Este trabajo fue financiado por el NIH Nacional del Corazón, los Pulmones y la Sangre subvención RO1 HL81248.

1. Cultivo de las células endoteliales aórticas bovinas (BAEC) <ol><li> Técnicas adecuadas de asepsia fueron seguidos. </li><li> En un baño de agua, medio caliente sin antibióticos a 37 ° C. </li></ol> Nota: El medio se compone de fenol libre Dulbecco modificado de Eagle (DMEM) con 4,5 g / l de D-glucosa, 4 mM de L-glutamina, 1% sin aminoácidos esenciales, suplementado con fetal al 10% de suero bovino (FBS) y 2,5 mg / l de factor de crecimiento endotelial. <ol start="3"><li> Retirar los T75 matraz que contenía las células de la incubadora y limpiar la superficie del matraz con etanol al 70% antes de colocarlo dentro de la campana. </li><li> Eliminar el medio de edad usando un aspirador y lavar dos veces con 5 ml de solución salina tampón de fosfato de Dulbecco (DPBS). </li><li> Añadir 2 ml de tripsina y esperar a 4-5 min para separar las células mientras que periódicamente inspeccionar bajo el microscopio. </li><li> Añadir 3 ml del medio y repetidamente mezclar utilizando una pipeta para separada tél las células y crean una suspensión homogénea. </li><li> Transferir 5 ml del medio con tripsina a un tubo de 15 ml y se centrifuga a 121 g durante 5 min. </li><li> Eliminar el sobrenadante utilizando un aspirador. Añadir 5 ml de DPBS y mezclar bien. Centrifugar a 121g durante 5 min. </li><li> Aspirar el sobrenadante y resuspender el sedimento celular mediante la adición de 6 ml del medio. </li><li> En una placa de 6 pocillos, añadir 1 ml de la suspensión de células a cada pocillo a continuación, añadir 1 ml del medio. Mezclar la suspensión con una pipeta. </li><li> Marque la placa y se incuba durante la noche a 37 ° C y 5% de CO 2. </li></ol> 2A. La detección de NO con la célula BAEC <ol><li> Técnicas adecuadas de asepsia fueron seguidos. </li><li> Retirar la placa de 6 pocillos de la incubadora y aspirar el medio desde el primer pocillo. Se lavan las células dos veces con 1 ml DPBS. </li><li> Añadir 210 l de 1,9 mM de sulfato de hierro heptahidratado (II) (FeSO 4 .7 H 2 O, prepararlos frescos mediante la disolución de 0,8 mg en1 DPBS ml con CaCl 2 y MgCl 2) y 210 l de amonio N-metil-D-glucamina ditiocarbamato (MGD, prepararlos frescos mediante la disolución de 2,7 mg en 500 ml con DPBS CaCl 2 y MgCl 2) con una proporción de 1:7 . (Nota:. Esta relación, cuando se utilice MGD exceso ha sido empleada convencionalmente para la preparación de Fe 2 +-MGD complejo debido al hecho de que el rendimiento para el Fe 3 +-mgd se maximiza en presencia de exceso de MGD La adición de ascorbato en solución para estabilizar el Fe 2 +-MGD no es necesario puesto que el NO 3-Fe +-mgd se forma endógenamente reduce a la detectable EPR NO-Fe 2 +-MGD por ascorbato, hidroquinona, o cisteína con una eficiencia de conversión de hasta a 99,9% .. El estado de espín diamagnético baja de Fe 2 + permite la detección de NO utilizando células plana o un tubo capilar sin la necesidad de un dispositivo de baja temperatura) 15. </li><li> Agitar la suspensión resultante bien y añadir 4,6 μl de ionóforo de calcio (ICa) (preparado a partir de una solución madre de 1,9 mM disolviendo 1 mg en 1 ml de DMSO). </li><li> Agitar la solución de nuevo y se incuba a 37 ° C durante 36 minutos a fin de permitir una mayor reducción de NO-Fe 3 +-MGD a la detectable EPR NO-Fe 2 +-MGD. </li><li> Recoger el sobrenadante (425 l) en un tubo Eppendorf y la transferencia a una celda EPR plana (o de un tubo capilar 50 l). </li><li> Parámetros de adquisición del EPR son los siguientes: la frecuencia de microondas: 9,8 GHz; el jardín central: 3427 G, la modulación de amplitud: 6,0 g; anchura de barrido: 100 g; ganancia del receptor: 1 x 10 5; potencia de microondas: 10 mW; número total de exploraciones: 121; tiempo de barrido: 10 s, y la constante de tiempo: 20 