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

Los estudios en animales se realizaron bajo un protocolo aprobado por el Cuidado de Animales institucional y el empleo Comisión en el Instituto Politécnico de Virginia y la Universidad Estatal. 1. Configuración (Tiempo: ~ 45 min) <ol><li> Tiendas Descongele de 0,25% de tripsina, Aislamiento Buffer de mitocondrias (IBM) 1 y IBM2 en un baño de agua a 37 ° C. </li><li> Enjuague la cristalería y los instrumentos de disección en etanol al 70%, seguido de agua de alta pureza. </li><li> Preparar una solución de tripsina al 0,05% desde 0,25% de tripsina stock de diluir 1 parte de tripsina en 4 partes IBM1. </li><li> Mezclar proteasa e inhibidor de la fosfatasa cóctel con tampón de lisis celular (1: 100 ratio) en un tubo de microcentrífuga de 1,5 ml con tapón de rosca. </li><li> Alícuota de 5 ml (5 ml / ratón) de 0,05% de tripsina en un tubo de plástico de 15 ml. </li><li> Conjunto motor impulsado homogeneizador de tejidos a 80 RPM. </li></ol> 2. Aislamiento de mitocondrias del músculo esquelético (Tiempo: ~ 90 min) <ol><li> La eutanasia a un ratón por el CO 2 </sub&gt; inhalación, seguido por dislocación cervical. </li><li> Eliminar músculo rojo del músculo gastrocnemio y cuádriceps, que incluye el sóleo (~ de 75-100 mg total) como se describe en los siguientes pasos: <ol><li> Pelar la piel hacia el ratón. </li><li> Retire la almohadilla de grasa por encima del punto de origen del cuádriceps. Cortar el tendón del cuádriceps que se adjunta a la rótula con unas tijeras de punta fina. Nota: El cuádriceps se identifica por su posición anatómica en la parte anterior del fémur proximal. </li><li> Lentamente cortar la aponeurosis entre el hueso y el cuádriceps, evitando al mismo tiempo la arteria femoral, para liberar el cuádriceps de la médula. Cortar el tendón con tijeras de punta fina en el punto de origen en el fémur para liberar el músculo cuádriceps y colocar el músculo cuádriceps en PBS enfriado. </li><li> Retire el tejido adiposo visible sobre los cuádriceps con tijeras. Voltear las cuádriceps de manera que la porción del músculo que se recubre el fémur se enfrenta up. Abra el músculo cuádriceps con fórceps en un movimiento en abanico. Retire las dos porciones musculares rojas visibles desde cada lóbulo con tijeras de punta fina y colocar en un vaso que contiene 5 ml de enfriado IBM1. Nota: del músculo cuádriceps aparecen como dos lóbulos con una tira de músculo rojo situado cerca del borde lateral de cada lóbulo. </li><li> Cortar la piel que recubre el tendón de Aquiles con punta fina tijera y la cáscara de la piel hacia el ratón. Cortar el tendón de Aquiles expuesto y pelar el músculo hacia el cuerpo del ratón. </li><li> Cortar el tendón en los cóndilos lateral y medial del fémur con tijeras de punta fina para liberar a los gemelos y colocar el gemelo unido a sus tendones en PBS frío. </li><li> Da la vuelta al gastrocnemio una y pelar el músculo sóleo y colocarlos en el vaso que contiene 5 ml de enfriado IBM1. Fan abrir el gastrocnemio. Retire los tres porciones visuales rojos musculares (dos tiras rojas laterales y uno medial superficiales) con tijeras de punta finas y les traslado al vaso que contiene 5 ml de enfriado IBM1. </li></ol></li><li> Retire otra ~ 75-100 mg de músculo rojo de la otra pata del ratón (como se describe en el paso 2.2) y ponerlo en un tubo de microcentrífuga de 1,5 ml con proteasa e inhibidor de la fosfatasa cóctel y tampón de