1. Tejido Colección: <ol><li> Utilizar ratones de entre 7 y 12 semanas de edad. Por lo general, al menos dos animales se utilizan: uno es la creación de la FACS de un solo color controles necesarios para la compensación y la fluorescencia-menos-uno muestras de control de isotipo. La otra es la muestra real para la clasificación. </li><li> La eutanasia a los ratones por inhalación de CO 2 o de acuerdo con un protocolo de ética de la elección. Ratones lugar en una toalla de papel, mirando hacia arriba, y rociar con etanol al 70% a fondo para evitar la contaminación con la piel del ratón. </li><li> Levante la piel en la región abdominal con una pinza, a continuación, hacer un pequeño corte longitudinal con tijeras afiladas. </li><li> Pelar los dos colgajos de piel en direcciones opuestas (arriba y abajo) para exponer completamente los músculos de las extremidades posteriores por debajo. </li><li> Extirpar el tejido muscular mediante la inserción y la ampliación de las tijeras a ambos lados de la tibia. </li><li> Repita el mismo proceso para el fémur para extraer el tejido muscular. </li><li> Coloque todos los tejidos del músculo extirpado en placas petri de 60 mm, utilizando un plato para el tejido de cada ratón. </li><li> Repita los pasos anteriores para todos los ratones. </li></ol> 2. Disociarse del tejido en células individuales por digestión enzimática: A lo largo de esta sección del protocolo, utilice los reactivos estériles y las herramientas y el trabajo en condiciones estériles. <ol start="9"><li> Añadir 2 ml de colagenasa II (Sigma, cat # 6885, 500u / mL) y 20 l de 250 mm de CaCl2 acciones a cada plato de Petri. </li><li> Romper el tejido muscular en trozos pequeños (alrededor de 1 mm 3) con dos pinzas (una sierra, una con 2 dientes, de las herramientas de Ciencias Artes, cat # 11051-10, 11154-10), prestando atención a retirar y desechar lo más contaminadas no tejido muscular como sea posible. </li><li> Cubra la placas Petri y se incuba a 37 grados centígrados en 30 minutos. </li><li> Recuperar el placas Petri y el uso de un émbolo de una jeringa de 3 ml a fondo el puré de trozos de tejido. Use un émbolo para cada muestra para evitar contaminaciones. </li><li> Agregue un poco de (~ 5 ml) PBS estéril fría a cada plato de Petri y la transferencia de los lodos de tejido en un tubo de 50 ml Falcon (enjuagar el plato de Petri, si es necesario para recuperar todo el tejido) </li><li> Agite el tubo Falcon con fuerza, llenar el tubo con PBS, @ 800rpm x 5 minutos (Eppendorf modelo de banco: 5810R), y aspirar el sobrenadante (repetir 3 veces) </li><li> Agregue la mezcla de 1 ml de colagenasa D (Roche, Cat # 11088882001, 1,5 U / ml) / Dispasa II (Roche, Cat #: 04942078001, 2.4u / mL) y 10μL de CaCl 2 (250 mm) a cada tubo, se incuba a 37 grados C con rotación durante una hora. </li><li> Añadir 5-10 ml de frío, PBS estéril, vortex y pipetear bien para homogeneizar el tejido. </li><li> Llenar los tubos con PBS y mezclar bien. </li><li> Trasvasar el líquido a un filtro de 40μm celular colocado en la parte superior de un tubo Falcon de 50 ml para filtrar el resto de piezas grandes de tejido. Divide cada muestra de manera uniforme en tres tubos de 50 ml Falcon. Llene cada uno de los tubos con PBS y centrifugar @ 1.600 rpm durante 5 minutos a 8 grados centígrados. </li><li> Eliminar el sobrenadante y recoger las células de la tinción. </li></ol> 3. La tinción de anticuerpos y clasificación: A lo largo de esta sección del protocolo, el uso FACS estéril y tampones de recogida y trabajar en condiciones estériles. Anticuerpos comerciales generalmente se consideran estériles si se maneja adecuadamente. <ol start="20"><li> De un solo color y las muestras de control de isotipo, volver a suspender la muestra en tampón FACS 1 mL y se dividen las células en suspensión en 1,5 ml nueve pre-etiquetados tubos Eppendorf (100 l cada tubo). <ol><li> Establecer manchas de color se mezcla única de acuerdo con la siguiente tabla: ATENCIÓN: La concentración final de anticuerpos isotipo de control de la mancha debe coincidir con el anticuerpo primario son el control de. Por ejemplo, si el anti-CEC-1 se utiliza para una μg/10 6 celdas, su isotipo correspondiente debe ser utilizado en la misma concentración. <table border="1"><tbody><tr><td> Tubo # </td><td> Nombre del tubo </td><td> Empresa </td><td> Cat # </td><td> clon </td><td> isotipo </td><td> dilución </td></tr><tr><td> 1 </td><td> No mancha </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> eBioscience </td><td> 11-0311-85 </td><td> 390 </td><td> IgG2a de la rata </td><td> 500 </td></tr><tr><td> Casa hecha </td><td></td><td> I3 / 2 </td><td> Rata IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> Sca1-PECY7 </td><td> eBioscience </td><td> 25-5981-82 </td><td> D7 </td><td> IgG2a de la rata </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> Casa hecha </td><td></td><td> R2F2 </td><td> Rata IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Establecer un control de isotipo tinción mezcla con la siguiente tabla: <table border="1"><tbody><tr><td> Tubo # </td><td> Nombre del tubo </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Esca- PE-Cy7 </td><td> A-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Iso- CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Iso- Esca- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Iso- α-7-APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Pipeta de un solo color y la mezcla de isótopos tinción de control (por lo general, ajustar la concentración de anticuerpos, por lo que entre el 5 y 10 l de la tinción de la mezcla se añadió a cada muestra) en la correspondiente tubo Eppendorf a partir del paso 20. Mezclar bien e incubar en hielo durante 1 hora. </li></ol></li><li> Tinción de las células de ratón muestra con 800μL del cóctel de anticuerpos (abajo) para la clasificación de células FACS. Mezclar bien e incubar en hielo durante 1 hora. Cóctel de anticuerpos: Hoechst, PI, CD31-FITC, CD45-FITC, Sca1-PECY7, α-7 APC ATENCIÓN: La relación óptima de anticuerpos contra las células deben ser determinados por