestra del software principale. Quindi, nella finestra &quot;bleaching&quot;, salvare il metodo appropriato fotoattivazione. Nel pannello regioni, scegliere il ROI per questa cella particolare, e aggiungere il ROI al Multitime scegliendo &quot;add area corrente alla lista ROI&quot; finestra. Assicurarsi di selezionare la stessa posizione nella &quot;ROI&quot; menu a discesa. </li></ol> Per la tempistica per funzionare correttamente e per ogni cella di avere un intervallo di 15 minuti, due metodi devono essere salvate. Nell&#39;elenco dei blocchi, il primo avrà la configurazione &quot;reale&quot; fotoattivazione, e il resto avrà una configurazione &quot;finto&quot; che non utilizza il laser a due fotoni. Il primo blocco sarà anche l&#39;unico blocco che non ha un ritardo (BKIntv = 0). Pertanto, il metodo inizia con una scansione di base, seguito da una scansione fotoattivazione. Il resto dei blocchi hanno un secondo BkIntv 900, ead ogni tempo ci sono due scansioni così come al tempo 0 sec, per mantenere la coerenza tempi. <ol start="7"><li> Per ciascuna delle celle 10 da seguire nel tempo, eseguire questa sequenza: Trova un PAGFP esprime cellule salvare il suo metodo di imaging Posizione pannello marcare la posizione stadio, specificare metodo di imaging Principale ROI pannello scegliere una regione di interesse per essere Bleach fotoattivati ​​pannello salvare ROI specifico e caricare anche nella casella ROI: Cancellare il ROI e ripristinare lo zoom scansione a 1 Trova la cella successiva e impostare lo zoom Ripetere la procedura per i prossimi 9 celle </li></ol> Controllare che ogni posizione stadio e tutti i blocchi hanno il metodo appropriato imaging (scan configurazione) associato. Assicurarsi inoltre che il primo blocco in ogni sede corrisponde al metodo fotoattivazione appropriato, mentre il resto contiene un metodo &quot;mock&quot;. Infine, selezionare &quot;Esegui&quot;. 5. Analizzare la PA-GFP intensità di segnale <ol><li&gt; Per le cellule in cui il fotoattivabile-GFP segnale colocalizza TMRE con il segnale rosso, sottrarre il fondo nelle immagini PAGFP (qui usiamo Metamorph). </li><li> Poi esportare i dati in Excel e calcolare l&#39;intensità media per z-stack in ogni punto. Eliminare sezioni ottiche che non hanno segnale. </li><li> Misurare la diluizione segnale in ciascun z-stack calcolando la percentuale del segnale originale fotoattivati. </li></ol> Ogni punto i dati vengono analizzati e registrata due volte, con conseguente duplicazione Z-Stack informazioni per ogni punto temporale. Verificare che i valori di Z-Stack d&#39;accordo. Se non, ciò è indicativo di un problema (ad esempio fuoco, il movimento delle cellule). 6. Risultati rappresentativi Quando un evento di fusione si verifica tra un attivato e non attivato, PA-GFP mitocondrio, il PAGFP nella matrice mitocondriale mescola con la matrice non marcato e si diluisce su un&#39;area più grande, diminuendo il segnaleintensità (Figura 1A). Nella cella INS1, una significativa diminuzione dell&#39;intensità del segnale avviene ogni 15 minuti, fino equilibrio della fusione mitocondriale è stato raggiunto (circa 1 ora). Si noti che la cella nella Figura 1B mostra colocalizzazione quasi completa della PA-GFP e segnali TMRE. In questi saggi una concentrazione molto bassa di TMRE (15 nM) è utilizzato per indirizzare la fotoattivazione di PAGFP, e anche per monitorare la salute delle cellule. Le cellule con un&#39;abbondanza di mitocondri depolarizzata avrà colocalizzazione incompleto di PAGFP e TMRE e non dovrebbe essere analizzato in quanto questo indica sia fototossicità, o cellule in uno stato di morte. Il tempo di equilibrazione di fusione mitocondriale è solitamente 1 ora in INS1 cellule, quando ~ 15% del volume mitocondriale è attivato. A volte, anche se una piccola area illuminata, la maggior parte dei mitocondri diventano fotoattivato a causa di essere altamente rete, nel qual caso fusione ulteriore è difficile da rilevare. Altri tipi di cellule possono presentare diversi tempi di equilibrazione e dovrebbe essere testato ad intervalli più brevi e per un periodo più lungo per caratterizzare le dinamiche mitocondriali. Per inibire la fusione mitocondriale, le cellule possono essere collocato all&#39;interno di un ambiente lipotoxic. È stato precedentemente dimostrato che 0,4 mM palmitato frammenti mitocondri, e inibisce fusione mitocondriale 9. Questo effetto può essere visto in Figura 2, dove vengono frammentati mitocondri, ma l&#39;intensità del segnale della mtPAGFP non cambia quanto in condizioni normali (figura 1). Pertanto, la diluizione del PA-GFP segnale è probabilmente dovuto alla fusione mitocondriale anziché fissione. In altri tipi di cellule, si consiglia di utilizzare altri metodi noti per indurre frammentazione mitocondriale, come silenziamento OPA1 che è necessaria per la fusione mitocondriale 14. <img alt="Figura 1" src="/files/ftp_upload/3991/3991fig1.jpg" /> Figura 1. </strong&gt; diluizione tipica del PA-GFP segnale dopo fotoattivazione durante un saggio di fusione mitocondriale. A. PA-GFP diventa progressivamente dimmer causa di eventi di fusione mitocondriali che portano alla diluizione della proteina su un&#39;area maggiore come si vede in queste immagini proiettate di 6 sezioni ottiche quantificato ogni 15 minuti. B. TMRE con PA-GFP mostra che i mitocondri sono attivi e non depolarizzato. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/3991/3991fig2.jpg" /> Figura 2. Fusione mitocondriale inibito con palmitato 0,4 mM diminuisce la diluizione della PA-GFP. Proiezioni A. 6 sezioni ottiche che mostrano i mitocondri a breve con una intensità di segnale immutabile nel tempo. Colocalizzazione B. PA-GFP con TMRE dimostra che i mitocondri non sono depolarizzata. <a href="http://www.jove.com/files/ftp_upload/3991/3991fig2large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> .

