Este trabajo fue apoyado por una subvención del Instituto Nacional del Ojo (R01 EY12998) para FH Andrade.

1. Preparación de tampones <ol><li> Encienda 5804R centrífuga y se puso a 4 ° C. A su vez en baño de agua Isotemp 3006D y se puso a 37 º C. </li><li> Prepare las siguientes soluciones antes de que el aislamiento del músculo: <ul><li> PBS: Disolver tampón fosfato salino (PBS) tabletas en agua destilada (5 comprimidos / litro). Mezclar bien. </li><li> PBS más 10 mM EDTA: Para preparar una solución de 100 ml, añadir 2 ml de 500 mM EDTA y 98 ml de PBS. </li><li> Buffer 8X mitocondrias: 10,28 g de sacarosa, para una concentración final de 0,6 M, 400 mg de ácidos grasos libres de albúmina sérica bovina (BSA) para una concentración final de 0,8%, 2,08 g de HEPES para una concentración final de 160 mM, pH a 7.4 y QS de 50 ml con agua destilada. </li><li> Buffer de aislamiento 1 (IB1): Añadir 200 l de 500 mM EDTA para una concentración final de 10 mM, 0,392 g de D-manitol para una concentración final de 215 mM, 1,25 ml de buffer 8X mitocondrias pH, a 7.4 y QS a 10 ml con agua destilada. </li><li> Buffer aislamiento 2 (IB2): Añadir 60 l de EGTA 500 mM para una concentración final de 3 mm, 0.392 g de D-manitol para una concentración final de 215 mM, 1,25 ml de buffer 8X mitocondrias pH, a 7.4 y QS a 10 ml con agua destilada. </li><li> Buffer experimental (EB): añadir 100 ml de 500 mM de MgCl 2 para una concentración final de 5 mM, 0,392 g de D-manitol para una concentración final de 215 mm, 25 l de 2,5 mM KH 2 PO 4 para una concentración final de 6,25 M, 2 l de 100 mM EGTA, para una concentración final de 20 mM, 1,25 ml de solución tampón mitocondrial, el pH a 7.4 y QS de 10 ml con agua destilada. </li></ul></li></ol> 2. Aislamiento de los músculos <ol><li> En un cubo de hielo, poner tres vasos de 10 ml, Potter-Elvehjem molinos tejidos y todo lo necesario de otros instrumentos / suministros en el hielo. Todo debe estar helado durante todo el experimento. </li><li> Lugar IB1 y IB2 en un cubo de hielo y EB en el baño de agua caliente cuando haya terminado. </li><li> En los tres vasos de 10 ml, las soluciones se debe agregar: <ul><li> Matraz de 1: 10 ml de PBS </li><li> Vaso 2: 10 ml de PBS / EDTA </li><li> Vaso de 3: 3 ml de IB1 </li></ul></li><li> Matar a una rata humana, aprobado por el local de Atención de Animales institucional y el empleo. </li><li> Aislar rápidamente 250-500 mg de músculo esquelético, enjuague en el vaso 1, entonces la transferencia de vaso 2. </li></ol> 3. Homogeneización / aislamiento mitocondrial <ol><li> Transferencia de músculo para vaso 3 y finamente picada el músculo con la tijera. </li><li> Transferir la solución a la homogeneizador Potter-Elvehjem. Homogeneizar el músculo con un mortero de motor (un taladro puede hacer el trabajo) 10 veces manteniendo el tubo en hielo en todo momento. </li><li> Transferencia de homogeneizado de pre-enfriado tubos de microcentrífuga de 2 ml y centrifugar a 700 g durante 10 minutos a 4 ° C. </li><li> Transferir el sobrenadante a un tubo nuevo de microcentrífuga de pre-enfriado. Deseche pellet. </li><li> Centrifugar el sobrenadante a 10.500 g durante 10 minutos a 4 ° C. </li><li> Transferir el sobrenadante a un tubo nuevo de microcentrífuga de pre-enfriado y SN1 etiqueta (número sobrenadante 1) y el tipo de músculo. </li><li> Vuelva a suspender el pellet en 500 l de IB2. </li><li> Centrifugar a 10.500 g durante 10 minutos a 4 ° C. </li><li> Transferir el sobrenadante a un tubo nuevo de microcentrífuga de pre-enfriado y SN2 etiqueta (número sobrenadante 2) y el tipo de músculo. </li><li> Suspender el precipitado final mitocondrial en 100 l de IB2. </li><li> Volver a girar la suspensión definitiva mitocondrial en minifuge durante unos segundos. Si hay una bolita, la transferencia de sobrenadante a un tubo nuevo de microcentrífuga de pre-enfriado y desechar el sedimento. </li><li> Determinar la concentración de proteína utilizando el ensayo de Bradford 8. </li></ol> 4. La calibración de electrodos NOTA: La calibración del electrodo durante el aislamiento completo mitocondrial. <ol><li> Añadir 100 ml de agua destilada al matraz de 250 ml. Agitar vigorosamente durante 20 minutos para equilibrar el agua con gas atmosférico. </li><li> Añadir unas gotas de 50% de KCl con el electrodo Oxygraph. </li><li> Coloque un pedazo pequeño de papel azul Rizla rodando en la parte superior del electrodo. </li><li> Coloque