titulación individual de cada anticuerpo, como Pueden existir diferencias significativas entre los lotes. La cantidad óptima de anticuerpos que se utilicen deben ser expresados ​​en μgs de anticuerpos por 10 6 células diana y se mantiene constante cuando se utiliza el mismo lote de anticuerpos. Claramente, si el número de células diana no cambia significativamente entre los experimentos, o si el volumen de tinción se escala con el número de células, una dilución fija de cada anticuerpo también puede ser utilizado. </li><li> Lavado: Añadir aproximadamente 1 ml de solución tampón FACS para cada uno de los nueve tubos de control, agregar aproximadamente 20 ml de solución tampón FACS para el tubo de muestra. </li><li> Centrifugar el tubos Eppendorf @ 3000rpm durante 5 minutos (centrífuga Eppendorf banco de la top model,: 5417C). Centrifugar el tubo de la muestra @ 1.600 rpm durante 5 minutos (banco de centrifugadora de Eppendorf, Modelo: 5810R). </li><li> Aspirar y desechar el sobrenadante de todos los tubos. </li><li> Resuspender las células en el tampón FACS (1 ml por cada uno de los controles, 4 ml para la muestra de la clasificación), las células con una pipeta en &quot;tubos FACS&quot; a través del filtro celular tapa (gato BD Falcon # 352235, 40μm de tamaño de poro) para eliminar los grupos restantes que pueden afectar el citómetro. </li><li> Establecer el Clasificador de FACS con los controles de un solo color y los controles de isotipo. </li><li> Muestras de células ordenados de acuerdo a la siguiente tabla en un buffer de recolección de 3,5 ml (95% DMEM, 5% de SFB) por separado. Normalmente, cuando se cosecha dos patas traseras, un solo ratón de tipo salvaje podría producir alrededor de 150.000 células progenitoras miogénica o 100.000 células progenitoras adipogénico, con la viabilidad por encima del 95%, a juzgar por volver a analizar las células ordenados por FACS en el final del procedimiento (Figura 1 ) Definición de los progenitores y miogénica adipogénico sobre la base de tinción de la superficie <table border="1"><tbody><tr><td> Anticuerpo / mancha </td><td> Progenitoras miogénica (MP) </td><td> Progenitoras adipogénico (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> Sca1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Trasplante: <ol start="28"><li> Centrifugar las células ordenados a 1500 rpm durante 5 minutos, eliminar las células sobrenadante y traslado al autoclave los tubos Eppendorf (1,5 ml), lavar las células con 500 l de PBS estéril (suero gratis!), Centrifugar @ 3000rpm durante 5 minutos. Mantener todos los reactivos en condiciones estériles y trabajar en una campana de cultivo de tejidos, resuspender progenitores miogénica en PBS, o progenitoras adipogénico en Matrigel (BD # 354234 gato) en aproximadamente 10 6 células / ml. </li><li> Trasplante de progenitores o progenitoras miogénica adipogénico a los ratones receptores. <ol><li> Anestesiar a los ratones con isoflouran de acuerdo con su política de la institución. </li><li> Afeitar el vello de la región de inyección y luego esterilizar la piel por el roce con el 70% de etanol </li><li> Inyectar 20μL de los progenitores o progenitoras miogénica adipogénico (= 20k células) con una jeringa CC 03.10 insulina (gato BD # 309300). </li></ol></li><li> Después de un tiempo apropiado (tres semanas funciona bien, pero miofibras expresar derivadas del donante marcadores transgénicos son evidentes dentro de una semana después del trasplante), anestesiar a los ratones receptores del paso 29 por inyección intraperitoneal de 400 l Avertin (Sigma Cat. # T04840-2, 25 mg / ml), luego perfundir transcardialmente con 50 ml de PBS (contiene 10 mM de EDTA), seguido por 15 ml de PFA al 4%. Recoger tejido diana, después de arreglar con un 4% PFA durante la noche si es necesario, a continuación, transferir el 20% de sacarosa durante la noche. Insertar en octubre, congelar y cryosection. </li></ol> 5. Los resultados representativos: Cuando se trasplantan MP en el músculo esquelético del ratón, que fácilmente se fusionan con pre-existentes miofibras. Por lo tanto, cualquier etiqueta genética presente en las células del donante será fácilmente detectable en las fibras que los recibió. La figura 2 muestra un ejemplo donde las células trasplantadas expresado phostphatase alcalinas humanos, reveló histológicamente. Cuando AP se trasplantan el resultado depende en gran medida el medio ambiente del sitio de trasplante: cuando se trasplantaron estas células por vía subcutánea dan origen a los adipocitos y miofibroblastos (ver figura 2). En muchos otros sitios, incluyendo los músculos, no sobrevivirá a menos que la degeneración grasa ha sido inducido 3. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Figura 1" /> Figura 1. Clasificación de la estrategia para el aislamiento de las poblaciones progenitoras de músculo esquelético (A) La estrategia de clasificación:. Las células viables fueron identificados sobre la base de dispersión hacia adelante y dispersión lateral. Tinción Hoechst se utilizó para excluir los residuos anucleadas y tinción con yoduro de propidio (PI) para excluir a las células muertas. Hematopoyéticas (CD45) y células endoteliales (CD31), las células fueron excluidos de las puertas de la clasificación. El α7 + Hoechst + PI - CD45 - CD31 - Scal - subconjunto contiene todos los progenitores miogénica (MP). El Scal + Hoechst + PI - CD45 - CD31 - α7 - población contiene progenitores adipogénico (AP). (B) de fluorescencia-menos-uno (FMO) controles de isotipo confirmar la especificidad de la mancha. (C) Pureza cheques de subconjuntos MP y AP después de la clasificación. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Figura 2" /> Figura 2. Los resultados representativos siguientes MP y el trasplante de AP AP. Fueron trasplantados por vía subcutánea (arriba) y MP por vía intramuscular (panel inferior). En ambos casos, las células del donante se originó a partir de un ratón transgénico que expresa la fosfatasa alcalina humana, identificados por la coloración marrón.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