ms (Nota: Ya que los parámetros pueden variar de un instrumento y las condiciones experimentales a otro, por lo tanto, sólo el centro del campo, la frecuencia y la modulación de amplitud son los parámetros más importantes a considerar.). </li><li> Registrar el espectro a temperatura ambiente y los espectros de 2-D se integnominal para reducir el ruido de fondo y la línea base corregida con Bruker WinEPR software de procesamiento de datos o cualquier otro software de procesamiento de datos. Para la cuantificación de la formación de aductos, parcelas estándar de la intensidad de las concentraciones en función de la señal (o área) puede ser construido con un SNAP donador de NO. </li></ol> 2B. eNOS desenganche Experimento <ol><li> Retirar la placa de 6 pocillos de la incubadora y aspirar el medio desde el segundo pozo y lavar dos veces con 1 ml de DPBS. </li><li> Un donante peroxinitrito, 5-amino-3-(4-morfolinil) -1,2,3-oxadiazolium cloruro (SIN-1) fue utilizado para desacoplar NOSe 16. Añadir 100 l de 0,5 mM SIN-1 (M r 206,6 g / mol, de 10 mM de solución madre recién preparada por disolución de 1 mg SIN-1 en 500 l de PBS sin iones Ca / Mg) y ml diluido a 2 con DPBS y 10% de FBS. </li><li> Incubar durante 2 horas a 37 ° C y 5% de CO 2. </li><li> Retirar la placa de la incubadora y se lava dos veces con DPBS. </li><li> Añadir 210 l de2,8 mM de FeSO 4 .7 H 2 O y 210 l de 19,6 mM MGD recién preparada de acuerdo con el procedimiento mencionado anteriormente. </li><li> Agitar la solución y añadir 4,6 l de 1,9 mM ICa. </li><li> Agitar la solución de nuevo y se incuba a 37 ° C durante 36 min. </li><li> Recoger el sobrenadante (425 l) en un tubo Eppendorf y la transferencia a una celda plana EPR. </li><li> Parámetros de adquisición del EPR son los siguientes: la frecuencia de microondas: 9,8 GHz; el jardín central: 3427 G, la modulación de amplitud: 6 G, anchura de barrido: 100 g, la ganancia del receptor: 1 x 10 5; potencia de microondas: 10 mW; número total de exploraciones: 121; tiempo de barrido: s 10, y la constante de tiempo: 20 ms. </li><li> Registrar el espectro y los espectros de 2-D fueron integrados para reducir el ruido de fondo y la línea base corregida como se mencionó anteriormente. </li></ol> 3. La detección de O 2 • - de los neutrófilos polimorfonucleares (PMN) <ol><li> Los neutrófilos fueron aisladas de muestras de sangre humana como se ha descrito previamente 17. </li><li> Hacer una solución madre de 1 M DMPO * en PBS que contenía 0,1 mM de ácido dietilentriaminapentaacético (DTPA). DMPO tiene un punto de fusión de 25-29 ° C por lo que es más conveniente para pipetear líquidos DMPO (densidad de ~ 1,02 g / ml a 25 ° C) en un frasco de vidrio (Nota: No utilizar los viales de plástico para el pesaje ya pura DMPO reacciona con plástico). Frozen DMPO se pueden fundir por el chorro de agua tibia en la que el vial (Nota: no deje correr el agua caliente como DMPO puede descomponerse). </li><li> Es importante usar alta pureza DMPO (&gt; 99%) ya que algunas de las trampas de espín comercialmente disponibles contienen impurezas paramagnéticas, y por lo tanto, es imperativo para ejecutar espectro EPR de la solución DMPO solos (10 mM en este caso). Es crítico que no hay señal de fondo es evidente (véase la Figura 3A). </li><li> Preparar solución madre (1 mg / ml) de forbol-12-miristato-13-acetato (PMA) en DMSO. Hacer alícuotas mediante la dilución de la solución a 10 mg / ml en PBS. </li><li> En una 1.5 ml tubo Eppendorf, preparar una solución con un volumen total de 0,6 ml, siguiendo la secuencia de adición: ~ 10 6 células por ml de PMN, D-glucosa (1 mg / ml) y la albúmina (1 mg / ml), 10 DMPO mM y 0,2 mg / ml de PMA. (Nota: PMA es el radical activador y se debe agregar pasado). </li><li> Transferir la solución a una célula del EPR plana. </li><li> EPR parámetros de adquisición de las condiciones son las