Agradecemos al Prof. A. Vinogradov y el Dr. Vera Grivennikova para la introducción de los autores en el campo de la biología mitocondrial y mucha información útil y consejos en este procedimiento. Los autores agradecen a la Sra. Amanda McKintock de la oficina de la Universidad de Queens medios de comunicación por la ayuda con la preparación de vídeo.

Introducción Las mitocondrias de corazón de rata es una de las preparaciones más comunes de los estudios anteriores y actuales del metabolismo celular. Se pueden obtener de forma rápida y fiable en gran cantidad de tipo silvestre o animales knock-out. El procedimiento general consiste en la digestión del tejido por la tripsina, fraccionamiento y centrifugación diferencial. Hay varias condiciones que han de mantenerse durante el aislamiento de las mitocondrias de varios tejidos, incluyendo el corazón (Fig. 1). <ul><li> El tejido tiene que ser bien picada, ya que afecta directamente el rendimiento de las mitocondrias obtenidas. El tejido del corazón que es mejor para picar con pequeñas tijeras curvas y puede ser seguido por el uso de un triturador de tejidos Latapie, o si un triturador de tejidos no está disponible, una prensa de ajo con una salida de diámetro de los agujeros de alrededor de 1,0 mm de diámetro. </li><li> Es necesario para mantener la temperatura de las soluciones a cada paso. Por lo tanto, todo el procedimiento ha de llevarse a cabo en una habitación fría y todos los instrumentos y las soluciones tienen que estar preparados y refrigerados con antelación. Si las condiciones de temperatura no se mantienen estables varias propiedades de la preparación se puede perder. La estructura mitocondrial es muy sensible a las heladas por lo overfreezing de la suspensión (por ejemplo durante la centrifugación) no está permitido, especialmente si los estudios de transporte activo son muy importantes. </li><li> Un parámetro importante es el tiempo de duración del procedimiento, el aislamiento debe ser realizado lo más rápidamente posible y la preparación obtenida debe ser utilizado inmediatamente. Por lo tanto, instrumentos quirúrgicos, soluciones y aparatos tienen que estar preparados de antemano. </li></ul> Materiales e instrumentos Instrumentos <ol><li> Tijeras rectas de operación, el punto fuerte </li><li> Tijeras curvas </li><li> Fórceps </li><li> Una caja de petri </li><li> Pequeño embudo + pedazo de nylon filtro (previamente lavada) </li><li> 6 vasos de 50 ml </li><li> Dos agitadores magnéticos y dos baños de hielo </li><li> Un gran baño de hielo </li><li> Dos barras magnéticas pequeñas </li><li> Espátulas para raspar pellet </li><li> Tubos de centrífuga </li><li> Prensa Latapie tejido (se puede sustituir con prensa de ajo) </li><li> Teflón-vidrio de 20 ml y homogeneizadores de un 2 ml- </li><li> Residuos vaso </li><li> Tubos Eppendorf de suspensión definitiva mitocondrial y muestras para la determinación de proteínas </li><li> Baños de hielo </li></ol> Soluciones (calculado por 2 ratas): <ol><li> Tampón de lavado: 0,3 M de sacarosa, 10 mM HEPES (pH = 7,2), 0,2 mM EDTA (1000 ml) </li><li> Aislamiento de amortiguación: 0,3 M de sacarosa, 10 mM HEPES (pH = 7,4), 0,2 mM EDTA, 1 mg / ml de BSA (Sigma A6003) (preparado a partir de la solución de lavado 100 ml). Albúmina sérica bovina puede ser sustituidos por otros menos caros de ácidos grasos libres análogos. El mismo tampón, o sus variantes isotónicas se puede utilizar como buffer de resuspensión. </li><li> Tripsina (Sigma T8003): Se 2.5mg/ml en 1 mM HCl (0,5 ml) </li><li> Inhibidor de tripsina de soja (Sigma T9128) 6,5 mg/10 ml de solución tampón de aislamiento </li></ol> Protocolo El esquema general del procedimiento se muestra en la figura. 1. Corazones deben ser enfriados y lavados en tampón de lavado, el tejido extirpado ventricular, picada y homogeneizada durante el tratamiento con tripsina fotolítica. La suspensión se centrifuga a baja velocidad y el sobrenadante obtenido se centrifugó de nuevo a una velocidad más alta para recoger las mitocondrias. En función de los experimentos previstos el buffer resuspensión puede ser sustituida por cualquier otra solución isotónica. Los animales fueron terminados de acuerdo con el Reino Unido &quot;Animals (Scientific Procedures) Act&quot;. por dislocación cervical seguido por decapitación. Es necesario extraer el corazón inmediatamente después