Damos las gracias a Laurie Destroismaisons y Sarah Peyrard de apoyo técnico excepcional y Dr.Alexandre Prat para el acceso al citómetro de flujo. También nos gustaría reconocer el Dr. Timothy Miller, por su contribución relativa a la retirada de la mielina de los preparativos. Esta labor fue apoyada por los Institutos Canadienses de Investigación en Salud (CIHR) Neuromuscular Asociación de Investigación de la Fundación Canadiense para la Innovación, Sociedad ALS de Canadá, la Fundación para la ELA Frick Investigación, Fundación CHUM y Fonds de la Recherche en Santé du Québec (CVV). Tanto CVV y NA son becarios de investigación del Fonds de la Recherche en Santé du Québec y CIHR Nuevos Investigadores. SP es apoyado por el Tim Noël Beca de la Sociedad ALS de Canadá.

Los animales utilizados en este estudio fueron tratados en estricta conformidad con un protocolo (N08001CVsr) aprobado por el l&#39;Université de Montréal (CRCHUM) Comité Centre de Recherche du Centre Hospitalier de de Institucional para la Protección de los Animales que sigue los estándares nacionales tal como se indica por la Canadian Consejo de los Animales (CCPA). Preparar todos los reactivos necesarios para llevar a cabo este protocolo (Tabla 1). Todos los demás detalles relacionados con el equipo, los suministros y proveedores se encuentran en la Lista de Materiales. <img alt="Tabla 1" fo:content-width="5in" src="/files/ftp_upload/51887/51887table1.jpg" width="500" /> Tabla 1. Buffer composiciones. 1. Colección de rata de la Médula Espinal <ol><li> Profundamente anestesiar la rata (Sprague Dawley) con 4% isoflorane. Verifique la anestesia por la falta de reflejo a pellizcos de jue pata. La eutanasia a las ratas por decapitación a través de la guillotina. Se prefiere este método de eutanasia sobre otros, que puedan distorsionar la médula espinal. </li><li> Cortar la piel de la espalda para exponer la columna vertebral. Cortar la columna vertebral con sus tijeras de hueso justo por encima del hueso de la pelvis. Visualice la apertura de la columna vertebral. </li><li> Inserte una jeringa de 10 ml, con una punta de pipeta de 200 l (unido a través de la fusión de un poco más de la llama), lleno de tampón fosfato salino (PBS), en la columna vertebral. </li><li> Enjuague la médula espinal mediante la aplicación de una cantidad media de la presión en el émbolo. </li><li> Si alguno de sangre está presente en la médula espinal, enjuagar con PBS antes de proceder al siguiente paso. NOTA: Este método también ha sido validado para el cerebro y el hígado. Si estos tejidos incluyendo, recoger media del cerebro de la rata sacrificados y un trozo de hígado igual en peso a la médula espinal. Todos los otros pasos permanecen idénticas. </li></ol> 2. Aislamiento de la médula espinal Mitochondria (Adaptado de Vande Velde et al. 4) <ol><li> Recoger toda la médula espinal y el lugar intacto en 5 ml homogeneizador de vidrio con 5 volúmenes (~ 3,25 ml) tampón de homogeneización (HB). Para una recuperación óptima de las mitocondrias aisladas, realizar todos los pasos en el hielo o en la cámara frigorífica. Homogeneizar el tejido a mano hasta que no hay grandes trozos de tejido permanecen, aproximadamente ocho golpes. Coloque homogeneizado en dos (2 ml) o tres (1,7 ml) tubos de microcentrífuga. Centrífuga 1300 xg durante 10 min a 4 ° C en una microcentrífuga de sobremesa. </li><li> Recuperar sobrenadante y colocar en un tubo de ultracentrífuga 5 ml. Añadir 750 l (~ 0,5 volúmenes) HB al tubo de microcentrífuga que contiene pellets y resuspender suavemente el pellet. Repita centrifugación y resuspensión pasos dos veces más. Piscina todos los sobrenadantes (S1a y S1b) en el mismo tubo de ultracentrífuga 5 ml por encima. Este paso sirve para eliminar los desechos pequeños. </li><li> Centrífuga agruparon S1 utilizando una ultracentrífuga equipado con una podredumbre basculanteo y centrifugar a 17.000 xg durante 15 min a 4 ° C. </li><li> Mantenga sobrenadante (S2) para su posterior procesamiento si la fracción citosólica es de interés (por ejemplo, para el análisis de