Name: measuring trans-plasma membrane electron transport by c2c12 myotubes
Uploaded: 2018-05-04T15:00:00.000+00:00
Duration: 10 min 27 s
Description: Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Vorremmo ringraziare Thomas Bell, Lyn Mattathil, Mark Mannino e Neej Patel per il loro supporto tecnico. Questo lavoro è stato supportato dal premio United States Public Health Service R15DK102122 dall'Istituto nazionale di diabete e digestivo e malattie renali (NIDDK) a Jonathan Fisher. Il contenuto del manoscritto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il punto di vista ufficiale il NIDDK o il National Institutes of Health.

Nota: Vedere la Figura 1 per una descrizione schematica di passaggi chiave.
1. analisi di riduzione WST-1
<ol><li>Crescere e differenziare cellule aderenti C2C12 con cella standard cultura procedure7 in una piastra a 96 pozzetti utilizzando righe A-F.<ol>
<li>Usare un mezzo di differenziazione consistente dell'Aquila per volta di Dulbecco Medium (DMEM) completati con 2% di siero equino, 100 U/mL di penicillina e 0,1 mg/mL di streptomicina. Incubare le cellule a 37 ° C con 5% CO2.</li>
		<li>Durante il monitoraggio di coinvolgimento di ascorbato in tPMET, supplemento media di differenziazione con l'acido ascorbico 100 µM. Consentire alle cellule di Incubare in media di differenziazione per ~ 24-48 h.</li>
	</ol>
</li>
	<li>Preparare soluzioni «stock» WST-1 e PMS.<ol>
<li>Per rendere uno stock di 10 mM di WST-1, sciogliere 0,033 g di WST-1 (Formula peso (FW): 651.34 g/mol) in 5 mL di fosfato tampone salino (PBS). Conservare a 4 ° C.</li>
		<li>Per rendere una scorta di 5 mM di PMS, sciogliere 0,0023 g di PMS (FW: 306.34 g/mol) in 1,5 mL di deionizzata H2O (diH2O). Conservare a-20 ° C e proteggere dalla luce.</li>
	</ol>
</li>
	<li>Aggiungi 0,0108 g di glucosio per soluzione salina tamponata con una concentrazione finale di 5 mM a 11,4 mL di PBS o HEPES (HBS; sale di sodio di 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2,5 mM MgSO4, 1mm CaCl2). µ L 480 di 10mm WST-1 per una concentrazione finale di 400 µM e 48 µ l di 5mm PMS per una concentrazione finale di 20 µM.</li>
	<li>Durante il monitoraggio di coinvolgimento di ascorbato in tPMET, è possibile dividere la soluzione in due aliquote di 6 mL. Ad un'aliquota, aggiungere 6 µ l di diH2O e l'altra aliquota aggiungere 6 µ l di AO 2 kU/mL per una concentrazione finale di 2 U/mL.</li>
	<li>Durante il monitoraggio di coinvolgimento di superossido in tPMET, è possibile dividere la soluzione in due aliquote di 6 mL. Ad un'aliquota, 55 µ l di tampone di4 KPO 0.1 M e l'altra aliquota aggiungere 55 µ l di SOD 6.5 kU/mL per una concentrazione finale di 60 U/mL.</li>
	<li>Lavare le cellule con PBS. Aspirare i media e aggiungere 150 µ l PBS. Poi aspirare il PBS. Nota: Il test è fatto con cellule placcate, allegate. Così, non esiste nessuna centrifugazione o uso della tripsina nel processo di lavaggio.</li>
	<li>Aggiungere 100 µ l di soluzione WST-1 (-) SOD o (-) AO a colonne 1-6 e aggiungere 100 µ l di soluzione WST-1 (+) SOD o (+) AO a colonne 7-12 la piastra a 96 pozzetti. Utilizzare righe G e H come sfondo controlli (vale a dire, per monitorare il cambiamento di assorbanza nel reagente solo nei pozzi senza cellule).</li>
	<li>Misurare i valori di assorbanza utilizzando lo spettrofotometro ogni 10 min per 1 h a 438 nm.</li>
	<li>Dopo la lettura, i media di aspirare e lavare ogni bene con 150 µ l di PBS. Poi aspirare il PBS.</li>
	<li>Calcolare la variazione di assorbanza di ciascun pozzetto sottraendo l'assorbanza iniziale per un pozzo dall'assorbanza in qualsiasi momento per bene. Correggere questo cambiamento per la variazione di assorbanza (se qualsiasi) osservata nei pozzetti sfondo (cioè, pozzetti con soluzione di saggio ma nessun cellule).</li>
	<li>Per l'analisi, normalizzare i dati di assorbanza per la misurazione di controllo 60 min o lavare i pozzetti con PBS o HBS e quindi eseguire un'analisi della proteina di bicinconinico acida (BCA).</li>