<ol>
	<li>Braschi, E., McBride, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+the+culture+of+the+Borg:+understanding+the+integration+of+mitochondrial+function+within+the+reticulum,+the+cell,+and+the+organism.">Mitochondria and the culture of the Borg: understanding the integration of mitochondrial function within the reticulum, the cell, and the organism.</a> Bioessays. 32, 958-966 (2010).</li><li>Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+dynamic+organelles+in+disease,+aging,+and+development.">Mitochondria: dynamic organelles in disease, aging, and development.</a> Cell. 125, 1241-1252 (2006).</li><li>Chan, D. C., Detmer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+and+dysfunctions+of+mitochondrial+dynamics.">Functions and dysfunctions of mitochondrial dynamics.</a> Nature Reviews Molecular Cell Biology. 8, 870-879 (2007).</li><li>Han, X. J., Tomizawa, K., Fujimura, A., Ohmori, I., Nishiki, T., Matsushita, M., Matsui, H. <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+mitochondrial+dynamics+and+neurodegenerative+diseases.">Regulation of mitochondrial dynamics and neurodegenerative diseases.</a> Acta Med Okayama. 65, 1-10 (2011).</li><li>Huang, H., Choi, S. Y., Frohman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+mitochondrial+fusion+using+Renilla+luciferase+complementation.">A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.</a> Mitochondrion. 10, 559-566 (2010).</li><li>Karbowski, M., Arnoult, D., Chen, H., Chan, D. C., Smith, C. L., Youle, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+mitochondrial+dynamics+by+photolabeling+of+individual+organelles+shows+that+mitochondrial+fusion+is+blocked+during+the+Bax+activation+phase+of+apoptosis.">Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis.</a> J. Cell. Biol. 164, 493-499 (2004).</li><li>Legros, F., Lombes, A., Frachon, P., Rojo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fusion+in+human+cells+is+efficient,+requires+the+inner+membrane+potential,+and+is+mediated+by+mitofusins.">Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins.</a> Mol. Biol. Cell. 13, 4343-4354 (2002).</li><li>Molina, A. J., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+mitochondrial+dynamics+with+photoactivatable+[corrected]+green+fluorescent+protein.">Monitoring mitochondrial dynamics with photoactivatable [corrected] green fluorescent protein.</a> Methods Enzymol. 457, 289-304 (2009).</li><li>Molina, A. J., Wikstrom, J. D., Stiles, L., Las, G., Mohamed, H., Elorza, A., Walzer, G., Twig, G., Katz, S., Corkey, B. E., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+networking+protects+beta-cells+from+nutrient-induced+apoptosis.">Mitochondrial networking protects beta-cells from nutrient-induced apoptosis.</a> Diabetes. 58, 2303-2315 (2009).</li><li>Otera, H., Mihara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+physiologic+functions+of+mitochondrial+dynamics.">Molecular mechanisms and physiologic functions of mitochondrial dynamics.</a> J. Biochem. 149, 241-251 (2011).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297, 1873-1877 (2002).</li><li>Schauss, A. C., Huang, H., Choi, S. Y., Xu, L., Soubeyrand, S., Bilodeau, P., Zunino, R., Rippstein, P., Frohman, M. A., McBride, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+cell-free+mitochondrial+fusion+assay+amenable+for+high-throughput+screenings+of+fusion+modulators.">A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators.</a> BMC Biol. 8, 100 (2011).</li><li>Sheridan, C., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fission/fusion+dynamics+and+apoptosis.">Mitochondrial fission/fusion dynamics and apoptosis.</a> Mitochondrion. 10, 640-648 (2010).</li><li>Twig, G., Elorza, A., Molina, A. J., Mohamed, H., Wikstrom, J. D., Walzer, G., Stiles, L., Haigh, S. E., Katz, S., Las, G., Alroy, J., Wu, M., Py, B. F., Yuan, J., Deeney, J. T., Corkey, B. E., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fission+and+selective+fusion+govern+mitochondrial+segregation+and+elimination+by+autophagy.">Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.</a> EMBO J. 27, 433-446 (2008).</li><li>Twig, G., Graf, S. A., Wikstrom, J. D., Mohamed, H., Haigh, S. E., Elorza, A., Deutsch, M., Zurgil, N., Reynolds, N., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tagging+and+tracking+individual+networks+within+a+complex+mitochondrial+web+with+photoactivatable+GFP.">Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP.</a> Am. J. Physiol. Cell Physiol. 291, C176-C184 (2006).</li><li>Twig, G., Hyde, B., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fusion,+fission+and+autophagy+as+a+quality+control+axis:+the+bioenergetic+view.">Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view.</a> Biochim. Biophys. Acta. 1777, 1092-1097 (2008).</li><li>Twig, G., Liu, X., Liesa, M., Wikstrom, J. D., Molina, A. J., Las, G., Yaniv, G., Hajnoczky, G., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+properties+of+mitochondrial+fusion+events+in+pancreatic+beta-cells+and+cardiac+cells+unravel+potential+control+mechanisms+of+its+selectivity.">Biophysical properties of mitochondrial fusion events in pancreatic beta-cells and cardiac cells unravel potential control mechanisms of its selectivity.</a> Am. J. Physiol. Cell. Physiol. 299, C477-C487 (2010).</li><li>Twig, G., Shirihai, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interplay+between+mitochondrial+dynamics+and+mitophagy.">The interplay between mitochondrial dynamics and mitophagy.</a> Antioxid. Redox Signal. 14, 1939-1951 (2011).</li></ol>