Western blot). Resuspender el precipitado (P2), la fracción mitocondrial crudo, en 4 ml HB + 50 mM de KCl. Centrifugar 17.000 xg durante 15 min a 4 ° C en un rotor de cubeta oscilante. Descartar el sobrenadante y resuspender suavemente el pellet (P3) en 800 l HB. NOTA: Las mitocondrias se lavaron con mM KCl + HB 50 para eliminar cualquier contaminante asociados mitocondrialmente no específicos. </li><li> En un nuevo tubo de ultracentrífuga 5 ml, añadir exactamente 800 l de precipitado resuspendido (P3). </li><li> A este tubo agregar, 200 l de iodixanol (medio de gradiente de densidad), creando así una concentración final de 12% Iodixanol. Mezcle el contenido del tubo suavemente, pero a fondo a través de pipeteo con un P1000. La adición de iodixanol primero, y después se resuspendieron pellets (P3) pueden ser preferibles para facilitar la mezcla completa. Se centrifuga en un ultracentrifuge equipado con un rotor basculante a 17.000 xg durante 15 min a 4 ° C. </li><li> NOTA: Hígado no contiene mielina y, por tanto, este paso no es necesario si se está procesando sólo el hígado. Sin embargo, si el hígado se está procesando al mismo tiempo que las mitocondrias del SNC, se recomienda para el tratamiento de todos los tejidos por igual. </li><li> Aspirar la capa de mielina en la parte superior del tubo y cuidadosamente retirar y desechar el sobrenadante. Pellet puede estar suelto. Resuspender el precipitado en 4 ml HB. Centrifugar de nuevo a 17.000 xg durante 10 min a 4 ° C. Descartar el sobrenadante y resuspender el precipitado en 4 ml HB. Repetir la centrifugación y eliminar el sobrenadante. </li><li> Resuspender el sedimento final (P7) en 100-200 l HB y transferir a un tubo de microcentrífuga de 1,7 ml. Esta muestra contiene mitocondrias aisladas. </li><li> Proceder a la cuantificación de proteínas. Diluir las muestras y la curva estándar en el 2% de dodecilsulfato de sodio (SDS) para asegurar la solubilización adecuada de duri mitocondriasng cuantificación de proteínas. </li></ol> 3. Immunolabeling de mitocondrias aisladas de Citometría de Flujo <ol><li> Para cada mezcla de tinción a ensayar, una pipeta 25 g de mitocondrias aisladas en un tubo de microcentrífuga de 1,7 ml. Incluya una muestra sin mancha en cada experimento; y para cada anticuerpo, asegúrese de incluir una muestra para el control de isotipo apropiado. </li><li> Centrifugar a 17.000 xg durante 2 min a 4 ° C en una microcentrífuga de sobremesa. </li><li> Eliminar el sobrenadante y resuspender las mitocondrias aisladas en 50 l Buffer mitocondrias (M Buffer) suplementado con BSA libre de ácidos grasos 10% durante 15 min a 4 ° C (etapa de bloqueo). NOTA: Durante el etiquetado, realizar incubaciones en un refrigerador a 4 ° C. </li><li> Añadir anticuerpo primario (anti-conejo Mfn2, 20 g por ml) al tubo y se incuba durante 30 min a 4 ° C. NOTA: Determine la concentración óptima de cada anticuerpo empíricamente por titulación. Debido a la variabilidad en la concentración y / o pureza, diferentes lotes del mismo anticuerpo del mismo fabricante pueden llevar a diferentes resultados; Por lo tanto, se necesita de titulación para cada nuevo lote de anticuerpos. </li><li> Lave anticuerpo primario no unido: Se centrifuga a 17.000 xg durante 2 min a 4 ° C. Eliminar el sobrenadante y resuspender suavemente el sedimento en 200 l M Buffer. Se centrifuga a 17.000 xg durante 2 min a 4 ° C. Aspirar el sobrenadante y resuspender el precipitado en 50 l M Buffer. </li><li> Añadir anticuerpo secundario (anti-conejo de burro IgG ficoeritrina (PE), 0,5 g por ml) al tubo e incubar las muestras durante 30 min a 4 ° C, protegido de la luz. </li><li> Lave anticuerpo secundario no unido: Se centrifuga a 17.000 xg durante 2 min a 4 ° C. Eliminar el sobrenadante y resuspender el sedimento en 200 l M Buffer. Se centrifuga a 17.000 xg durante 2 min a 4 ° C. Aspirar el sobrenadante y resuspender el sedimento en 500 l M Buffer. </li><li> Para asegurarse de eventos están en la mitocondria hecho, mancha aislada mitocondrias con unamitocondrias colorante fluorescente específico para 15 