El objetivo de este protocolo es establecer un equilibrio razonable entre alto rendimiento y alta viabilidad de las células purificadas. El paso más crítico para asegurar que las células sanas se recuperan es, como era previsible, la disociación enzimática del tejido de partida. El manejo de los tejidos deben ser especialmente cuidadoso, lo cual es difícil de demostrar o aprecia incluso en un formato audiovisual. Otro factor clave es la duración del procedimiento. Cuanto más tiempo se tarda en ir desde el donante hasta el receptor de los animales, menor será la viabilidad y por lo tanto, la eficiencia de injerto. Si hubiera alguna duda sobre la viabilidad de que no es inmediatamente contestada por mirar a la frecuencia de eventos PI positivos en las muestras de células purificadas después de la clasificación, se sugiere que un ensayo de dilución límite para medir clonogenicidad se lleva a cabo 3. Clase típica de los músculos en buen estado habitualmente producen alrededor de un 15-20 en células capaces de iniciar colonias miogénica o fibro / adipogénico in vitro. Mientras que prácticamente el 100% de las colonias de los diputados iniciaron contienen miotubos diferenciados, sólo una tercera parte de las colonias obtenidas a partir de FAP contiene adipocitos, además de suave miofibroblastos actina de músculo positivo.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Colagenasa II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 U / ml </td></tr><tr><td> Colagenasa D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 U / ml </td></tr><tr><td> Plegar II </td><td> Roche </td><td> 04942078001 </td><td> 2.4 u / ml </td></tr><tr><td> células colador </td><td> BD </td><td> 352340 </td><td> 40 m </td></tr><tr><td> Tubo con filtro celular </td><td> BD </td><td> 352235 </td><td> 40 m </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2.5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Clon 390 </td></tr><tr><td> CD45-FITC </td><td> Hecho en casa </td><td></td><td> Clon I3 / 2 </td></tr><tr><td> Sca1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Clon D7 </td></tr><tr><td> α-integrina 7 APC </td><td> Hecho en casa </td><td> Consulte con Rossi laboratorio </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