de la decapitación del animal, de modo que no hay retraso entre la terminación de los animales y la extracción de corazón. Si el aislamiento paralelo de mitocondrias de hígado se espera que las ratas deben estar en ayunas durante la noche antes del experimento. <ol><li> Preparar tres vasos de precipitado que contiene 40 ml de Tampón de Lavado. Enfriar en un baño de hielo y sal de 3 a 5 min antes de proceder al siguiente paso. Asegúrese de que no los overfreeze y utilizar inmediatamente justo después de las nubes de cristales de hielo comienzan a aparecer. </li><li> Abrir el tórax de la rata decapitado y extraer el corazón. Hacen pequeñas incisiones y luego transferir inmediatamente el corazón a la solución enfriada con hielo en el vaso de primera. Apriete el corazón para eliminar la sangre y la puso en el segundo vaso. Repita esta operación para cada corazón. </li><li> Seca el corazón en el papel de filtro. Eliminar todas las grasas en la sangre, coagulada, aurículas y la fascia y la piscina del tejido ventricular. </li><li> Picar el tejido combinado con pequeñas tijeras curvas en la placa de Petri en el hielo durante unos 5 minutos hasta que el tamaño de las partículas es de unos 1-2 mm. </li><li> Coloque el tejido picado en la prensa del tejido y pasartravés de él. Tenga en cuenta que una cantidad sustancial de material puede permanecer en la prensa para recoger todos los tejidos del corazón de las paredes y el fondo de la prensa. </li><li> Transferir el material en el vaso con 50 ml de Tampón de Lavado y revuelva. Después se filtra la suspensión a través de un filtro de nylon. Lave el tejido picado en el filtro 3 veces con el tampón de lavado y descartar el filtrado. </li><li> La transferencia de los tejidos lavados en 20 ml de tampón de lavado y colocarlo en el baño de hielo en el agitador magnético. Añadir 0,5 ml de solución de tripsina con agitación constante y en marcha el temporizador. Se prepara otro agitador magnético, baño de hielo y un segundo vaso de 50 ml. </li><li> En el minuto 4 brevemente homogeneizar la parte de suspensión por parte de utilizar una pequeña muy ajustada de vidrio Teflón homogeneizador (10 y 15 ml) con varios golpes arriba y abajo para dispersar a la suspensión. Después de la homogeneización de transferencia de la suspensión en el segundo vaso. Repita el procedimiento en el minuto 9 º, por lo que se realiza la homogeneización de la suspensión de dos veces en total. Después de 15 minutos de incubación, agregar 10 ml de aislamiento de un tampón que contiene inhibidores de la tripsina y se incuba durante 1 min. </li><li> Filtrar la suspensión una vez más a través de un filtro de nylon y guardar el líquido filtrado. Recoger la fracción de partículas a la izquierda en un filtro y se homogeneiza durante 1 minuto en 15-20 ml de tampón de lavado. </li><li> Combine el homogeneizado con el filtrado y centrifugado durante 15 minutos a 600g. </li><li> Cuidadosamente filtra el sobrenadante resultante en un tubo de centrífuga de nuevo. Evitar la contaminación por el sedimento y se centrifuga de nuevo durante 15 min a 8500g. </li><li> Después de la centrifugación de alta velocidad descartar el sobrenadante y lavar el pellet 2-3 veces con 1 ml de buffer de descartar la mullida capa blanca del borde exterior. Romper la pastilla con la espátula, sino evitar la mancha roja de los eritrocitos en la parte inferior. Si las células sanguíneas se loos, eliminar los bits de color rojo de la suspensión antes de la homogeneización. </li><li> Resuspender el precipitado con un homogeneizador de pequeñas o bien mediante pipeteo suave. Diluir la suspensión con más Isolationbuffer y centrifugar de nuevo durante 15 min a 8500g. </li><li> Descartar el sobrenadante, resuspender el precipitado en la resuspensión de búfer (un tampón isotónico de elección) y se centrifuga de nuevo. </li><li> El marrón resultante pastilla contiene lavado mitocondrias intactas. Resuspender el precipitado en un volumen muy pequeño de tampón isotónico de su elección. </li></ol><img src="/files/ftp_upload/2202/2202fig1.jpg" alt="Figura 1" /> Figura 1. Esquema que demuestra el procedimiento paso a paso de aislamiento de las mitocondrias de corazón de rata. Parte superior, Etapas 1-9: alteración del tejido, el tratamiento de la tripsina y la homogeneización; parte inferior, las etapas 10-15: centrifugación diferencial.