min a RT, protegido de la luz. Si se desea la tinción de otros parámetros funcionales (potenciales transmembrana mitocondrial o de producción de superóxido), continúe con el paso 4 Si no es así, continúe con el paso 5 para la adquisición. NOTA: Es importante verificar que el espectro de emisión del anticuerpo secundario es compatible con la de los colorantes funcionales. Por ejemplo, si la verificación de la pureza mitocondrial con un colorante comercial con propiedades espectrales similares a FITC y potencial transmembrana con éster metílico de tetrametilrodamina (TMRM), un anticuerpo secundario viable sería aloficocianina (APC: Ex 650 nm / Em 660 nm). Añadir compensaciones controles, es decir, una muestra immunolabeled o teñido con un solo fluoróforo, cuando sea aplicable. </li><li> Transferir a un tubo adecuado para el flujo de carga citómetro. (Para facilitar el tamaño de muestra pequeño, un tubo de microtitulación se coloca dentro de 3 ml citómetro de flujo del tubo.) Mantener las muestras en hielo y se procederá de inmediato acitómetro de flujo para la adquisición. </li></ol> 4. Dosificación mitocondrial potencial transmembrana mitocondrial de superóxido y de Producción por Citometría de Flujo <ol><li> Verificar que las mitocondrias aisladas tienen un potencial transmembrana intactas mediante tinción con 100 nM TMRM (Ex 548 nm / Em 574 nm) 5, en el paso 3.8, durante 15 min a RT, protegido de la luz. Para la comparación de potencial transmembrana entre las muestras y las poblaciones, el uso de concentraciones menores / no extinción de la TMRM (1 a 30 nM), puede ser más apropiado 6. <ol><li> Como control para la tinción TMRM, aislado mancha mitocondrias con 100 nM TMRM en presencia de 100 mM fenil cianuro de carbonilo m-cloro hidrazina (CCCP), un desacoplador mitocondrial que despolarizar las mitocondrias. La concentración de CCCP requerido para despolarizar las mitocondrias puede ser menor si se utilizan concentraciones más bajas de tinte. </li></ol></li><li> Verifique que las mitocondrias aisladas producen superóxido mitocondrial por staining con un indicador de superóxido mitocondrial apropiado 7, también en el paso 3.8, durante 15 min a RT, protegido de la luz. <ol><li> Como control para la producción de superóxido mitocondrial, aislado mancha mitocondrias con el tinte en presencia de 10 mM antimicina A, un inhibidor del complejo III de la cadena respiratoria que aumentar la producción de superóxido mitocondrial. </li></ol></li></ol> 5. Adquisición y Análisis de immunolabeled mitocondrias aisladas por citometría de flujo <ol><li> Instrumento creado: tensiones cambia de lineal a modo de registro para facilitar el análisis de las mitocondrias aisladas y voltajes establecidos (FSC: 450; SSC: 250). Asegúrese de que los eventos se recogen en el modo de FSC-A (área), así como FSC-W (ancho) y FSC-H (altura), para poder excluir dobletes (es decir, dos acontecimientos, que pasan por el detector al mismo tiempo ) en la recopilación de post-análisis de datos. Establecer el número de eventos que se percibe para 100.000. Adquirir los controles de compensación, En su caso. </li><li> Adquisición de Datos: Antes de la adquisición de datos, evitar vortex muestras. En lugar mezclar golpeando suavemente el tubo. Inicialmente recopilar eventos en una baja presión, durante gating. Puerta en la población total. Ajuste tensiones de histogramas en consecuencia, por lo general el pico de la muestra sin teñir corresponderá a la segunda década (10 2). Una vez establecidas las puertas, y se están procesando las muestras, la presión puede ser cambiado a alta. </li><li> Análisis: Visualice dobletes trazando FSC W frente a FSC. Identificar singletes y dobletes. Puerta en interiores. Seleccione la población mitocondrial, colocando puertas en los eventos que se tiñen positivamente con un tinte mitocondrial selectiva. </li><li> Determinar el etiquetado de fondo de control de isotipo. Usando la muestra de control de isotipo, determinar el porcentaje de la población mitocondrial etiquetado positivo para el anticuerpo Mfn2. </li></ol>