El músculo esquelético contiene múltiples poblaciones progenitoras de distintos orígenes embrionarios y potencial de desarrollo. Progenitores miogénica, por lo general residen en una &quot;posición de las células satélite&quot; entre la membrana plasmática miofibrilar y la membrana basal laminina-ricos que se ensheaths, se auto-renovación de células que son los únicos comprometidos con el 1,2 linaje miogénico. Recientemente hemos descrito una segunda clase de progenitores buques asociados que pueden generar miofibroblastos y los adipocitos blancos, que responde a los daños causados ​​por la proliferación de entrar de manera eficiente y proporciona soporte trófico de las células miogénica en la regeneración de tejidos 3,4. Una de las pruebas más fiables para determinar el potencial de desarrollo y regeneración de una población determinada célula depende de su aislamiento y el trasplante de 5-7. Con este fin, hemos optimizado los protocolos para su purificación mediante citometría de flujo del músculo disociadas enzimáticamente, que vamos a esbozar en este artículo. Las poblaciones obtenidas con este método se contienen células colonia miogénica o fibro / adipogénico formación: no otros tipos de células son capaces de expandir in vitro o in vivo sobrevivir en la entrega. Sin embargo, cuando estas poblaciones se utilizan inmediatamente después de la clasificación para el análisis molecular (por ejemplo, QRT-PCR) se debe tener en cuenta que los subconjuntos recién ordenados pueden contener otras células contaminantes que carecen de la capacidad de formar colonias o injerto destinatarios.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Método para la disociación enzimática, el etiquetado de superficie y la purificación por citometría de flujo de fibro / adipogénico y progenitores miogénica del músculo esquelético murino.

Purificación de los progenitores del músculo esquelético

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

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K vistas.  University of British Columbia. Método para la disociación enzimática, el etiquetado de superficie y la purificación por citometría de flujo de fibro / adipogénico y progenitores miogénica del músculo esquelético murino.

Video: Purification of Progenitors 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

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 ...

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

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

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

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

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

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido lesiones de músculo esquelético embrionario de pez cebra

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 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 ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identificación, aislamiento y caracterización de progenitores fibroadipogénicos (FAP) y progenitores miogénicos (MP) en el músculo esquelético en la rata

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

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.