The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

Gli studi su animali sono stati effettuati nel quadro di un protocollo approvato dalla cura degli animali istituzionale e utilizzano Comitato al Virginia Polytechnic Institute e State University. 1. Setup (Tempo: ~ 45 min) <ol><li> Scongelare depositi congelati di 0,25% tripsina, isolamento tampone per mitocondri (IBM) 1 e IBM2 in un bagno d&#39;acqua a 37 ° C. </li><li> Sciacquare vetreria e strumenti di dissezione in etanolo al 70% e poi con acqua ad elevata purezza. </li><li> Preparare la soluzione tripsina 0,05% dal 0,25% tripsina magazzino diluendo 1 parte di tripsina in 4 parti IBM1. </li><li> Mescolare proteasi e fosfatasi inibitore cocktail con tampone di lisi cellulare (1: 100 ratio) in una provetta da 1,5 ml microcentrifuga con tappo a vite. </li><li> Aliquote 5 ml (5 ml / topo) di 0,05% tripsina in un tubo di plastica da 15 ml. </li><li> Impostare motorizzato omogeneizzatore tessuto a 80 giri. </li></ol> 2. Isolamento del muscolo scheletrico mitocondri (Tempo: ~ 90 min) <ol><li> L&#39;eutanasia di un mouse da CO 2 </sub&gt; inalazione, seguita da dislocazione cervicale. </li><li> Rimuovere muscolo rosso dal quadricipite e del muscolo gastrocnemio, che comprende il soleo (~ 75-100 mg totale) come descritto nei seguenti passaggi: <ol><li> Sbucciare la pelle verso il mouse. </li><li> Rimuovere il cuscinetto di grasso sopra il punto di origine del quadricipite. Tagliare il tendine del quadricipite che è collegato alla rotula con sottili forbici a punta. Nota: Il quadricipite è identificato dalla sua posizione anatomica sulla parte anteriore del femore prossimale. </li><li> Lentamente tagliare l&#39;aponeurosi tra l&#39;osso e il quadricipite, evitando l&#39;arteria femorale, per liberare i quadricipiti dall&#39;osso. Tagliare il tendine con sottili forbici a punta in corrispondenza del punto di origine sul femore per liberare il muscolo quadricipite e posizionare il muscolo quadricipite in refrigerate PBS. </li><li> Rimuovere il tessuto adiposo visibile sopra il quadricipite con le forbici. Vibrazione quadricipiti sopra in modo che la porzione del muscolo che è stato sovrapposto il femore sta affrontando up. Aprire il muscolo quadricipite con una pinza in un movimento rotatorio. Rimuovere le due porzioni muscolari rosse visibili da ogni lobo con ammenda forbici a punta e mettere in un bicchiere contenente 5 ml di acqua refrigerata IBM1. Nota: muscolo quadricipite appaiono come due lobi con una striscia di muscolo rosso si trova vicino al bordo laterale di ciascun lobo. </li><li> Tagliare la pelle sovrastante il tendine di Achille con ammenda punta forbici e sbucciare la pelle verso il mouse. Tagliare il tendine di Achille esposto e staccare il muscolo verso il corpo del mouse. </li><li> Tagliare il tendine ai condili mediale e laterale del femore con sottili forbici a punta per liberare il gastrocnemio e collocare il gastrocnemio attaccato ai suoi tendini in refrigerata PBS. </li><li> Capovolgere il gastrocnemio sopra e staccare il muscolo soleo e mettere nel bicchiere contenente 5 ml di acqua refrigerata IBM1. Fan aprire il gastrocnemio. Rimuovere le tre porzioni visive rosse muscolari (due strisce rosse laterali e uno mediale superficiale) con multa forbice a puntas e trasferirli al bicchiere contenente 5 ml di acqua refrigerata IBM1. </li></ol></li><li> Rimuovere un&#39;altra ~ 75-100 mg di muscolo rosso dalla altra gamba del mouse (come descritto al punto 2.2) e posto in una provetta da 1,5 ml microcentrifuga con proteasi e fosfatasi inibitore cocktail e tampone di lisi cellulare (50 mM Tris-HCL, 1 mM EDTA, NaCl 150 mm, 1% dodecil solfato di sodio, 0,5% deossicolato di sodio, 1% Poliossietilene (9) nonylphenylether, ramificata. Subito lampeggiare-freeze questo secondo campione in azoto liquido per un uso successivo a assorbente occidentale (vedi punto 6.1). </li><li> Mettere una superficie di plastica piatta pre-raffreddata su ghiaccio in un grande secchio. Versare una goccia di IBM1 sulla superficie di plastica e utilizzare pinzette per mettere tutti del muscolo rosso sezionato (passo 2.2.4 e 2.2.7) in IBM1 goccia. Tritare il tessuto muscolare per 2 minuti utilizzando 3 singole lame di rasoio bordo cambiando rasoi ogni 40 sec. </li><li> Trasferire il tessuto tritato in un becher con 5 ml di IBM1 fresco tenendo superficie plastica sopra the becher e raschiare il muscolo nel becher con una lama di rasoio. Prendete questa soluzione e di scarico attraverso un colino cella di 100 micron collocato su un tubo da 50 ml. </li><li> Tamponare il tessuto con un tergicristallo compito delicato e poi trasferisce in 5 ml della soluzione di tripsina 0,05%. Utilizzare pinzette per rimuovere il tessuto dal tergicristallo compito. </li><li> Incubare il tessuto muscolare in ghiaccio per 30 minuti in soluzione tripsina 0,05%. </li><li> Gira le tubo da 15 ml miscela contenente tripsina / muscolo a 200 xg per 3 min a 4 ° C. </li><li> Versare il surnatante tripsina in un contenitore per rifiuti e risospendere il pellet con 3 ml di ghiaccio freddo IBM1. Trasferire il tessuto 45 ml tubo omogeneizzatore