Ce travail a été financé par le NIH Cœur nationale, Lung, and Blood Institute subvention RO1 HL81248.

1. Culture de cellules endothéliales aortiques bovines (CEAB) <ol><li> Des techniques d&#39;asepsie ont été respectées. </li><li> Dans un bain d&#39;eau, milieu chaud sans antibiotiques à 37 ° C. </li></ol> Remarque: Le milieu est constitué de phénol milieu exempt de Eagle modifié par Dulbecco (DMEM) avec 4,5 g / L D-glucose, 4 mM de L-glutamine, 1% non-acides aminés essentiels, supplémenté avec 10% de sérum bovin (FBS) et 2,5 mg / L facteur de croissance endothélial. <ol start="3"><li> Retirer les cellules T75 flacon contenant de l&#39;incubateur et nettoyer la surface de la fiole avec de l&#39;éthanol 70% avant de le placer à l&#39;intérieur du capot. </li><li> Retirez l&#39;ancien milieu à l&#39;aide d&#39;un aspirateur et laver deux fois avec 5 ml de solution saline de Dulbecco tampon phosphate (DPBS). </li><li> Ajouter 2 ml de trypsine et d&#39;attendre 4-5 min pour les cellules à se détacher tout inspecter périodiquement sous le microscope. </li><li> Ajouter 3 ml du milieu et à plusieurs reprises mélanger à l&#39;aide d&#39;une pipette de séparer til cellules et créer une suspension même. </li><li> Transférer 5 ml du milieu avec de la trypsine dans un tube de 15 ml et centrifuger à 121 g pendant 5 min. </li><li> Eliminer le surnageant à l&#39;aide d&#39;un aspirateur. Ajouter 5 ml de DPBS et bien mélanger. Centrifuger à 121g pendant 5 min. </li><li> Aspirer le surnageant et remettre en suspension le culot cellulaire en ajoutant 6 ml du milieu. </li><li> Sur une plaque à 6 puits, ajouter 1 ml de la suspension cellulaire dans chaque puits, puis ajouter 1 ml du milieu. Mélanger la suspension en utilisant une pipette. </li><li> Etiqueter la plaque et laisser incuber une nuit à 37 ° C et 5% de CO 2. </li></ol> 2A. Détection de NO avec la revue Cell BAEC <ol><li> Des techniques d&#39;asepsie ont été respectées. </li><li> Retirer la plaque de 6 puits de l&#39;incubateur et aspirer le milieu à partir du premier puits. Laver les cellules deux fois avec 1 ml de DPBS. </li><li> Ajouter ul 210 de 1,9 mm de fer heptahydraté sulfate (II) (FeSO 4 .7 H 2 O, fraîchement préparer en dissolvant 0,8 mg de1 ml DPBS avec CaCl 2 et MgCl 2) et 210 de pl d&#39;ammonium N-méthyl-D-glucamine dithiocarbamate (MGD, préparer fraîchement en dissolvant 2,7 mg dans 500 ul DPBS avec CaCl 2 et MgCl 2) en utilisant un ratio de 1:7 . (Note:. Ce rapport, où MGD excès est utilisé a été classiquement utilisé pour la préparation de Fe 2 +-MGD complexe en raison du fait que le rendement de Fe 3 +-MGD est maximisée en présence d&#39;excès de MGD L&#39;ajout de ascorbate en solution pour stabiliser le Fe 2 +-MGD n&#39;est pas nécessaire que l&#39;3 N-Fe +-MGD formé de façon endogène est réduit à l&#39;EPE détectable NO-Fe 2 +-MGD par l&#39;ascorbate, l&#39;hydroquinone, ou la cystéine avec de l&#39;efficacité de conversion jusqu&#39;à à 99,9% .. l&#39;état bas spin diamagnétique en Fe 2 + permet la détection de NO en utilisant la cellule plate ou capillaire sans la nécessité d&#39;un dispositif de basse température) 15. </li><li> Swirl la suspension résultante et ajouter 4,6 μl de calcium ionophore (CAI) (préparé à partir d&#39;une solution stock de 1,9 mM