Este trabajo fue apoyado por el Instituto Nacional del Envejecimiento (AG04418), Instituto Nacional sobre Abuso de Drogas, Instituto Nacional de Salud-Karolinska Institutet Programa de Postgrado asociaciones, Karolinska Institutet, Consejo de Investigación sueco, el poder del cerebro de Suecia, y la Fundación Cerebro de Suecia. Muchas gracias a Mattias Karlen y el Dr. Giuseppe Coppotelli de apoyo creativo con la Figura 1 y 2, respectivamente; Karin Pernold de asistencia técnica, y los Dres. Barry J. Hoffer, Lars Olson, y Nils-Göran Larsson para muchos consejos útiles y la discusión.

1. Preparación de tejidos para muestras criostáticas <ol><li> El sacrificio del animal por dislocación cervical o decapitación, en conformidad con el permiso ético disponibles. </li><li> Rápidamente recoger tejidos de interés (por ejemplo. Cerebral) y la velocidad de congelación del hielo seco (tejidos puede requerir congelación en isopentano o propano refrigerado con nitrógeno líquido para obtener la morfología óptima). Tejidos almacenar en papel de aluminio a -80 ° C hasta que esté listo a la sección. </li><li> Insertar tejido congelado en la preparación de muestras criostáticas. </li><li> Recoger de 14 micras secciones congeladas a -21 ° C (puede ser necesario ajustar la temperatura de ± 2.1 ° C). Secciones de deshielo en las diapositivas con un calentador, y se desliza tienda sin cubreobjetos a -20 ° C hasta su utilización. </li></ol> 2. COX histoquímica <ol><li> Permitir que se sequen a temperatura ambiente durante 1 hora. Poner los portaobjetos en una cámara de diapositivas-tinción con papel de filtro húmedo, cortada en tiras. Para obtener consistenciaresultados t en cada experimento, se recomienda para procesar un máximo de diez diapositivas por experimento para reducir al mínimo los retrasos. </li><li> Prepare en una campana química 1X DAB, 100 mM citocromo c en PBS 0,1 M pH = 7,0. Vortex rápidamente. </li><li> Añadir 2 g de bovino catalasa (2 mg ml -1 o IU aproximadamente 4 ml -1). Mezclar adecuadamente por agitación para romper todos los granos de la catalasa. </li><li> Aplicar 150 a 200 l de medio de incubación en cada diapositiva, utilice punta de la pipeta para repartir uniformemente en todas las secciones. </li><li> Incubar los portaobjetos durante 40 minutos a 37 º C. </li><li> Retire el exceso de solución de las diapositivas. Lavar los portaobjetos 4 veces, 10 minutos cada vez, en PBS 0,1 M pH = 7,0. </li><li> Volver a la cámara de diapositivas diapositivas-tinción con tiras de papel mojado. </li></ol> 3. SDH histoquímica <ol><li> Prepare en una campana química 1,5 mM de NBT, 130 mM succinato de sodio, 0,2 mM de PMS, y 1,0 mM de azida sódica en PBS 0,1 M pH = 7,0. Tome la precaución de proteger a losPMS de la luz. Vortex rápidamente. </li><li> Aplicar 150-200 L de medio de incubación en cada diapositiva, utilice punta de la pipeta para repartir uniformemente en todas las secciones. </li><li> Incubar los portaobjetos durante 40 minutos a 37 º C. </li><li> Retire el exceso de solución de las diapositivas. Lavar los portaobjetos 4 veces, 10 minutos cada vez, en PBS 0,1 M pH = 7,0. </li><li> Deshidratar las diapositivas durante 2 minutos en las siguientes concentraciones de etanol: 70%, 70%, 95%, 95%, el 99,5%. A continuación, espere 10 minutos en un paso adicional del 99,5%. </li><li> Coloque los portaobjetos en xileno durante 10 minutos. Montar con Entellan y cubreobjetos. Deje que el que se sequen horas de la noche, o 2.1 por lo menos en un área ventilada. </li></ol> 4. Determinación de la disfunción mitocondrial <ol><li> El monto de la disfunción mitocondrial se indica por la cantidad de manchas azules celular. Para semi-cuantificar estas cantidades, las