vetro. Sciacquare i tubo da 15 ml con un altro 1,5 ml IBM1 raccogliere qualsiasi campione rimanente e aggiungere questo al tubo omogeneizzatore vetro. </li><li> Posizionare il tubo di omogeneizzatore vetro in un bicchiere o contenitore di plastica pieno di ghiaccio a metà strada quindi l&#39;omogeneizzatore vetro si muove minimamente all&#39;interno del bicchiere. </li><li&gt; Omogeneizzare la miscela di tessuti / IMB1 con il pestello in PTFE collegati ad un comando a motore omogeneizzatore tessuto con 10 passaggi a 80 giri al minuto. Tenere la parte inferiore di ogni pass per ~ 2 sec. </li><li> Trasferire il tessuto omogenato di un nuovo tubo da 15 ml conica e sciacquare il tubo omogeneizzatore vetro con 6,5 ml di IBM1. Versare il IBM1 nel tubo da 15 ml contenente tessuto omogenato. </li><li> Spin tubo conico da 15 ml a 700 g per 10 minuti a 4 ° C. Delicatamente versare il surnatante in una provetta da centrifuga ad alta resistenza vetro e scartare il pellet. </li><li> Spin il supernatante dal passo a 8000 xg per 10 min a 4 ° C. </li><li> Rimuovere il surnatante IBM1 e risospendere il pellet aggiungendo lentamente 500 ml di IBM2. Tagliare la fine di un puntale e omogeneizzare delicatamente il pellet in IBM2 con miscelazione e agitazione movimenti. Aggiungere altri 4,5 ml di IBM2 alla miscela dopo il pellet è completamente sospesa. Nota: Evitare l&#39;eccessiva triturazione mentre rompendo pezzi pellet più grandi come tla sua può danneggiare le membrane mitocondriali. </li><li> Spin l&#39;omogeneizzato / tessuto IBM2 a 8000 xg per 10 minuti a 4 ° C. Nota: Questo passaggio può essere ripetuto in una provetta pulita centrifuga ad alta resistenza per più precisa quantificazione delle proteine ​​di mitocondri isolati al punto 4 dal residuo BSA può contribuire al contenuto totale di proteine. </li><li> Rimuovere il surnatante e delicatamente, ma completamente risospendere il pellet con l&#39;aggiunta di due incrementi 25 microlitri di IBM2 con un puntale con il suo punto di taglio. Mescolare delicatamente e miscelare il pellet dopo ogni aggiunta di 25 ml di IBM2. Mettere questo stock mitocondriale su ghiaccio. </li></ol> 3. omogeneizzazione di tutto il tessuto lisato (Tempo: ~ 45 min; può essere effettuato durante Passo 7) <ol><li> Rimuovere il muscolo da che è stato posto in azoto liquido dal punto 2.3 e incubare a temperatura ambiente fino completamente scongelati. </li><li> Tritare il tessuto per ~ 30 secondi sul ghiaccio con pregiati forbici a punta. </li><li> Aggiungere due cucchiai 100 microlitri di ZircoPerline in ossido di zirconio alla provetta da 1,5 ml microcentrifuga con tappo a vite dal punto 3.2 che contiene il tessuto tritato. Omogeneizzare il tessuto in un omogeneizzatore tessuto mulino tallone con due o tre cicli di 5 min ad una impostazione di velocità di 4-6 (regolazione media). </li><li> Spin la miscela dal punto 3.3 a 12.000 xg per 10 min a 4 ° C. Rimuovere il surnatante con una pipetta 1000 ml punta dopo lo spin e il trasferimento in una provetta da 1,5 ml microcentrifuga. Eliminare il pellet e posizionare il surnatante su ghiaccio. </li></ol> 4. Determinazione delle proteine ​​(Tempo: ~ 30 min) <ol><li> Determinare la concentrazione di proteine ​​di tutto il lisato tissutale (punto 3.4) e magazzino mitocondriale (passo 2.17) utilizzando il kit di analisi delle proteine ​​BCA secondo le specifiche del costruttore. </li></ol> 5. I mitocondri Membrana di integrità; Citrato sintasi (CS) Attività (Tempo: ~ 15 min) <ol><li> Fare due separati 1: 20 diluizioni dello stock mitocondriale in acqua ad elevata purezza. Aggiungere0,1% di Triton 100-X per un campione e sonicare è a un livello basso (765 watt, l&#39;ampiezza di 2). Lasciare l&#39;altra soluzione sul ghiaccio. Riportare il campione ghiaccio dopo sonicazione. </li><li> Eseguire un test citrato sintasi sia con il non-sonicato e sonicato diluizioni mitocondriali utilizzando un saggio spettrofotometrico precedentemente descritto 11,12. </li><li> Determinare la percentuale delle membrane mitocondriali intatte in base alle seguenti equazioni: (Attività CS non sonicato ÷ sonicato CS attività) * 100 </li></ol> 100- N (cento raggiunto da sopra) 6. I mitocondri isolamento di qualità; Western Blotting (Tempo: Secondo Lab Protocol) <ol><li> Eseguire Western blotting su tutta lisato tissutale e mitocondri isolati come descritto in precedenza 12. Nota: Utilizzare anticorpi primari per GAPDH (1: 1.000 diluizione 5% BSA / TBS-T) e COXIV (1: 1.000 diluizione 5% senza grassiLatte / TBS-T), seguita da anti-capra, e anticorpi secondari di coniglio, rispettivamente, (1: 10.000). </li></ol> 7. respirometrici Assay Run (Tempo: ~ 90 min) <ol><li> Effettuare test respirometrici desiderati usando basato O 2 tecnologia di misurazione del consumo di micropiastre come descritto in precedenza (vedi articolo correlato e Rogers et al 8). </li><li> Determinare il rapporto di controllo respiratorio (RCR) dividendo Stato 3 per Stato 4o (RCR) o dividere 3u Stato per Stato 4o. Utilizzare il mezzo (media) il punto, o il più alto (Stato 3) e tariffe più basse (4o Stato) dal punto a punto le tracce per determinare RCR. </li></ol>