siguientes: la frecuencia de microondas: 9,8 GHz; el jardín central: 3486 G, la modulación de amplitud: 0,5 G, anchura de barrido: 100 g, la ganancia del receptor: 5 x 10 5, el número total de análisis: 10, el tiempo de barrido: 30 s ; potencia de microondas: de 20 MW, y la constante de tiempo: 81 ms. Para la cuantificación de la formación de aducto, parcelas estándar de intensidad frente a la concentración de la señal (o área) puede construirse utilizando los nitróxidos estables tales como TEMPO o 3-carboxílico PROXYL-. </li></ol> * Nota importante sobre el uso de DMPO: Mediante el uso de una celda plana, se puede aumentar la densidad celular y aumentar así la señalintensidad del aducto giro pero la vida media de O 2 • - aducto de DMPO es corto (t 1/2 ~ 1 min) que se descompone a DMPO-OH. DMPO puede sustituirse utilizando las mismas concentraciones de EMPO, BMPO o DEPMPO que están disponibles comercialmente para la estabilidad aducto aumento con t 1/2 ~ 8 y 14 min, respectivamente. 4. Los resultados representativos Haga girar la captura de ningún radical se realizó con Fe 2 +-MGD. Figura 2A y 2B muestran ninguna señal de EPR de Fe 2 +-MGD o una mezcla de Fe 2 +-MGD con el CAI, lo que indica que no hay ninguna señal de fondo NO proviene de estos reactivos. BAEC a la estimulación con ICa libera NO que reacciona con el Fe 2 +-MGD para formar el aducto giro, el NO-Fe 2 +-MGD, y muestra una señal triplete característico con hiperfina constante (HFSC) un valor de N = 12,66 G y factor g de g = 2,040. (Figura 2C). El HFsc valor se determinó usando el programa de simulación winsim que puede ser descargado desde sitio web de base de datos de NIEHS EPR Software. El HFSC experimental es consistente con el valor de la bibliografía de una N G = 12,70 g = 2,041 y 18 para el NO Fe-2 + DGM-aducto. De manera similar, el efecto de SIN-1 en BAEC bajó la producción de NO debido a NOSe desacoplamiento como se muestra en la Figura 2D. DMPO trampa de giro se utiliza para O 2 • - la detección. DMPO por sí solo no dar una señal como se muestra en las figura 3A que confirman que la trampa giro está libre de impurezas paramagnéticas. Figura 3B es el espectro de DMPO y PMN solamente, y de manera similar, no hay ninguna señal detectable que sugiere que el DMPO no causa activación de la enzima NADPH oxidasa. Figura 3C muestra la señal observada EPR a la estimulación por PMA de PMN. Los valores de HFSC para esta señal se determina que es un N = 14,71 g, y unabeta;-H = 11,40 G y un γ-H = 1,25 G, y son consistentes con los valores de la literatura de un N = 14,3 g, una β-H = 11,7 T y un γ-H = 1,3 G 19 para DMPO-S 2 H aducto. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama de flujo para la detección de los radicales de los neutrófilos y BAEC utilizando atrapamiento giro EPR. (A) PMN fueron mezclados con DMPO y PMA, y la mezcla resultante transferido a una célula EPR plana para la adquisición de datos. (B) BAEC fueron cultivadas en una placa, y se lavó con DPBS. La trampa de la vuelta Fe (MGD) 2 fue introducido junto con el CAI. La solución se mezcló a fondo y se incuba. La mezcla se transfirió a una celda EPR plana para EPR adquisición de datos. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="Figura 2" /> Figura 2. EPR detección de NO de BAEC. (A) Espectro de Fe 2 +-MGD sólo (B) Espectro de Fe 2 +-MGD + ICa solamente. (C) Espectro trío ocasionados por la ausencia de captura por Fe 2 +-MGD utilizando CAI-estimulado las células. (D) Espectro mostrando disminución en la producción de NO debido a 0,5 mM de SIN-1 tratamiento de las células. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Figura 3" /> Figura 3. EPR detección de DMPO-O 2 H desde los neutrófilos activados. (A) Espectro de DMPO 10 mM solamente. (B) Espectro de PMN solo en la presencia de DMPO 10 mM. (C) Espectro de PMN activado por PMA en presencia de 10 mM de DMPO.