diapositivas debe ser codificado y se visualizó bajo el microscopio de campo brillante. Semi-cuantificación se debe realizar en una persiana<html> base en una escala, por ejemplo, 0-4 (0, ausencia de manchas azules, 4, sólo la tinción de azul). Lo mejor es realizar este tipo de semi-cuantificación de varias secciones de un tema determinado / animal para calcular un valor medio para cada sujeto / animal. </li><li> Las estadísticas se debe realizar mediante una prueba no paramétrica, como la de Mann-Whitney o Kruskal-Wallis. </li></ol> 5. Controles apropiados especificidad <ol><li> Para los controles de la especificidad de la actividad COX, repita &quot;COX histoquímica&quot; pasos, y añadir 2,5 mM de azida sódica, un inhibidor de la cadena respiratoria terminal. </li><li> Para los controles de la especificidad de la actividad SDH, repita &quot;SDH histoquímica&quot; medidas con la eliminación de succinato de sodio y la adición de 50 mM malonato, un inhibidor competitivo de la SDH. </li><li> Lave y deshidratar las secciones en una serie de etanol, y luego montar y cubreobjetos las diapositivas tal como se describe en los pasos 3,4 a 3,6. </li></ol> 6. Los resultados representativos: tienda de campaña &quot;&gt; El esquema general de los ejemplos representativos COX / SDH doble etiquetado ensayo histoquímico se ilustra en la Figura 2. apropiado de la COX / SDH doble etiquetado histoquímica en secciones de cerebro de tipo salvaje y envejecimiento prematuro de los ratones mutadores ADNmt se muestra en la Figura 3. Las manchas de color marrón oscuro en ratones de tipo salvaje (Figura 3, panel izquierdo) mostraron actividad normal de la COX. Las células con deficiencias de la cadena respiratoria, indicada por la tinción de azul, se dieron a conocer en 12 semanas de edad, los ratones mutadores ADNmt, y se convirtió en estas deficiencias más amplia que los ratones mutadores ADNmt entre a 46 semanas (Figura 3, el centro y el panel de la derecha). Ejemplos de inadecuado COX / SDH de doble etiquetado en las secciones de cerebro de ratones de tipo salvaje debido a la falta de etiquetado COX se muestran en la Figura 4. Tiempo de incubación inadecuados para la demostración de la actividad de la COX, o la reducción de la disponibilidad de oxígeno molecular por el cubreobjetos de la diapositiva durante la incubación, se tradujo en una reducción de la deposición de la reacción DABproducto de iones, y por lo tanto permite la formación del producto final azul formazán durante la incubación SDH. Actividades COX y SDH también puede ser investigado por separado (Figura 5, izquierda y centro), sin embargo, el etiquetado secuencial es útil para identificar las células con deficiencias de la COX, debido a la formación del precipitado de color azul durante la incubación SDH (Figura 3, el centro y derecha). Los controles de la especificidad de las actividades COX y SDH también se puede hacer (Figura 5, derecha). <img alt="Figura 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> Figura 1. Respiratoria mitocondrial IV complejos. La cadena respiratoria mitocondrial se encuentra dentro de la membrana interna e incluye cinco complejos. El propósito de la cadena respiratoria es el transporte de electrones del Complejo I a IV, y al hacerlo se crea un gradiente de protones a través de la membrana interna utilizada por el complejo V (ATPasa) para producir ATP. Hexágonos represe Rednt subunidades codificadas por el ADNmt. Hexágonos blancos representan las subunidades codificadas por el ADN nuclear (tenga en cuenta que el complejo II está completamente codificada del genoma nuclear). Por lo tanto, las mutaciones en el genoma mitocondrial pueden causar una disfunción de la cadena respiratoria