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Los autores no tienen nada que revelar.

Es cada vez más evidente que las mitocondrias son actores clave en la fisiología normal y la enfermedad. Mientras inmunotransferencia puede determinar qué proteínas se encuentran dentro de las mitocondrias o en la superficie mitocondrial en una cierta condición, este método informes sobre la media de toda la población. Este método no puede dar información sobre la abundancia relativa de las subpoblaciones o subconjuntos mitocondriales. Si bien ha sido asumido previamente que todas las mitocondrias son iguales, ...

Las mitocondrias son orgánulos muy dinámicos que se someten a múltiples rondas de la fisión y la fusión, son transportados a sitios de alta demanda energética y responder rápidamente a los estímulos fisiológicos 1. Dado que se reconoce cada vez más que las mitocondrias dentro de los diferentes tejidos, incluso diferentes compartimentos celulares, tienen perfiles funcionales distintos, se necesitan nuevos métodos para identificar estos subconjuntos mitocondriales distintos. Microscopía proporciona un medio por el cual las mitocondrias individuales se pueden visualizar y la presencia de una proteína en, o en las mitocondrias puede ser determinada por inmunofluorescencia 2. Sin embargo, el análisis cuantitativo por este método es laborioso y es más adecuado para experimentos utilizando líneas celulares inmortalizadas o primarios. El estudio de las mitocondrias individuales derivados de tejido es significativamente más difícil y la mayoría de los métodos no permiten una fácil identificación de subconjuntos mitocondriales simultáneamente con la evaación de la función mitocondrial 3. Para hacer frente a este obstáculo, un nuevo método para inmunomarcador mitocondrias aisladas de tejidos de roedores y posteriormente analizadas por citometría de flujo se ha desarrollado. Esto permite la rápida detección y cuantificación de proteínas localizadas en la membrana mitocondrial externa, que en comparación con el análisis por microscopía, es mucho menos mano de obra intensiva y permite el análisis de miles de mitocondrias en una sola muestra. Este ensayo se puede aplicar para controlar el destino y la cantidad relativa de proteínas de la membrana externa mitocondrial que se cree que son constitutivamente presente en la mitocondria, el reclutamiento de proteínas a la superficie mitocondrial, o la detección de proteínas mislocalized a la mitocondria en condiciones patológicas. Por otra parte, la incorporación de colorantes indicadores fluorescentes convencionales permite la evaluación simultánea de ciertos aspectos de la función mitocondrial en distinta mitocondrialsubpoblaciones.

<table border="0" cellpadding="0" cellspacing="0" width="1276">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Name of Material/ Equipment</td>
			<td style="width:349px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:543px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:267px;">Rats</td>
			<td style="width:349px;">Charles River</td>
			<td style="width:119px;">Strain code 400</td>
			<td style="width:543px;">Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dounce homogenizer</td>
			<td style="width:349px;">Kontes Glass Co.&#160;</td>
			<td style="width:119px;">885450-0022</td>
			<td>Duall 22</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Microcentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall Legend Micro 17 R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Ulara-Clear Ultracentrifuge tubes</td>
			<td style="width:349px;">Beckman Coultier</td>
			<td style="width:119px;">344057</td>
			<td>Transparent, thin walled&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sorvall Ultracentrifuge</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>Sorvall WX UltraSeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">AH-650 rotor and buckets&#160;&#160;</td>
			<td style="width:349px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:267px;">Opti-prep</td>
			<td style="width:349px;">Axis-Shield</td>
			<td style="width:119px;">1114542</td>
			<td>Iodixanol, density gradient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Fatty acid free Bovine Serum Albumin</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8806</td>
			<td>Must be fatty acid free for mitochondria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Sodium succinate dibasic hexahydrate&#160;&#160;</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">S9637</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:267px;">Rabbit anti-Mitofusin2 antibody</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">M6319</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:267px;">Rabbit IgG</td>
			<td style="width:349px;">Jackson Immuno Research&#160;</td>
			<td style="width:119px;">011-000-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Anti-Rabbit IgG PE</td>
			<td style="width:349px;">eBioscience</td>
			<td style="width:119px;">12-4739-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Micro titer tube</td>
			<td style="width:349px;">Bio-Rad</td>
			<td style="width:119px;">223-9391</td>
			<td>For sample acquisition by flow cytometry&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoTrackerGreen (MTG)</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M7514</td>
			<td>100 nM: Ex490 nm/Em516 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">TMRM</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">T668</td>
			<td>100 nM: Ex548 nm/Em574 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CCCP</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">C2759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MitoSOX Red</td>
			<td style="width:349px;">Invitrogen</td>
			<td style="width:119px;">M36008</td>
			<td>5 &#181;M: Ex540 nm/Em600 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Antimycin A</td>
			<td style="width:349px;">Sigma</td>
			<td style="width:119px;">A8874</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">LSR II flow cytometer</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">BS FACSDiva Software</td>
			<td style="width:349px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FlowJo</td>
			<td style="width:349px;">TreeStar Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Software used for analysis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:267px;">BCA protein assay kit&#160;&#160;</td>
			<td style="width:349px;">Pierce/Thermo Scientific</td>
			<td style="width:119px;">23225</td>
			<td style="width:543px;">Bradford assay is not recomended as it is not compatible with high concentrations of SDS</td>
		</tr>
	</tbody>
</table>

immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria

Métodos para detectar y controlar los componentes de proteína de membrana externa mitocondrial en los tejidos animales son vitales para estudiar la fisiología mitocondrial y la fisiopatología. Este protocolo describe una técnica donde las mitocondrias aisladas de tejido de roedores se immunolabeled y se analizaron por citometría de flujo. Las mitocondrias se aislaron a partir de roedores médulas espinales y se sometieron a una etapa de enriquecimiento rápido a fin de eliminar la mielina, un contaminante principal de las fracciones mitocondriales preparadas a partir de tejido nervioso. Mitocondrias aisladas se marcaron a continuación con un anticuerpo de elección y un anticuerpo secundario conjugado con fluorescencia. Análisis por citometría de flujo verifica la pureza relativa de las preparaciones mitocondriales por tinción con un colorante específico mitocondrial, seguido por la detección y cuantificación de la proteína immunolabeled. Esta técnica es rápida, cuantificable y de alto rendimiento, lo que permite el análisis de cientos de miles de mitocondrias por muestra. Es aplicable para evaluar novela proteins en la superficie mitocondrial en condiciones fisiológicas normales, así como las proteínas que pueden convertirse en mislocalized a este orgánulo durante la patología. Es importante destacar que este método puede ser acoplado a colorantes indicadores fluorescentes para informar sobre ciertas actividades de las subpoblaciones mitocondriales y es factible para las mitocondrias del sistema nervioso central (cerebro y médula espinal), así como el hígado.

Las mitocondrias derivadas de médula espinal de rata se pueden immunolabeled con un anticuerpo dirigido a Mitofusin2 (Mfn2), una proteína implicada en la fusión de la membrana externa de las mitocondrias 8. Tras el aislamiento y el etiquetado con un anticuerpo específico Mfn2 y un anticuerpo secundario conjugado con fluorescencia, las mitocondrias son procesados ​​por citometría de flujo (Figura 1). Después de la adquisición de datos, las muestras se analizaron mediante citometría...

Watch this Scientific Journal Video about Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria at JoVE.com

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Se describe aquí es un método para detectar y cuantificar proteínas de la membrana externa mitocondrial por inmunomarcaje de las mitocondrias aisladas de tejido de roedores y análisis por citometría de flujo. Este método puede ampliarse para evaluar los aspectos funcionales de subpoblaciones mitocondriales.

La inmunodetección de la membrana externa proteínas por Citometría de Flujo de mitocondrias aisladas

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

immunodetection-outer-membrane-proteins-flow-cytometry-isolated

Research

JoVE Journal

Biology

15.4K vistas.  Université de Montréal, CRCHUM.  Se describe aquí es un método para detectar y cuantificar proteínas de la membrana externa mitocondrial por inmunomarcaje de las mitocondrias aisladas de tejido de roedores y análisis por citometría de flujo. Este método puede ampliarse para evaluar los aspectos funcionales de subpoblaciones mitocondriales. 

Video: Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP-FCM: inmunoprecipitación detectada por citometría de flujo

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Investigación

Biología

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

La medición cuantitativa de la translocación de GLUT4 a la membrana plasmática por citometría de flujo

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

Citometría de flujo de purificación de las células de ratón meiótica

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Recolección y enfriamiento Cryo-cristales de proteínas de membrana cultivadas en mesofases lipídicas para la Determinación de la estructura por cristalografía macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parámetro de medición de la apertura del poro de transición de permeabilidad en el corazón del ratón mitocondrias aisladas

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimizado manchas y Proliferación métodos de modelización para la vigilancia del uso de tintes División Celular Celulares de seguimiento

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles.
Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.

Here, a protocol (MitoCeption) is presented to transfer mitochondria, isolated from human mesenchymal stem cells (MSC), to glioblastoma stem cells (GSC), with the goal of studying their biological effects on GSC metabolism and functions. A similar protocol can be adapted to transfer mitochondria between other cell types.

MitoCeption: Transferencia hermoso humano MSC mitocondrias de las células madre de glioblastoma

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...