un pedazo de membrana PTFE en la parte superior del papel de fumar. </li><li> Aplicar el anillo interior, utilizando el aplicador de anillo y coloque el anillo exterior en la ranura de los electrodos. </li><li> Conectar los electrodos y montar el resto del equipo: más grande base de anillo de plástico, cámara de las mitocondrias, y mangueras de agua. </li><li> A su vez en cajas de Oxygraph y comenzar software Oxygraph. </li><li> Agregue el agua a la cámara de equilibrio mitocondrial. </li><li> Añadir una barra de agitación y gire a una velocidad de 60. </li><li> Comenzar un nuevo experimento. </li><li> Haga clic en calibrar y calibración de fase líquida para el Cuadro 1. Los cambios de temperatura a 37 ° C y haga clic en &quot;Aceptar&quot; dos veces. </li><li> Cuando &quot;Override&quot; cambia a &quot;OK&quot; haga clic en él y abrir el tanque de gas nitrógeno. </li><li> Coloque la punta conectadosal tanque de nitrógeno en la cámara y establecer el oxígeno cero. Haga clic en &quot;Aceptar&quot; cuando se cambia de &quot;anulación&quot;. </li><li> Aspirar el agua y añadir 500 l de tampón EB a las cámaras de las mitocondrias. </li><li> Junta de la cámara con el émbolo. </li><li> Si utiliza varias cajas, repita los pasos 11-15 para la calibración de la caja 2. </li></ol> 5. La respiración mitocondrial <ol><li> Iniciar un nuevo experimento, se deja correr durante 1 minuto para estabilizar la señal. </li><li> Añada el volumen necesario de la suspensión mitocondrial (paso 3.11) por 150 g en cada cámara, evento marca, y se deja correr durante 1 minuto. </li><li> Añadir 10 l de agua tibia 250 mM/125 mM de glutamato / malato para una concentración final de 5 mM mM/2.5. Mark y deje correr durante 1 minuto. Esta se define como la etapa 2. </li><li> Añadir 7,5 l de 10 mM de ADP, para una concentración final de 150 M, la marca, y dejar funcionar durante 30 segundos. Esta se define como Estado 3. </li><li> Añadir otro 7,5 l de 10 mM de ADP, la marca, y deje correr durante 1,5 minutos. </li><li> Añadir 0,5 l de oligomicina frío de 10 mm para una concentración final de 1 M, marca, y dejar funcionar durante 3 minutos. Esta se define como Estado 4. </li><li> Añadir 1 l de frío mM FCCP 0.1 para una concentración final de 0,2 M, marca, y dejar funcionar durante 3 minutos. Esta se define como Estado 5. </li><li> Cuando haya terminado, al final experimento y guardar. </li><li> Adquirir las tasas de respiración utilizando el software Oxygraph. </li><li> Introduzca factor de normalización: si la adición de 150 mg de proteína mitocondrial, el factor de normalización será 0,15. </li><li> Calcular las tasas de respiración normalizado para los estados de respiración diferente. </li><li> Calcular la relación de control respiratorio (RCR), dividiendo por el Estado 3 Estado 4. </li></ol> 6. Los resultados representativos: <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="Figura 1" /> Figura 1. Muestra un representante de seguimiento del consumo de oxígeno por las mitocondrias del músculo esquelético. Después de la señal del electrodo se haya estabilizado, la muestra de las mitocondrias se añade a la cámara de Oxygraph. Estado 2 de respiración comienza con la adición de glutamato y malato. Además de ADP aumenta el consumo de oxígeno, la definición de estado de tres respiración. ATP sintasa está bloqueada por la adición de oligomicina para obtener el estado 4 de respiración. Por último, FCCP se añade a desacoplar la respiración mitocondrial (estado 5). La Tabla 1 muestra las tasas de consumo de oxígeno representante de los estados 2, 3, 4 y 5. La relación del control respiratorio (RCR) se calcula para cada experimento. RCR ≥ 4 se considera como evidencia de una preparación de mitocondrias viable. <table border="1"><tbody><tr><td> Estados </td><td> Tarifas normalizado </td></tr><tr><td> El estado 2 </td><td> 34.74 </td></tr><tr><td> Estado 3 </td><td> 153,40 </td></tr><tr><td> El estado 4 </td><td> 16.49 </td></tr><tr><td> Estado 5 </td><td> 143,70 </td></tr><tr><td> RCR </td><td> 9.30 </td></tr></tbody></table> Tabla 1. Las tasas de respiración mitocondrial. Representativeoxygen las tasas de consumo de las mitocondrias del músculo esquelético. Los valores se normalizan a la cantidad de mitocondrias añadido a la cámara. La relación del control respiratorio (RCR) se determina dividiendo el estado 3 en el estado 4. Un RCR ≥ 4 representa una preparación de mitocondrias viable.