debida a mutaciones en las subunidades de los complejos de la cadena respiratoria. <img alt="Figura 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> Figura 2. Diagrama de flujo del ensayo de COX / SDH de doble etiquetado de histoquímica. Diseccionar los órganos de interés, congelar rápidamente los tejidos en hielo seco, y almacenarlas a - 80 ° C. Recoger secciones de criostato y mantener a - 20 ° C hasta su uso. Permitir que las secciones se sequen al aire a temperatura ambiente durante 1 hora. Preparar el medio de incubación para la histoquímica de la COX, que se aplican a las diapositivas, y se incuba durante 40 minutos a 37 º C. Lavar las secciones en PBS 4 veces durante 10 minutos cada lavado. Preparar el medio de incubación para la histoquímica SDH, que se aplican a la slides, y de nuevo se incuba durante 40 minutos a 37 ° C. Lavar las secciones de nuevo en PBS, se deshidrata en una serie de etanol, y luego montar y cubreobjetos de las diapositivas. La COX / SDH de doble etiquetado secciones está listo para ver en el microscopio de campo brillante en 1-2 horas. <img alt="Figura 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> Figura 3. Ejemplos representativos de la COX / SDH de doble etiquetado. Secciones de cerebro de tipo salvaje y envejecimiento prematuro de los ratones mutadores ADNmt se etiquetarán secuencialmente para las actividades de la COX y SDH. (Barra de escala:. 200 micras) normal actividad de la COX (indicado por el color marrón oscuro) se muestra en el hipocampo de ratones de tipo salvaje (izquierda). Las deficiencias de la COX (indicado por el color azul) se dieron a conocer en el hipocampo de los ratones mutadores ADNmt (centro y derecha). Hubo una nueva disminución en la actividad de la COX por 46 semanas de edad en el ADNmt ratones mutadores, lo que sugiere la exacerbación de la disfunción generalizada de la cadena respiratoria. El mito observadoLa disfunción mitocondrial en los ratones mutadores ADNmt 12 es causada por niveles altos de mutaciones puntuales del mtDNA, así como mayores niveles de eliminaciones lineal 5. <img alt="Figura 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> Figura 4. Ejemplos de inadecuado COX / SDH de doble etiquetado. Secciones de cerebro de ratones de tipo salvaje fueron etiquetados de forma secuencial para las actividades de la COX y SDH. (Barra de escala:. 200 micras) veces inadecuada de incubación (10 y 25 minutos) para la demostración de la actividad COX dio lugar a una reducción de la deposición del producto de reacción marrón DAB, en comparación con el tiempo de incubación de 40 minutos (izquierda y centro). El acortar los tiempos de incubación permitió la formación del producto final azul formazán durante la incubación SDH, engañosamente que sugiere la presencia de células con deficiencias de la COX. Cubreobjetos las diapositivas durante la incubación de la COX también dio lugar a la formación de inexacta y la deposición de la reacción DABiones producto (derecha). <img alt="Figura 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> Figura 5. INDIVIDUALES COX y SDH etiquetado y el control de la especificidad. Secciones de cerebro de ratones de tipo salvaje fueron etiquetados por separado para las actividades COX y SDH, indicado por el color marrón oscuro y el color azul, respectivamente (izquierda y centro). Aunque las actividades de la COX y SDH puede ser etiquetados individualmente, el etiquetado secuencial ha demostrado ser ventajoso en la localización de las células con la disfunción mitocondrial. Un ejemplo de un control específico de la COX y SDH actividades en el cerebro de un ratón de tipo salvaje mostró ausencia de etiquetado (derecha). (Barra de escala:. 200 micras)