lisis celular (50 mM Tris-HCl, 1 EDTA mM, NaCl 150 mM, 1% de dodecilsulfato de sodio, 0,5% de desoxicolato de sodio, 1% de polioxietileno (9) nonilfeniléter, ramificada. Inmediatamente FLASH-congelar esta segunda muestra en nitrógeno líquido para su uso posterior en Western Blot (véase el paso 6.1). </li><li> Coloque una superficie de plástico pre-enfriado, plana sobre hielo en un gran cubo. Vierta una gota de IBM1 sobre la superficie de plástico y utilizar pinzas para colocar toda la músculo rojo seccionado (paso 2.2.4 y 2.2.7) en IBM1 gotas. Picar el tejido muscular durante 2 minutos con 3 cuchillas de afeitar borde individuales cambiando las maquinillas de afeitar cada 40 seg. </li><li> Transferir el tejido picado a un nuevo vaso de precipitados con 5 ml de IBM1 fresco mediante la celebración de la superficie de plástico sobre el the vaso de precipitados y raspando el músculo en el vaso de precipitados con una hoja de afeitar. Tome esta solución y drenarla a través de un filtro de células 100 micras colocado sobre un tubo cónico de 50 ml. </li><li> Seque el tejido con un limpiador tarea delicada y luego transferirlo a 5 ml de la solución de tripsina 0,05%. Utilice pinzas para retirar el tejido del limpiaparabrisas tarea. </li><li> Incubar el tejido muscular en hielo durante 30 min en solución de tripsina 0,05%. </li><li> Haga girar los 15 ml tubo cónico de mezcla que contiene tripsina / muscular a 200 xg durante 3 min a 4 ° C. </li><li> Verter el sobrenadante de tripsina en un contenedor de residuos y resuspender el precipitado con 3 ml de hielo frío IBM1. Transferir el tejido a un tubo de vidrio de 45 ml homogeneizador. Enjuague los 15 ml tubo cónico con otro 1,5 ml IBM1 reunir cualquier muestra restante y agregar esto a el tubo homogeneizador de vidrio. </li><li> Coloque el tubo homogeneizador de vidrio en un lleno hasta la mitad vaso o recipiente de plástico con hielo por lo que el homogeneizador de vidrio se mueve mínimamente dentro del vaso de precipitados. </li><li&gt; Homogeneizar la mezcla de tejidos / IMB1 con las maja PTFE unido a un motor impulsado homogeneizador de tejidos con 10 pases a 80 rpm. Sostenga la parte inferior de cada pasada por unos 2 seg. </li><li> Transferir el homogeneizado de tejido a un nuevo tubo cónico de 15 ml y enjuagar el tubo homogeneizador de vidrio con 6,5 ml de IBM1. Verter la IBM1 en el tubo cónico de 15 ml que contenía homogeneizado de tejido. </li><li> Haga girar el tubo cónico de 15 ml a 700 g durante 10 min a 4 ° C. Vierta suavemente el sobrenadante en un tubo de centrífuga de alta resistencia vidrio y desechar el sedimento. </li><li> Girar el sobrenadante de la etapa anterior a 8000 xg durante 10 min a 4 ° C. </li><li> Eliminar el sobrenadante IBM1 y volver a suspender el sedimento añadiendo lentamente 500 l de IBM2. Corte el extremo de una punta de pipeta y homogeneizar suavemente el pellet en IBM2 con mezcla y agitación movimientos. Añadir otros 4,5 ml de IBM2 a la mezcla después el sedimento está totalmente suspendida. Nota: Evite la trituración excesiva mientras que romper pedazos de pellets de mayor tamaño como tsu puede dañar las membranas mitocondriales. </li><li> Haga girar el homogeneizado / tejido IBM2 a 8.000 xg durante 10 min a 4 ° C. Nota: Este paso puede repetirse en un tubo de centrífuga de alta resistencia limpia para la cuantificación de proteínas más precisa de mitocondrias aisladas en el paso 4 ya BSA residual puede contribuir al