<ol>
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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

I metodi qui presentati forniscono una descrizione dettagliata di una procedura di isolamento mitocondriale da quantità minime (~ 75-100 mg) di topo muscoli scheletrici. Questa procedura di isolamento è in grado di produrre ad alto funzionamento, i mitocondri puro (~ 450 mg) come evidenziato da O 2 tassi di consumo, valori RCR, la massima attività citrato sintasi e di espressione proteica da immunoblotting. È importante sottolineare che i mitocondri isolati da questa procedura può essere utilizzato per p...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table><tbody><tr><td>Essentially Fatty</td><td>Sigma Aldrich</td><td>A6003</td><td>N/A</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Tris/HCl</td><td>Promega</td><td>H5123</td><td>N/A</td></tr><tr><td>KCL</td><td>Sigma Aldrich</td><td>P9541</td><td>N/A</td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td>N/A</td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>E6511</td><td>N/A</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>N/A</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>N/A</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>N/A</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermo Scientific</td><td>25200-056</td><td>N/A</td></tr><tr><td>Sodium Chloride&lt;br/&gt; White Crystals or Crystalline Powder&lt;br/&gt; &amp;ge;99.0 %</td><td>Fisher Scientific</td><td>BP3581</td><td>N/A</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L3771&amp;nbsp;</td><td>N/A</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich</td><td>D6750&amp;nbsp;</td><td>N/A</td></tr><tr><td>Polyoxyethylene (12) nonylphenyl ether, branched</td><td>Sigma Aldrich</td><td>238651</td><td>N/A</td></tr><tr><td>Single Edge Razor Blades</td><td>Fisher Scientific</td><td>12-640</td><td>N/A</td></tr><tr><td>Falcon- 100 uM Nylon Cell Strainers</td><td>Fisher Scientific</td><td>352360</td><td>N/A</td></tr><tr><td>Halt Protease &amp;amp; Phosphatse Inhibitor Cocktail</td><td>Thermo Scientific</td><td>1861284</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes with screw cap</td><td>Thermo Scientific</td><td>3474</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Zirconium Oxide beads</td><td>Fisher Scientific</td><td>C9012112</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>GAPDH antibody (1D4)</td><td>Santa Cruz Biotechnology</td><td>sc-59540</td><td>N/A</td></tr><tr><td>Anti- COXIV antibody</td><td>Cell Signaling</td><td>4844s</td><td>Any mitochondrial inner membrane protein will suffice</td></tr><tr><td>Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>711-035-152</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td><td>Jackson ImmunoResearh</td><td>115-001-003</td><td>N/A</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Triton-X100</td><td>Sigma Aldrich</td><td>X100</td><td>N/A</td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C,pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2-8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C, to 7.4 with KOH prior to use in respirometric assay</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at room temperature</td></tr></tbody></table>

isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

Disfunzionali mitocondri dei muscoli scheletrici hanno un ruolo nel metabolismo alterato osservata con l&#39;invecchiamento, l&#39;obesità e il diabete di tipo II. Saggi respirometrici mitocondriali da isolate preparati mitocondriali consentono la valutazione della funzione mitocondriale, nonché la determinazione del meccanismo (s) d&#39;azione dei farmaci e proteine ​​che modulano il metabolismo. Procedure di isolamento attuali richiedono spesso grandi quantità di tessuto per produrre mitocondri di alta qualità necessari per le analisi respirometrici. I metodi qui presentati descrivono come di alta qualità mitocondri purificato (~ 450 mg) può essere isolato da quantità minime (~ 75-100 mg) di topo muscolo scheletrico per l&#39;uso in misure respiratorie throughput elevato. Abbiamo determinato che il nostro metodo di isolamento si ottiene il 92,5 ± 2,0% mitocondri intatti misurando l&#39;attività citrato sintasi spettrofotometricamente. Inoltre, l&#39;analisi Western Blot in mitocondri isolati portato alla debole espressione del cytosoproteine ​​lic, GAPDH, e il robusto espressione della proteina mitocondriale, COXIV. L&#39;assenza di una banda di GAPDH prominente nei mitocondri isolati è indicativo di poco la contaminazione da fonti non-mitocondriali durante la procedura di isolamento. Soprattutto, la misura di O 2 tasso di consumo con tecnologia basata micropiastra e determinare il rapporto di controllo respiratorio (RCR) per saggi respirometrici accoppiati mostra altamente accoppiate (RCR;&gt; 6 per tutti i saggi) e mitocondri funzionali. In conclusione, l&#39;aggiunta di una fase di macinazione separata e riducendo significativamente motorizzato velocità omogeneizzazione di un metodo riportato in precedenza ha permesso l&#39;isolamento di alta qualità e mitocondri purificati da piccole quantità di topo muscoli scheletrici che si traduce nei mitocondri altamente accoppiate che respirano con alta funzione durante micropiastra saggi respirometirc.

Attività citrato sintasi serve come misura per l&#39;integrità della membrana dal citrato sintasi si trova nella membrana mitocondriale interna, e quindi non dovrebbero essere presenti nelle sospensioni di mitocondri con membrane intatte. La Figura 1 rappresenta l&#39;attività citrato sintasi in campioni non sonicato mitocondriali rispetto sonicato campioni dello stesso isolamento. Sonicating i risultati dei mitocondri in un aumento statisticamente significativo nell&#39;attività ci...

Watch this Scientific Journal Video about Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements at JoVE.com

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K Views.  Virginia Tech. Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Video: Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

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

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function. Therefore, protein import serves as a barometer of cellular health.

Misurazione della capacità di importazione di proteine dei mitocondri del muscolo scheletrico

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, ...

Biochimica

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 ...

Medicina

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Biologia

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Isolamento dei mitocondri dal muscolo scheletrico

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificazione di progenitori dal muscolo scheletrico

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

<meta charset="utf8"></head><body>Isolamento mitocondriale dai roditori per studi bioenergetici di alta qualità

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

<meta charset="utf8"></head><body>Autore in primo piano: Scoprire il ruolo del fosfato di calcio mitocondriale nell'insufficienza cardiaca e nella bioenergetica

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Robusto isolamento mitocondriale dal tessuto cardiaco dei roditori 

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

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 ...

L'isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

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Capitoli in questo video

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

Misurazione della capacità di importazione di proteine dei mitocondri del muscolo scheletrico

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

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Isolamento dei mitocondri dal muscolo scheletrico

Purificazione di progenitori dal muscolo scheletrico

Robusto isolamento mitocondriale dal tessuto cardiaco dei roditori

Robusto isolamento mitocondriale dal tessuto cardiaco dei roditori

Robusto isolamento mitocondriale dal tessuto cardiaco dei roditori

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