<ol>
	<li>Jacobus, W. E., Saks, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creatine+kinase+of+heart+mitochondria:+changes+in+its+kinetic+properties+induced+by+coupling+to+oxidative+phosphorylation.">Creatine kinase of heart mitochondria: changes in its kinetic properties induced by coupling to oxidative phosphorylation.</a> Arch. Biochem. Biophys. 219, 167-178 (1982).</li><li>Gostimskaya, I. S., Grivennikova, V. G., Zharova, T. V., Bakeeva, L. E., Vinogradov, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+assay+of+the+intramitochondrial+enzymes:+use+of+alamethicin+for+permeabilization+of+mitochondria.">In situ assay of the intramitochondrial enzymes: use of alamethicin for permeabilization of mitochondria.</a> Anal. Biochem. 313, 46-52 (2003).</li></ol>

Protocolo detallado rápido para el aislamiento de las mitocondrias intactas con un alto grado de acoplamiento se describe. La preparación obtenida puede ser utilizada para la investigación funcional o estructural de la bioenergética celular, estudios de proteómica o análisis de ADN mitocondrial y el contenido de lípidos. Estudios biofísicos del transporte activo de iones y los intermediarios metabólicos también se puede realizar. Es posible que otro proceso de la preparación mitocondrial con el fin de aislar partículas submitocondriales, la membrana externa o purificar los componentes separados de la fosforilación oxidativa. El método descrito es una modificación del protocolo estándar de aislamiento por Jacobus y Saks 1. El rendimiento esperado de las mitocondrias preparado es de aproximadamente 10-15 mg de proteína mitocondrial por el corazón y la razón de control de la respiración en malato / glutamato de esta preparación varía entre 8 y 12 con respiración IV Estado de alrededor de 0,12 mmol O 2 -1 proteína xmin XMG -1 a 23 ° C en el medio que comprende 0,25 M de sacarosa, 10 mM KCl, 0,2 mM EDTA, 5 mM fosfato de potasio (pH 8,0) con 150 mM de ADP para la iniciación de la respiración del Estado III (por las condiciones exactas experimental y adiciones, ver ref . 2). Se debe prestar especial atención a varios detalles técnicos antes del inicio del procedimiento de aislamiento. Es crucial para el uso limpio tubos de policarbonato o polipropileno centrífuga, por lo tanto, los tubos deben lavarse con un cepillo limpio y agua caliente, sin embargo, el tratamiento con detergente debe mantenerse al mínimo. Además, es conveniente recoger toda la cristalería y los instrumentos necesarios en la cámara fría un día antes del aislamiento. Durante el aislamiento, cuando la transferencia de buffers de los frascos de vasos, la atención а debe prestarse especial atención para evitar que las gotas de agua helada en las soluciones. Al manipular los tejidos hay que destacar que la extracción del corazón deben llevarse a cabo tan pronto como sea posible, ya que incluso a corto tiempo de retardo entre la decapitación del animal y el enfriamiento del tejido se traduciría en las mitocondrias parcialmente desacoplada. El enfriamiento rápido y un lavado profundo de los corazones eliminado es esencial para obtener la preparación estándar, libre de hemoglobina y eritrocitos NADase. Tripsina se utiliza para la degradación de las proteínas conectivo para aumentar la susceptibilidad del tejido a fuerza de corte durante la homogeneización. La exposición prolongada de los tejidos de la proteasa se debe evitar, ya que los resultados en las mitocondrias de calidad inferior. Después de centrifugaciones a alta velocidad de las paredes de los tubos de centrifugación deben limpiarse con un pañuelo de papel para eliminar los depósitos de material de grasa acumulada en las paredes. Para la resuspensión final es mejor utilizar un volumen bajo de la amortiguación final con el fin de minimizar la pérdida de las funciones mitocondriales durante el almacenamiento (150 a 200 l de tampón por el corazón). Para los estudios de transporte de cationes bivalentes no agentes quelantes deben estar presentes en la preparación final de lo que las mitocondrias se deben lavar varias veces con EGTA medio libre y utilizado en las primeras horas después de su aislamiento.