	<li>Aggiungere standard di albumina di siero bovino (BSA) 2 mg/mL (intervallo da 0,5-2 µ l per la curva standard, a seconda del grado di confluenza delle cellule) ai pozzetti vuoti (righe G e H), quindi aggiungere il reagente BCA in tutti i pozzetti.</li>
	<li>Quantificare i dati via nmol di WST-1 riduzione per µ g di proteina. Utilizzare 37 mM-1 cm-1 22 come il coefficiente di estinzione per ridotta WST-1 a 438 nm.</li>
</ol>2. analisi di riduzione DPIP
<ol><li>Crescere e differenziare le cellule aderenti C2C12 con la stessa procedura come passo 1.1.</li>
	<li>Preparare soluzione DPIP stock 10 mM come segue. Sciogliere 0,029 g di DPIP (FW: 290.08 g/mol) in 10 mL di diH2O. Confirm la concentrazione di DPIP misurando l'assorbanza a 600 nm con uno spettrofotometro. Utilizzare 1 mM-1 cm-1 23 come il coefficiente di estinzione per DPIP ridotta a 600 nm. Conservare a 4 ° C.</li>
	<li>Aggiungere 0,0108 g di glucosio 11,880 mL di PBS per una concentrazione finale di 5 mM. Aggiungere 120 µ l di 10 mM DPIP per una concentrazione finale di 100 µM.</li>
	<li>Durante il monitoraggio di coinvolgimento di ascorbato in tPMET, è possibile dividere la soluzione in due aliquote di 6 mL. Ad un'aliquota, aggiungere 6 µ l di diH2O e l'altra aliquota aggiungere 6 µ l di AO 2 kU/mL per una concentrazione finale di 2 U/mL.</li>
	<li>Durante il monitoraggio di coinvolgimento di superossido in tPMET, è possibile dividere la soluzione in due aliquote di 6 mL. Ad un'aliquota, 55 µ l di tampone di4 KPO 0.1 M e l'altra aliquota aggiungere 55 µ l di SOD 6.5 kU/mL per una concentrazione finale di 60 U/mL.</li>
	<li>I media di aspirare e lavare le cellule in 150 µ l di PBS. Aspirare il PBS e aggiungere 100 µ l di soluzione di DPIP (-) SOD o (-) AO a colonne 1-6 e aggiungere 100 µ l di soluzione DPIP (+) SOD o (+) AO a colonne 7-12 la piastra a 96 pozzetti. Utilizzare righe G e H volontà come sfondo controlli (vale a dire, per monitorare il cambiamento di assorbanza nel reagente solo nei pozzi senza cellule).</li>
	<li>Misurare l'assorbanza a 600 nm mediante spettrofotometro ogni 10 min per 1 h. quantificare la variazione di assorbanza relativo al controllo al min 60 simile procedura 1,9-1.13.</li>
</ol>3. determinazione di se ridotta accettori sono substrati per AO o SOD
<ol><li>5,436 mL di PBS o HBS, aggiungere 240 µ l di 10 mM WST-1 per una concentrazione finale di 400 µM e 24 µ l di 5mm PMS per una concentrazione finale di 20 µM.<ol>
<li>Per DPIP, aggiungere 60 µ l di 10 mM DPIP, per una concentrazione finale di 100 µM, a 5,940 mL di PBS o HBS.</li>
	</ol>
</li>
	<li>Aggiungere 100 µ l di soluzione di ogni bene in una piastra a 96 pozzetti a fondo piatto in assenza di cellule e misurare l'assorbanza a uno spettrofotometro a 438 nm, per WST-1, o 600 nm, per DPIP.</li>
	<li>Aggiungere 1 µ l di ascorbato di 10 mM per la metà dei pozzi per una concentrazione finale di 100 μM. Monitorare l'assorbanza finché non si stabilizza.</li>
	<li>Al momento di stabilizzazione, aggiungere 1 µ l di 200 U/mL AO per una concentrazione finale di 2 U/mL ad ogni pozzetto o aggiungere 1 µ l di 6 kU/mL SOD per una concentrazione finale di 60 U/mL a ciascun bene e monitorare l'assorbanza per 1 h.</li>
</ol>

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

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Abbiamo presentato due metodi per utilizzare extracellulare accettori, WST-1 e DPIP, nelle analisi spettrofotometriche per monitorare tPMET in miotubi C2C12. Con la crescita di linee cellulari nelle procedure standard di cultura e di un lettore di piastra spettrofotometro, è possibile monitorare tPMET con questi accettori in un'analisi semplice micropiastra. WST-1 riduzione è riproducibile dal pozzetto a altro all'interno di un test, ma non c'è variabilità giorno per giorno. Il quotidiano coefficiente di variazione (...