<ol>
	<li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2, 287-295 (2007).</li><li>Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+mitochondria+in+health+and+disease.">Roles of mitochondria in health and disease.</a> Diabetes. 53, S96-S102 (2004).</li><li>Johannsen, D. L., Ravussin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+health+and+disease.">The role of mitochondria in health and disease.</a> Curr Opin Pharmacol. , (2009).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 65, 1-35 (2001).</li><li>Gamboa, J. L., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+content+and+distribution+changes+specific+to+mouse+diaphragm+after+chronic+normobaric+hypoxia.">Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia.</a> Am J Physiol Regul Integr Comp Physiol. , (2009).</li><li>Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+respiratory+capacity+in+extraocular+muscle+mitochondria:+evidence+for+intrinsic+differences+in+mitochondrial+composition+and+function.">Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.</a> Invest Ophthalmol Vis Sci. 50, 180-186 (2009).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

Se presenta un protocolo para aislar viable mitocondrias de los músculos esqueléticos. Si el rendimiento es un problema, el protocolo puede ser modificado mediante la incubación de los músculos aislados en 5 ml de tripsina PBS/10mM EDTA/0.01% durante 30 minutos en hielo. Para asegurar la digestión completa del músculo con tripsina, el músculo tiene que ser totalmente picada. Después de la incubación de 30 minutos, la solución PBS/10mM EDTA/0.01% de tripsina debe ser reemplazado por completo con 3 ml de tampón...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>95% CO2 / 5% O2 mix</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2754</td>
<td>&nbsp;</td>
<tr>
<td>Blue Rizla Paper</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>890101</td>
<td>&nbsp;</td>
<tr>
<td>Bradford protein assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Compressed nitrogen</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
<tr>
<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
<tr>
<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
<tr>
<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
<td>&nbsp;</td>
<tr>
<td>Isotemp 3006D</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Male Sprague Dawley Rats</td>
<td>&nbsp;</td>
<td>Harlan</td>
<td>300-500g</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
<td>&nbsp;</td>
<tr>
<td>Minifuge</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C1301P</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen electrode disc</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S1</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph Plus V1.01 Software</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pH-meter</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>1225506149</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8416</td>
<td>&nbsp;</td>
<tr>
<td>Potter-Elvehjem homogenizers</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-414-14A</td>
<td>&nbsp;</td>
<tr>
<td>PTFE (0.0125mm &#215; 25mm) membrane</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S4</td>
<td>&nbsp;</td>
<tr>
<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>		</tbody>
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mitochondrial isolation from skeletal muscle