contenido total de proteínas. </li><li> Eliminar el sobrenadante y suavemente, pero totalmente volver a suspender el sedimento añadiendo dos incrementos de 25 l de IBM2 con una punta de pipeta con su punto de corte. Revuelva suavemente y mezclar la pastilla después de cada adición de 25 l de IBM2. Coloca esta población mitocondrial en hielo. </li></ol> 3. La homogeneización del Plenario del tejido lisado (Tiempo: ~ 45 min; se puede hacer durante el paso 7) <ol><li> Retire el músculo de que se colocó en el nitrógeno líquido desde el paso 2.3 y se incuba a temperatura ambiente hasta que esté completamente descongelado. </li><li> Picar el tejido de ~ 30 seg en el hielo con unas tijeras de punta fina. </li><li> Añadir dos cucharadas de 100 l de Zircoperlas de óxido nio al tubo de microcentrífuga de 1,5 ml con tapón de rosca desde el paso 3.2 que contiene el tejido picado. Homogeneizar los tejidos en un homogeneizador de tejidos molino de bolas utilizando dos o tres ciclos de 5 min con un ajuste de velocidad de 4-6 (posición media). </li><li> Gira la mezcla de la etapa 3.3 a 12.000 xg durante 10 min a 4 ° C. Eliminar el sobrenadante usando una punta de pipeta de 1.000 l después del giro y la transferencia en un tubo de microcentrífuga de 1,5 ml. Deseche la pastilla y colocar el sobrenadante en hielo. </li></ol> 4. Determinación de proteínas (Tiempo: ~ 30 min) <ol><li> Determinar la concentración de proteínas de todo el lisado de tejido (paso 3.4) y de la mitocondrial (paso 2,17) utilizando el kit de ensayo de proteína BCA de acuerdo con las especificaciones del fabricante. </li></ol> 5. Las mitocondrias integridad de la membrana; Citrato sintasa (CS) Actividad (Tiempo: ~ 15 min) <ol><li> Haga dos separadas 1: 20 diluciones de la población mitocondrial en agua de alta pureza. Añadir0,1% de Triton X-100 a una muestra y sonicar que en un ajuste bajo (765 Watts, amplitud de 2). Deje el otro dilución en el hielo. Devuelva la muestra en hielo después de la sonicación. </li><li> Realizar un ensayo de la citrato sintasa tanto con la no sonicado y se sonicó diluciones mitocondriales usando un ensayo espectrofotométrico como se describió previamente 11,12. </li><li> Determinar el porcentaje de membranas de mitocondrias intactas mediante el uso de las siguientes ecuaciones: (Actividad CS no sonicado ÷ sonicada CS actividad) * 100 </li></ol> 100- N (porcentaje alcanzado desde arriba) 6. Las mitocondrias Calidad de aislamiento; Western Blot (Hora: De acuerdo con el Protocolo de Laboratorio) <ol><li> Realizar transferencia de Western en el lisado de tejido y mitocondrias aisladas como se ha descrito previamente 12. Nota: Utilice los anticuerpos primarios para GAPDH (1: 1000 dilución en el 5% de BSA / TBS-T) y COXIV (1: 1000 dilución en el 5% sin grasaleche / TBS-T) seguido de anti-cabra y conejo anticuerpos secundarios, respectivamente (1: 10.000). </li></ol> 7. respirometría Ensayo Run (Tiempo: ~ 90 min) <ol><li> Realizar ensayos respirométricos deseados utilizando la tecnología de medición de consumo basado en una microplaca de O 2 como se ha descrito anteriormente (véase el artículo de compañía y Rogers et al 8). </li><li> Determinar la relación de control respiratorio (RCR) dividiendo Estado 3 por 4o Estado (RCR) o dividir 3u Estado por Estado 4o. Utilice el punto medio (promedio), o el más alto (estado 3) y tarifas más bajas (4o Estado) a partir de los restos de punto a punto en la determinación de RCR. </li></ol>