La capacità delle membrane plasmatiche purificate per ridurre accettori ha condotto alla vista che la membrana plasmatica ha un redox intrinseca capacità1. Precedentemente visto in funghi, piante e animali, tPMET è un processo comune a più organismi2,3,4,5. In particolare, questo processo è stato dimostrato in Saccharomyces cerevisiae, cellule di carota, eritrociti, linfociti, osteosarcoma, il melanoma, macrofagi, muscolo scheletrico e neutrofili2,3, 4 , 5 , 6 , 7. in un processo che trasporta gli elettroni attraverso la membrana plasmatica per ridurre extracellulari ossidanti, tPMET è coinvolto in molte funzioni cellulari tra cui: cella crescita5,8, cellula attuabilità9, Ferro da stiro metabolismo10,11,12,13e protezione da ossigeno reattivo specie12,14,15di segnalazione delle cellule. A causa del coinvolgimento di tPMET in molte funzioni cellulari, uno squilibrio di tPMET è stata supposta per contribuire allo sviluppo di alcune condizioni di salute gravi, tra cui cancro16, malattia cardiovascolare17e metabolica la sindrome18.
Ci sono diversi modi per monitorare il trasferimento di elettroni attraverso la membrana plasmatica, ma la tecnica più ampiamente usata è quello di valutare la riduzione di accettori extracellulari attraverso saggi colorimetrici. Accettori extracellulari comunemente utilizzati sono sali di tetrazolio, DPIP e FeCN ferricitocromo c19,20. Il sale di tetrazolio più comunemente usato è un noto sale seconda generazione come WST-119. Questo composto è più facile da utilizzare in saggi colorimetrici rispetto ai sali di tetrazolio di prima generazione a causa di due gruppi solfonato, che aumentano la sua acqua solubilità21. WST-1, in concomitanza con l'intermedio elettrone accettore 1-metossi-fenazina methosulfate (MPM), è ridotto in eventi di due trasferimento singolo elettrone. Questa riduzione cambia la forma ossidata debolmente colorato di WST-1 a un più intenso, giallo formazan20,22. WST-1 ha un coefficiente di estinzione molare alta di 37 x 103 M-1cm-1, portando a un dosaggio ad alta sensibilità21,22. DPIP viene anche utilizzato come un ricettore dell'elettrone extracellulare per monitorare tPMET. È stato dimostrato che DPIP può essere ridotto extracellularly di tPMET senza l'ausilio di intermedi elettroni accettori23,24. A causa della mancanza di accettori intermedi, DPIP può direttamente gli elettroni di pick-up dalla membrana del plasma, a differenza di WST-124. Simile a DPIP, FeCN ha dimostrato di essere ridotto extracellularly a ferrocianuro di tPMET senza l'ausilio di intermedi elettroni accettori19,24. A differenza di WST-1 e DPIP, FeCN ha un coefficiente di estinzione molare basso che conduce a un più basso dosaggio sensibilità9. Un altro accettore di elettroni extracellulare comunemente usate per monitorare tPMET è ferricitocromo c. simile a WST-1, ferricitocromo c riduzione aumenta con l'uso di accettore di elettroni intermedi, mPMS22. A differenza di WST-1 però, il metodo di ferricitocromo c è meno sensibile a causa di un'alta priorità bassa e un coefficiente di estinzione molare basso22.
Qui presentiamo un metodo per l'analisi in tempo reale di tPMET attraverso analisi spettrofotometriche. Il metodo utilizzato l'accettori extracellulari WST-1 e DPIP, dato che entrambi hanno un coefficiente di estinzione molare alta mentre essendo meno costoso rispetto agli altri comunemente usato accettori extracellulari quali ferricitocromo c. Abbiamo utilizzato fenazina methosulfate (PMS) invece di mPMS hanno una composizione chimica simile e PMS è molto meno costoso. mPMS è fotochimicamente stabile che è una caratteristica importante per un kit commerciale che ha bisogno di una lunga shelf life. Tuttavia, facciamo PMS fresco per ogni dosaggio, quindi stabilità non dovrebbe essere un problema. Presentiamo anche un metodo per valutare le possibili interazioni enzimatiche (Vedi Figura 1) tra il ricettore dell'elettrone extracellulare ed enzimi che possono essere utilizzati per caratterizzare ulteriormente il processo di tPMET. In particolare, gli enzimi AO e SOD può essere usato determinano quale parte del tPMET è dovuto al trasporto dell'ascorbato o rilascio extracellulare del superossido, due metodi comuni per gli elettroni essere trasportato attraverso la membrana plasmatica.