Las mitocondrias son orgánulos que controlan la vida y la muerte de la célula. Participan en las principales reacciones metabólicas, síntesis de la mayor parte de la ATP, y regular una serie de cascadas de señalización 2,3. Los investigadores del pasado y actuales tienen mitocondrias aisladas de tejidos de ratas y ratones, como el hígado, cerebro y corazón 4,5. En los últimos años, muchos investigadores se han centrado en estudiar la función mitocondrial de los músculos esqueléticos. Aquí se describe un método que hemos utilizado con éxito para el aislamiento de las mitocondrias de los músculos del esqueleto 6. Nuestro procedimiento requiere que todos los tampones y reactivos son frescas y la necesidad de 250-500 mg de músculo esquelético. Hemos estudiado las mitocondrias aisladas de gastrocnemio de ratas y ratones y el diafragma y los músculos extraoculares rata. Concentración de proteína mitocondrial se mide con el ensayo de Bradford. Es importante que las muestras se mantendrán mitocondrial helado durante la preparación y que los estudios funcionales se realizará en un plazo relativamente corto (aproximadamente 1 hora). Respiración mitocondrial se mide utilizando la polarografía con un electrodo tipo Clark (sistema Oxygraph) a 37 ° C 7. La calibración del electrodo de oxígeno es un paso clave en este protocolo y debe realizarse diariamente. Mitocondrias aisladas (150 mg) se añadió a 0,5 ml de solución tampón experimental (EB). Estado 2 de respiración comienza con la adición de glutamato (5 mm) y malato (2,5 mM). Entonces, la adenosina difosfato (ADP) (150 M) se añade al estado de inicio 3. Oligomicina (1 M), un bloqueador de la sintasa de ATP-asa, se utiliza para estimar el estado 4. Por último, el cianuro carbonilo p-[trifluorometoxi]-fenil-hidrazona (FCCP, 0,2 M) se añade a measurestate 5, o no enganchados respiración 6. La relación del control respiratorio (RCR), la relación entre el estado 3 al estado 4, se calcula después de cada experimento. Un RCR ≥ 4 se considera como evidencia de una preparación de mitocondrias viable. En resumen, se presenta un método para el aislamiento de las mitocondrias viable de los músculos esqueléticos que pueden ser utilizados en estudios bioquímicos (por ejemplo, la actividad enzimática, inmunodetección, proteómica) y funcionales (respiración mitocondrial).

Watch this Scientific Journal Video about Mitochondrial Isolation from Skeletal Muscle at JoVE.com

Mitochondrial Isolation from Skeletal Muscle

Este protocolo describe un procedimiento para estudiar la respiración de las mitocondrias aisladas de los músculos esqueléticos. Este método fue adaptado de Scorrano<em&gt; Et al.</em&gt; (2007). El procedimiento de aislamiento mitocondrial requiere aproximadamente 2 horas. La respiración mitocondrial se puede completar en aproximadamente 1 hora.

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 ...

mitochondrial-isolation-from-skeletal-muscle

Research

JoVE Journal

Biology

28.8K vistas.  University of Kentucky. Este protocolo describe un procedimiento para estudiar la respiración de las mitocondrias aisladas de los músculos esqueléticos. Este método fue adaptado de Scorrano Et al. (2007). El procedimiento de aislamiento mitocondrial requiere aproximadamente 2 horas. La respiración mitocondrial se puede completar en aproximadamente 1 hora.

Video: Mitochondrial Isolation from Skeletal Muscle

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

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 ...

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

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

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 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 Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Aislamiento de cardiomiocitos ventriculares humanos de rebanadas de miocardio de corte de vibratome

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Aislamiento de mitocondrias del músculo esquelético de ratón para ensayos respirométricos

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.