Produzione e l&#39;accesso gratuito a questo articolo è sponsorizzato da Zeiss, Inc.

Questo metodo consente l&#39;imaging di circa 10 celle per volta, se l&#39;acquisizione avviene ogni 15 minuti dopo fotoattivazione. Il numero esatto delle cellule dipende da quanto velocemente si è in grado di individuare e marcare le cellule che esprimono mtPAGFP all&#39;interno della capsula di Petri, e quanto velocemente si possono impostare tutti i parametri del software. Per rendere l&#39;automazione senza intoppi uno strato uniforme di cellule devono essere utilizzati perché i designati Z-Stack margini si applica per tutte le celle. Le dimensioni di questa area fotoattivazione iniziale governare il tempo di equilibrio. Per poter misurare la fusione mitocondriale, è importante photoactivate solo il 10-20% della rete, in modo tale che la diffusione del segnale al resto della rete può essere monitorata nel tempo. Se troppo della rete è fotoattivato, è possibile che la fusione completa si verifica troppo rapidamente, e l&#39;evento non sarà catturato. Estrema cura deve essere presa aregolare la potenza del laser di due fotoni laser, nonché la concentrazione TMRE fototossicità per evitare che porta alla depolarizzazione mitocondriale. Garantire che il segnale mtPAGFP colocalizza con il segnale TMRE può aiutare nella valutazione fototossicità e la salute delle cellule in generale 8,15. Illuminazione con luce epifluoerescent deve essere evitato. Durante la ricerca di cellule che esprimono mtPAGFP, il foro deve essere di massima apertura durante la scansione con eccitazione 488nm bassa potenza. Regolazione due fotoni potenza del laser photoactivate sufficiente PA-GFP per misurare il segnale in 1 ora, ma non saturare eccessivamente qualsiasi delle cellule può essere difficile 8. Tuttavia, il tempo dovrebbe essere speso in questa fase di ottimizzazione perché il programma automatico, una volta avviato, è noioso per fermarlo, per selezionare altre celle, e riprendere. Per il controllo della qualità, l&#39;acquisizione di un contrasto di interferenza differenziale (DIC) l&#39;immagine (o luce trasmessa) per monitorare l&#39;attenzione sulle cellule possono essere moltoutile e anche un buon modo per rilevare bolle formatesi nella immersione in olio durante la scansione, questo accade a volte dai movimenti del palcoscenico. Anche se questo metodo mtPAGFP raccoglie dati sul movimento unidirezionale di proteine ​​della matrice mitocondriale mitocondri fotoattivato a quelli che non sono etichettati, è concepibile utilizzare questa tecnica per studiare altri processi. Ad esempio, fluorocromi specifici può essere collegato a proteine ​​di membrana per osservare il loro movimento specifico durante eventi di fusione, come è stato mostrato per ABC-me, la fusione di che si verifica su una scala di tempo differente dalla miscelazione di proteine ​​di matrice solubili 15.