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Atrapamiento EPR giro ha sido empleado en una amplia gama de aplicaciones biomédicas para la cuantificación y la identificación de los radicales libres. Atrapamiento Spin es altamente sensible, capaz de detectar los radicales en concentraciones que van desde nM a mM tanto por lo que es adecuado para aplicación en los sistemas biológicos. La formación del aducto paramagnético, NO-Fe 2 +-MGD, es la base de NO detección a través de EPR. Fe 2 +-MGD reacciona con rapidez NO 18</su...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Medio DMEM libre de fenol Alta glucosa en 1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% de tripsina-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-glutamina </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM aminoácidos no esenciales </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Suero bovino fetal </td><td> Atlanta Productos Biológicos </td><td> S11550 </td><td></td></tr><tr><td> Factor de crecimiento endotelial </td><td> Millipore </td><td> 02-102 </td><td></td></tr><tr><td> ICa </td><td> Enzo Ciencias de la Vida </td><td> A-23187 </td><td> Disolver en DMSO </td></tr><tr><td> SIN-1 </td><td> Enzo Ciencias de la Vida </td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> Dojindo Laboratories </td><td> D048-10 </td><td></td></tr><tr><td> FeSO 4 .7 H 2 O </td><td> Sigma Aldrich </td><td> 215422-250G </td><td> Disolver en PBS con Ca y Mg </td></tr><tr><td> MGD </td><td> Enzo Ciencias de la Vida </td><td> ALX-400-014-M050 </td><td> Disolver en PBS con Ca 2 + y Mg 2 + </td></tr><tr><td> Células BAEC </td><td> Sistemas de células </td><td> 2B2-C75 </td><td></td></tr><tr><td> DMSO </td><td> Fisher Scientific </td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td> Sigma Aldrich </td><td> D8537 </td><td></td></tr><tr><td> DPBS con CaCl 2 y MgCl 2 </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Forbol-miristato acetato (PMA) </td><td> Sigma Aldrich </td><td> 79346-1mg </td><td></td></tr></tbody></table>

detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps

El nitrógeno reactivo / especies de oxígeno (ROS / RNS) a bajas concentraciones jugar un papel importante en la regulación de la función celular, de señalización, y la respuesta inmune, pero en concentraciones no regulados son perjudiciales para la viabilidad celular 1, 2. Mientras que los sistemas vivos han evolucionado con los mecanismos de defensa antioxidantes endógenos y la dieta para regular la generación de ROS, ROS se producen continuamente, como subproductos naturales del metabolismo normal del oxígeno y puede causar daño oxidativo a las biomoléculas que resultan en la pérdida de función de las proteínas, ADN división, o de los lípidos peroxidación de los tres, y en última instancia con el estrés oxidativo que conduce a la lesión celular o la muerte 4. Radical anión superóxido (O 2 • -) es el principal precursor de algunas de las especies más altamente oxidantes conocidos que existen en los sistemas biológicos, tales como peroxinitrito y el radical hidroxilo. La generación de O 2 • - señala el primer signo de ruptura oxidativa, y por lo tanto, ict detección y / o retención en los sistemas biológicos es importante. En esta demostración, O 2 • - fue generada a partir de los neutrófilos polimorfonucleares (PMN). A través de la estimulación quimiotáctica con forbol-12-miristato-13-acetato (PMA), PMN genera O 2 • - a través de la activación de la nicotinamida adenina dinucleótido fosfato (NADPH) oxidasa 5. El óxido nítrico (NO) sintasa, que se presenta en tres isoformas, como inducible, neuronal y NOS endotelial, o iNOS, nNOS o eNOS, respectivamente, cataliza la conversión de L-arginina a L-citrulina, utilizando NADPH para producir N º 6 . Aquí, NO generado a partir de células endoteliales. Bajo condiciones de estrés oxidativo, NOSe por ejemplo puede cambiar de la producción de NO a O 2 • - en un proceso llamado desacoplamiento, que se cree que está causada por la oxidación del grupo hemo 7 o el factor de co-, tetrahidrobiopterina (BH 4) 8. Hay sólo unos pocosmétodos fiables para la detección de radicales libres en los sistemas biológicos, pero están limitadas por la especificidad y sensibilidad. Atrapamiento centrifugado se utiliza comúnmente para la identificación de los radicales libres e implica la reacción de adición de un radical a una trampa de giro formando un aducto giro persistente que puede ser detectado por resonancia paramagnética electrónica (EPR), espectroscopía. Los aductos de varios radicales presentan espectro característico que puede ser utilizado para identificar a los radicales que se generan y pueden proporcionar una gran cantidad de información acerca de la naturaleza y la cinética de la producción de radicales 9. Los nitronas cíclicos, 5,5-dimetil-pirrolina-N-óxido, DMPO 10, la fosforil-sustituido DEPMPO 11, y el éster-sustituido, EMPO 12 y BMPO 13, han sido ampliamente empleado como atrapadores - el giro último trampas exhibe ya las vidas medias de O 2 • - aducto. Hierro (II)-N-metil-D-glucamina ditiocarbamato, Fe (MGD) 2 &lt;/&gt; Sub se utiliza comúnmente para atrapar NO debido a la alta tasa de formación de aducto y la alta estabilidad del aducto giro 14.

Watch this Scientific Journal Video about Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps at JoVE.com

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Resonancia de electrones paramagnético (EPR), espectroscopía fue empleado para detectar óxido nítrico a partir de células endoteliales aórticas bovinas y aniones radicales superóxido de los neutrófilos humanos utilizando hierro (II)-N-metil-D-glucamina ditiocarbamato, Fe (mgd)<sub&gt; 2</sub&gt; Y 5,5-dimetil-1-pyroroline-N-óxido, DMPO, respectivamente.

La detección de óxido nítrico y el radical anión superóxido por espectroscopía de resonancia paramagnética de electrones de las células utilizando atrapadores

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

Research

JoVE Journal

Biology

18.9K vistas.  The Ohio State University. Resonancia de electrones paramagnético (EPR), espectroscopía fue empleado para detectar óxido nítrico a partir de células endoteliales aórticas bovinas y aniones radicales superóxido de los neutrófilos humanos utilizando hierro (II)-N-metil-D-glucamina ditiocarbamato, Fe (mgd) 2 Y 5,5-dimetil-1-pyroroline-N-óxido, DMPO, respectivamente.

Video: Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

Resonancia de spin electrónico micro imágenes en vivo de las especies para la asignación de oxígeno

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment ...

Investigación

Biología

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Experimento perfil bioenergético utilizando células C2C12 mioblastos

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

La concentración de los metabolitos de las comunidades planctónicas de baja densidad para la metabolómica ambiental utilizando espectroscopia de Resonancia Magnética Nuclear

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

La detección de vivo<em&gt; Escherichia coli</em&gt; O157: H7 células por PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

De dos fotones de imágenes de Ca intracelular<sup&gt; 2 +</sup&gt; Manejo y producción de óxido nítrico en el endotelio y las células del músculo liso de la aorta de rata aislada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

En Detección de la cara del óxido nítrico y superóxido en la capa endotelial de las arterias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desarrollo y caracterización de<em&gt; In Vitro</em&gt; Red de microvasos y mediciones cuantitativas de endotelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Y la producción de óxido nítrico

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

La detección de formas modificadas de la citosina Uso Sensitive Inmunohistoquímica

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

La medición de nitritos y nitratos, los metabolitos en el Oxido Nítrico, en Materiales Biológicos utilizando el método de quimioluminiscencia

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Methods Article

La detección de óxido nítrico y el radical anión superóxido por espectroscopía de resonancia paramagnética de electrones de las células utilizando atrapadores