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

Los métodos presentados en este documento proporcionan una descripción detallada de un procedimiento de aislamiento mitocondrial a partir de cantidades mínimas (~ de 75-100 mg) de músculo esquelético de ratón. Este procedimiento de aislamiento es capaz de producir de alto funcionamiento, las mitocondrias pura (~ 450 g) como lo demuestra O 2 tasas de consumo, valores RCR, máxima actividad de la citrato sintasa y de expresión de proteínas de inmunotransferencia. Es importante destacar que las mitocondr...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</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 
			White Crystals or Crystalline Powder 
			&#8805;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&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</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 rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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 rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</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&#160;</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&#176;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, &#8805;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 rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">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>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</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

Mitocondrias del músculo esquelético disfuncionales juegan un papel en el metabolismo alterado observado con el envejecimiento, la obesidad y la diabetes tipo II. Ensayos respirométricos mitocondrial de preparaciones mitocondriales aislados permiten la evaluación de la función mitocondrial, así como la determinación del mecanismo (s) de acción de los fármacos y las proteínas que modulan el metabolismo. Procedimientos de aislamiento actuales requieren a menudo grandes cantidades de tejido para producir las mitocondrias de alta calidad necesarias para los ensayos de respirométricos. Los métodos presentados en este documento describen cómo de alta calidad mitocondrias purificadas (~ 450 g) se puede aislar a partir de cantidades mínimas (~ de 75-100 mg) de músculo esquelético de ratón para su uso en mediciones respiratorias de alto rendimiento. Se determinó que nuestro método de aislamiento produce 92,5 ± 2,0% mitocondrias intactas midiendo la actividad de la citrato sintasa espectrofotometría. Además, el análisis Western blot en mitocondrias aisladas resultó en la expresión débil del cytosoproteína lic, GAPDH, y la expresión robusta de la proteína mitocondrial, COXIV. La ausencia de una banda de GAPDH prominente en las mitocondrias aisladas es indicativo de poca contaminación de fuentes no mitocondriales durante el procedimiento de aislamiento. Lo más importante, la medición de la tasa de consumo de O 2 con tecnología basada en micro-placa y la determinación de la relación del control respiratorio (RCR) para los ensayos de respirométricos acoplado muestra altamente acoplado (RCR;&gt; 6 para todos los ensayos) y las mitocondrias funcionales. En conclusión, la adición de una etapa de picar carne separada y reduciendo significativamente impulsado por motor de velocidad homogeneización de un método reportado previamente ha permitido el aislamiento de alta calidad y mitocondrias purificada a partir de cantidades más pequeñas de ratón músculo esquelético que da como resultado en las mitocondrias altamente acoplados que respiran con alta función durante ensayos respirometirc basado en una microplaca.

La actividad de la citrato sintasa sirve como medida para la integridad de la membrana desde la citrato sintasa se ​​encuentra en la membrana mitocondrial interna, y por lo tanto no debe estar presente en las suspensiones de las mitocondrias con membranas intactas. La figura 1 representa la actividad de la citrato sintasa en muestras no se sonicaron mitocondriales en comparación con sonicado muestras de la misma aislamiento. Sonicando los resultados mitocondrias en un aumento estadísticamente sign...

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.

Aislamiento de mitocondrias a partir de cantidades mínimas de ratón músculo esquelético para mediciones de alto rendimiento de microplacas Respiratorias

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 vistas.  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

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.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

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

Investigación

Biología

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.

Aislamiento mitocondrial del músculo esquelético

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.

Purificación de los progenitores del músculo esquelético

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

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Aislamiento de Vaso sanguíneo derivado Multipotentes Precursores de músculo esquelético humano

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Aislamiento de mitocondrias intactas de músculo esquelético por centrifugación diferencial para Medidas respirometría de alta resolución

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Aislamiento y caracterización de exosomas de fibroblastos del músculo esquelético

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

Preparación de homogeneizados del músculo esquelético de rata para mediciones de nitratos y nitritos

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...