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> COXIII adenovirale-PA-GFP </td><td> Dr. Lippincott-Schwartz </td><td></td></tr><tr><td> TMRE </td><td> Invitrogen </td><td> T669 </td></tr><tr><td> Confocale Zeiss LSM 710 </td><td> Zeiss </td><td></td></tr></tbody></table>

a faster, high resolution, mtpa-gfp-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.

Watch this Scientific Journal Video about A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy at JoVE.co

A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

Fusione mitocondriale è stato misurato mediante il monitoraggio di equilibrare le photoconverted matrice mirata GFP attraverso la rete mitocondriale nel corso del tempo. Finora, una sola cella può essere sottoposto ad un&#39;ora lunga analisi cinetica alla volta. Vi presentiamo un metodo che misura contemporaneamente più celle, in modo da accelerare il processo di raccolta dati.

Una più veloce, ad alta risoluzione, MTPA-GFP-based test Fusion mitocondriale Acquisizione dati cinetici di più celle in parallelo con microscopia confocale

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in ...

a-faster-high-resolution-mtpa-gfp-based-mitochondrial-fusion-assay

Research

JoVE Journal

Biology

16.4K Views.  Tufts School of Medicine. Fusione mitocondriale è stato misurato mediante il monitoraggio di equilibrare le photoconverted matrice mirata GFP attraverso la rete mitocondriale nel corso del tempo. Finora, una sola cella può essere sottoposto ad un'ora lunga analisi cinetica alla volta. Vi presentiamo un metodo che misura contemporaneamente più celle, in modo da accelerare il processo di raccolta dati.

Video: A Faster, High Resolution, mtPA-GFP-based Mitochondrial Fusion Assay Acquiring Kinetic Data of Multiple Cells in Parallel Using Confocal Microscopy

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.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

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

Ricerca

Biologia

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

Analisi delle SNARE-mediata fusione della membrana L&#39;utilizzo di un test enzimatico fusione cellulare

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine ​​fluorescenti che utilizzano confocale e microscopia Multiphoton

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

Ad alta risoluzione Time-lapse imaging e analisi automatizzata dei microtubuli Dynamics in Living ombelicale umano cellule endoteliali Vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

Ad alta risoluzione respirometria mitocondriale per valutare la funzione in permeabilizzate e cellule intatte

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combinazione di sincronizzazione cellulare mitotica e microscopia confocale ad alta risoluzione per studiare il ruolo delle proteine del ciclo cellulare multifunzione durante la mitosi

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

Respirometry ad alta risoluzione per misurare la funzione mitocondriale di cellule Beta intatte in presenza di composti naturali

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Analisi cinetica ad alta velocità ad alto rendimento parallela per cleavage del DNA specifico del sito

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Analisi in tempo reale della bioenergetica in cellule epiteliali pigmentate primarie della retina umana mediante respirometria ad alta risoluzione

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

Quantificazione del potenziale di membrana mitocondriale e dei livelli di superossido

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Autore in primo piano: Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Quantificazione basata sulla fluorescenza del potenziale di membrana mitocondriale e dei livelli di superossido utilizzando l'imaging dal vivo nelle cellule HeLa

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...