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La combinación de la COX / SDH método histoquímico permite la visualización de las células con la disfunción mitocondrial. Esta técnica, con los primeros estudios se remontan a 1968, sigue siendo popular, con muchos teniendo en cuenta el &quot;gold standard&quot; para la identificación de las enfermedades mitocondriales en los pacientes 14,19,26,27. Ahora se utiliza con frecuencia para investigar ADNmt mutación impulsada por el envejecimiento y el envejecimiento de los trastornos relacionados con <su...

<table border="1"><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> De hielo seco </td><td> AGA Gas AB </td><td> bloque de forma </td><td></td></tr><tr><td> Isopentano (2-metilbutano) </td><td> Sigma-Aldrich </td><td> 277258 CAS: 78-78-4 </td><td></td></tr><tr><td> Cyrostat solución incrustación </td><td> Sakura Finetek </td><td> Tissue Tek 4583 </td><td></td></tr><tr><td> Criostato </td><td> Microm </td><td> Microm modelo HM 500M </td><td></td></tr><tr><td> Diapositivas </td><td> Thermo Scientific </td><td> Además súper helada Menzel Gläser J1800AMWZ </td><td></td></tr><tr><td> Cubreobjetos Vidrio de borosilicato </td><td> VWR International </td><td> 16004-098 </td><td> 24 x 50 mm </td></tr><tr><td> El filtro de papel </td><td> Munktell filtro AB </td><td> Calidad: 1350 ArtículoNúmero: 242 001 </td><td> 430 x 430 mm </td></tr><tr><td> 3,3 &#39;-diaminobenzidina tetrahidrocloruro (DAB) </td><td> Sigma-Aldrich </td><td> Sigma sistema de sustrato líquido, D7304 </td><td></td></tr><tr><td> Citocromo c (tipo III, del corazón de equino) </td><td> Sigma-Aldrich </td><td> C2506 CAS: 9007-43-6 </td><td></td></tr><tr><td> Bovina catalasa (de hígado) </td><td> Sigma-Aldrich </td><td> C9322 CAS: 9001-05-2 </td><td></td></tr><tr><td> Nitroazul de tetrazolio (NBT) </td><td> Sigma-Aldrich </td><td> N6876 CAS: 298-83-9 </td><td></td></tr><tr><td> Succinato sódico </td><td> Sigma-Aldrich </td><td> S2378 CAS: 6106-21-4 </td><td></td></tr><tr><td> Fenazina metosulfato (PMS) </td><td> Sigma-Aldrich </td><td> P9625 CAS: 299-11-6 </td><td> PMS es sensible a la luz. Escudo de la luz. </td></tr><tr><td> Azida de sodio </td><td> Sigma-Aldrich </td><td> S8032 CAS: 26628-22-8 </td><td></td></tr><tr&gt; <td> Xileno </td><td> VWR International </td><td> EM-XX0060-4 </td><td></td></tr><tr><td> Entellan </td><td> VWR International </td><td> 100503-870 </td><td></td></tr><tr><td> Malonato (Ácido malónico) </td><td> Sigma-Aldrich </td><td> M1296 CAS: 141-82-2 </td><td></td></tr></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