en dissolvant 1 mg dans 1 ml de DMSO). </li><li> Swirl à nouveau la solution et incuber à 37 ° C pendant 36 min afin de continuer à permettre la réduction des NO-Fe 3 +-MGD à l&#39;EPR détectable NO-Fe 2 +-MGD. </li><li> Recueillir le surnageant (425 pi) dans un tube Eppendorf et le transfert à un EPR cellule plate (ou à un 50 tube capillaire ul). </li><li> Les paramètres d&#39;acquisition EPR sont: la fréquence micro-ondes: 9,8 GHz; centre du terrain: 3427 G; modulation d&#39;amplitude: 6,0 G; largeur de balayage: 100 G; gain du récepteur: 1 x 10 5; puissance micro-onde: 10 mW, le nombre total de balayages: 121; temps de balayage: 10 s et la constante de temps: 20 ms (Remarque: Depuis paramètres varient d&#39;un instrument et les conditions expérimentales à l&#39;autre, par conséquent, seul le centre du terrain, la fréquence et l&#39;amplitude de modulation sont les paramètres les plus importants à considérer.). </li><li> Enregistrez les spectres à température ambiante, et les spectres 2-D ont été Integnominale pour réduire le bruit de fond et de référence corrigée à l&#39;aide du logiciel Bruker WinEPR traitement des données ou tout autre logiciel de traitement de données. Pour la quantification de la formation d&#39;adduits, parcelles standards de l&#39;intensité du signal par rapport à la concentration (ou région) peut être construit en utilisant un composant logiciel enfichable donneur de NO. </li></ol> 2B. eNOS Dételage Expérience <ol><li> Retirer la plaque de 6 puits de l&#39;incubateur et aspirer le milieu de la deuxième puits et laver deux fois avec du DPBS ml 1. </li><li> Un donneur de peroxynitrite, 5-amino-3-(4-morpholinyl) -1,2,3-oxadiazolium chlorure de (SIN-1) a été utilisé pour découpler eNOS 16. Ajouter 100 ul de 0,5 mM sin-1 (M r 206,6 g / mol, de 10 mM solution mère fraîchement préparée en dissolvant 1 mg SIN-1 dans 500 ul de PBS sans ions Ca / Mg) et diluée à 2 ml avec du DPBS et 10% de FBS. </li><li> Incuber pendant 2 h à 37 ° C et 5% de CO 2. </li><li> Retirer la plaque de l&#39;incubateur et laver deux fois avec du DPBS. </li><li> Ajouter ul 210 de2,8 mM FeSO 4 .7 H 2 O et de 19,6 pi 210 mM MGD fraîchement préparée selon le procédé mentionné ci-dessus. </li><li> Swirl la solution et ajouter 4,6 ul de 1,9 mM CaI. </li><li> Swirl à nouveau la solution et incuber à 37 ° C pendant 36 min. </li><li> Recueillir le surnageant (425 pi) dans un tube Eppendorf et le transfert dans une cellule EPR plat. </li><li> Les paramètres d&#39;acquisition EPR sont: la fréquence micro-ondes: 9,8 GHz; centre du terrain: 3427 G; modulation d&#39;amplitude: 6 G; largeur de balayage: 100 G; gain du récepteur: 1 x 10 5; puissance micro-onde: 10 mW, le nombre total de balayages: 121; temps de balayage: 10 s et la constante de temps: 20 ms. </li><li> Enregistrez les spectres et les spectres 2-D ont été intégrées pour réduire le bruit de fond et de référence corrigée comme indiqué ci-dessus. </li></ol> 3. Détection de O 2 • - de polynucléaires neutrophiles (PMN) <ol><li> Les neutrophiles ont été isolés à partir du sang humain comme décrit précédemment 17. </li><li> Préparer une solution mère de 1 M DMPO * dans du PBS contenant 0,1 mM diéthylènetriamine-pentaacétique (DTPA). DMPO a un point de fusion de 25-29 ° C de sorte qu&#39;il