preparation of highly coupled rat heart mitochondria

La función de la mitocondria en la generación de ATP celular en el proceso de fosforilación oxidativa es ampliamente reconocida. Durante las últimas décadas ha habido avances significativos en nuestra comprensión de las funciones de otros mitocondrias que la generación de energía. Estos incluyen su papel en la apoptosis, actuando como una señal de orgánulos, desarrollo de los mamíferos y el envejecimiento, así como su contribución a la coordinación entre el metabolismo celular y la proliferación celular. Nuestra comprensión de los procesos biológicos modulada por las mitocondrias, se basa en métodos robustos para el aislamiento y la manipulación de las mitocondrias intactas de tejidos de los animales de laboratorio. Las mitocondrias de corazón de rata es una de las preparaciones más comunes de los estudios anteriores y actuales del metabolismo celular incluyendo estudios sobre animales knock-out. A continuación se describe un método detallado rápido para el aislamiento de las mitocondrias intactas con un alto grado de acoplamiento. Tal preparación de corazón de rata mitocondria es un objeto ideal para la investigación funcional y estructural de la bioenergética celular, el transporte de biomoléculas, los estudios proteómicos y análisis de ADN mitocondrial, las proteínas y los lípidos.

Watch this Scientific Journal Video about Preparation of Highly Coupled Rat Heart Mitochondria at JoVE.com

Preparation of Highly Coupled Rat Heart Mitochondria

Se describe el protocolo а para el aislamiento de corazón puro, una rata muy unido las mitocondrias de los estudios funcionales o estructurales de la bioenergética celular, mediciones biofísicas, la proteómica y el ADN mitocondrial y el análisis de los lípidos.

Preparación de corazón de rata altamente acoplado mitocondrias

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

preparation-of-highly-coupled-rat-heart-mitochondria

Research

JoVE Journal

Biology

22.7K vistas.  University of Manchester. Se describe el protocolo а para el aislamiento de corazón puro, una rata muy unido las mitocondrias de los estudios funcionales o estructurales de la bioenergética celular, mediciones biofísicas, la proteómica y el ADN mitocondrial y el análisis de los lípidos.

Video: Preparation of Highly Coupled Rat Heart Mitochondria

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantación de la ingeniería tisular en el corazón de rata

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Investigación

Biología

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultivo primario de miocitos adultos de corazón de rata

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificación de las mitocondrias de las células de levadura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Preparación de los tendones Cola de Rata de Estudios biomecánicos y Mechanobiological

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...

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 ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagen confocal de Soltero mitocondriales superóxido Parpadea en corazón intacto o<em&gt; En Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparación de suero de rata Adecuado para mamíferos Whole cultivo de embriones

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Plataforma modular de selección automatizados de alto rendimiento de exopolisacáridos Junto con el análisis de carbohidratos de la huella digital de alta sensibilidad

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

Los métodos para la determinación de las tasas de glucosa y la oxidación de ácidos grasos en el corazón de rata aislado de Trabajo

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...