El ADN mitocondrial (ADNmt) los defectos son una causa importante de enfermedad y puede ser la base del envejecimiento y el envejecimiento, alteraciones 1,2. La teoría mitocondrial del envejecimiento sugiere un papel de mutaciones en el ADNmt, que pueden alterar la homeostasis de la bioenergética y la función celular, en el proceso de envejecimiento 3. Una gran cantidad de pruebas ha sido elaborado en apoyo de esta teoría de 1,4, siendo un ejemplo el ADN mitocondrial mutador ratón 5, sin embargo, el papel preciso de daño mitocondrial en el envejecimiento no es completamente entendido 6,7. Observando la actividad de las enzimas respiratorias es un método directo para la investigación de la disfunción mitocondrial. Complejo IV o citocromo c oxidasa (COX), es esencial para la función mitocondrial. Las subunidades catalíticas de la COX son codificadas por el ADNmt y son esenciales para el ensamblaje del complejo (Figura 1). Por lo tanto, la síntesis y funcionamiento se basan principalmente en la integridad del ADNmt 2.Aunque otros complejos respiratorios podrían ser investigados, complejos IV y II son los más susceptibles de examen histoquímico 8,9. Complejo II o succinato deshidrogenasa (SDH), es totalmente codificada por el ADN nuclear (Figura 1), y su actividad no suele ser afectada por un deterioro del ADNmt, a pesar de un aumento puede indicar la biogénesis mitocondrial 10-12. El ADNmt deterioro observado en las enfermedades mitocondriales, el envejecimiento y las enfermedades relacionadas con la edad a menudo conduce a la presencia de células con actividad de la COX bajo o ausente 2,12-14. Aunque las actividades de la COX y SDH pueden ser investigados de forma individual, la secuencial de doble etiquetado método de 15,16 ha demostrado ser ventajoso en la localización de las células con la disfunción mitocondrial 12,17-21. Muchas de las constituciones óptimo del test, se han determinado, como por ejemplo la concentración de sustrato, receptores de electrones / donantes, intermediarios transportadores de electrones, la influencia del pH, y la reacción time 9,22,23. 3,3 &#39;-diaminobencidina (DAB) es un donador de electrones eficaz y fiable 22. En las células con el funcionamiento de la COX, la indamine marrón polímero producto va a localizar en crestas mitocondriales y saturar las células 22. Aquellas células que tienen disfuncionales COX por lo tanto no estar saturado por el producto DAB, lo que permite la visualización de la actividad SDH por la reducción de nitroazul de tetrazolio (NBT), un receptor de electrones, a un producto final azul formazán 9,24. Citocromo c y sustratos succinato sódico son añadidos para normalizar los niveles endógenos entre el control y los tejidos enfermos / mutante 9. Catalasa se ​​añade como medida de precaución para evitar posibles reacciones contaminantes de la actividad de peroxidasa 9,22. Metosulfato fenazina (PMS), un transportador de electrones intermedia, se utiliza en conjunción con azida sódica, un inhibidor de la cadena respiratoria, para aumentar la formación de los productos de reacción final de 9,25. A pesar de este informeción, algunos detalles importantes que afectan el resultado de este ensayo sencillo decorosa, además de los controles de especificidad y los avances en la técnica, aún no se han presentado.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

El citocromo c oxidasa / deshidrogenasa de sodio (COX / SDH) de doble etiquetado método permite la visualización directa de las deficiencias de las enzimas respiratorias mitocondriales en secciones de tejido fresco congelado. Esta es una técnica histoquímica directa y es útil en la investigación de las enfermedades mitocondriales, el envejecimiento y los trastornos relacionados con el envejecimiento.

La visualización de la función respiratoria mitocondrial utilizando citocromo C Oxidasa / Succinato Deshidrogenasa (COX / SDH) de doble etiquetado Histoquímica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Research

JoVE Journal

Biology

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

36.5K vistas.  Karolinska Institutet. El citocromo c oxidasa / deshidrogenasa de sodio (COX / SDH) de doble etiquetado método permite la visualización directa de las deficiencias de las enzimas respiratorias mitocondriales en secciones de tejido fresco congelado. Esta es una técnica histoquímica directa y es útil en la investigación de las enfermedades mitocondriales, el envejecimiento y los trastornos relacionados con el envejecimiento.

Video: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generación de transgénicos estable C. elegans con microinyección

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Investigación

Biología

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

La activación de la apoptosis mediante microinyección citoplasmática del citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

La Ex vivo Preparación aislada Microvessel esquelético para la Investigación de la reactividad vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Evaluación de las funciones mitocondriales y la viabilidad celular renal de células que sobreexpresan la proteína quinasa C Isoenzimas

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualización de Desarrollo Craneofacial en los SOX10: Kaede pez cebra transgénico Línea Usando Time-lapse microscopía confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Derribo Gene a gran escala en<em&gt; C. elegans</em&gt; Utilizar bibliotecas de alimentación de dsRNA para generar sólidas fenotipos de pérdida de función

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

Medición de la función respiratoria en ratones usando irrestricta pletismografía corporal total

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

-Alta resolución respirometría para evaluar la función mitocondrial en células intactas y permeabilizadas

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Estudiar mitocondrial Estructura y función en<em&gt; Drosophila</em&gt; Los ovarios

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.