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

measuring trans-plasma membrane electron transport by c2c12 myotubes

Trans-plasma membrana electron transport (tPMET) svolge un ruolo nella protezione delle cellule dallo stress riduttivo intracellulare così come protezione da danni da ossidanti extracellulari. Questo processo di trasporto di elettroni da riducenti intracellulare agli ossidanti extracellulari non è ben definito. Qui vi presentiamo le analisi spettrofotometriche di miotubi C2C12 per monitorare tPMET utilizzando gli accettori extracellulari: sale di tetrazolio solubile in acqua-1 (WST-1) e 2,6-dichlorophenolindophenol (DPIP o DCIP). Attraverso la riduzione di questi accettori, siamo in grado di monitorare questo processo in un'analisi in tempo reale. Con l'aggiunta di enzimi come la superossido dismutasi (SOD) per l'analisi e l'ascorbato ossidasi (AO), possiamo determinare quale parte del tPMET è a causa della produzione di superossido o esportazione dell'ascorbato, rispettivamente. Mentre WST-1 è stato indicato per produrre risultati stabili con fondo basso, DPIP è stato in grado di essere ri-ossidato dopo l'aggiunta di AO e SOD, che è stata dimostrata con analisi spettrofotometriche. Questo metodo dimostra un'analisi spettrofotometrica in tempo reale, multi-pozzetto e veloce con vantaggi rispetto ad altri metodi utilizzati per il monitoraggio tPMET, come ferricianuro (FeCN) e ferricitocromo c riduzione.

Le statistiche sono state effettuate con ANOVA con misure ripetute utilizzando RStudio software statistico25. Dimensioni del campione sono indicate nelle leggende figura.
Per monitorare tPMET, miotubi C2C12 sono stati utilizzati insieme accettori extracellulari, WST-1 e DPIP. AO è stato usato per determinare quale parte del WST-1 e DPIP riduzione era a causa del deflusso dell'ascorbato e SOD è stato uti...

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

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trasporto dell'elettrone di miotubi C2C12 Trans-Plasma membrana di misura

L'obiettivo del presente protocollo è di monitorare spettrofotometricamente trans-plasma membrana elettrone trasporto utilizzando accettori extracellulari e di analizzare le interazioni enzimatiche che possono verificarsi con questi accettori extracellulari.

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

6.9K Views.  Saint Louis University. L'obiettivo del presente protocollo è di monitorare spettrofotometricamente trans-plasma membrana elettrone trasporto utilizzando accettori extracellulari e di analizzare le interazioni enzimatiche che possono verificarsi con questi accettori extracellulari.

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

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

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

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

Utilizzo di mitocondri isolati da minime quantità di muscolo scheletrico del mouse per High micropiastre rendimento respiratorie Misure

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

Ricerca

Biologia dello sviluppo

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

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

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

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

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

Medicina

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

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

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

Misurazione dei complessi di trasferimento di elettroni mitocondriali nel tessuto cardiaco precedentemente congelato dalla prole della scrofa: un modello per valutare i cambiamenti bioenergetici mitocondriali indotti dall'esercizio

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

Biochimica

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

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

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

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

La sensibilità della membrana mitocondriale interna a Na+ rivela pool di CoQ funzionali parzialmente segmentati

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

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

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

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

Preparazione e respirometrici valutazione dei mitocondri isolati dal tessuto muscolare scheletrico Ottenuto dalla biopsia percutanea

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

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

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

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

Profilo Esperimento bioenergetica utilizzando cellule dei mioblasti C2C12

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

Biologia

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

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

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

Modellazione Distrofia miotonica 1 nelle cellule dei mioblasti C2C12

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

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

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

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

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 ...

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 ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...