est plus commode de pipeter liquide DMPO (densité ~ 1,02 g / ml à 25 ° C) dans un flacon en verre (Note: ne pas utiliser les flacons en plastique pour la pesée depuis pur DMPO réagit avec du plastique). Frozen DMPO peut être fondu par l&#39;eau courante tiède sur le flacon de (Note: ne pas laisser couler l&#39;eau chaude que DMPO peut se décomposer). </li><li> Il est important d&#39;utiliser une grande pureté DMPO (&gt; 99%) étant donné que certains des pièges de spin disponibles dans le commerce contiennent des impuretés paramagnétiques, et, par conséquent, il est impératif d&#39;exécuter spectre RPE d&#39;un peu de la solution DMPO seul (10 mM dans ce cas). Il est essentiel qu&#39;aucun signal de fond est évident (voir figure 3A). </li><li> Préparer une solution mère (1 mg / ml) de phorbol-12-myristate-13-acétate (PMA) dans le DMSO. Faire aliquotes en diluant la solution à 10 pg / ml dans du PBS. </li><li> Dans un 1.5 ml tube Eppendorf, préparer une solution avec un volume total de 0,6 ml en suivant la séquence d&#39;addition: ~ 10 6 cellules par ml de PMN, D-glucose (1 mg / ml) et de l&#39;albumine (1 mg / ml), 10 mM DMPO et 0,2 ug / ml de PMA. (Remarque: PMA est le radical activateur et devrait être ajouté en dernier). </li><li> Transférer la solution dans une cellule EPR plat. </li><li> EPR paramètres d&#39;acquisition conditions sont les suivantes: la fréquence micro-ondes: 9,8 GHz; centre du terrain: 3486 G; modulation d&#39;amplitude: 0,5 G; largeur de balayage: 100 G; gain du récepteur: 5 x 10 5, le nombre total de balayages: 10; temps de balayage: 30 s ; puissance micro-onde: 20 mW, et la constante de temps: 81 ms. Pour la quantification de la formation d&#39;adduits, parcelles types d&#39;intensité de signal la concentration en fonction (ou de la zone) peut être construit en utilisant les nitroxydes stables tels que TEMPO ou 3-carboxylique-PROXYL. </li></ol> * Note importante sur l&#39;utilisation de DMPO: En utilisant une cellule à plat, on peut augmenter la densité cellulaire et augmenter ainsi le signal del&#39;intensité de l&#39;adduit de spin, mais la demi-vie de 2 O • - produit d&#39;addition de DMPO est court (t 1/2 ~ 1 min) qui se décompose pour DMPO-OH. DMPO peut être substitué en utilisant les mêmes concentrations de EMPO, BMPO ou DEPMPO qui sont disponibles dans le commerce pour la stabilité accrue avec adduit t 1/2 ~ 8 et 14 min, respectivement. 4. Les résultats représentatifs Spin piégeage du NO radicale a été réalisée en utilisant Fe 2 +-MGD. Figure 2A et 2B montrent aucun signal EPR de Fe 2 +-MGD ou un mélange de Fe 2 +-MGD avec Cai, indiquant qu&#39;aucun signal de fond NO provient de ces réactifs. BAEC lors de la stimulation avec Cai libère du NO qui réagit avec Fe 2 +-MGD pour former l&#39;adduit de spin, NO-Fe 2 +-MGD, et montre un signal triplet caractéristique avec la séparation hyperfine constante (HFSC) la valeur d&#39;un N = 12,66 G et g-facteur de g = 2,040. (Figure 2C). Le hfvaleur a été déterminée en utilisant sc le programme de simulation WINSIM qui peut être téléchargé à partir du NIEHS EPR site Base de données du logiciel. Le HFSC expérimentale est conforme à la valeur de la littérature d&#39;un G N = 12,70 g = 2,041 et 18 pour NO 2 + Fe-MGD-adduit. De même, l&#39;effet de SIN-1 sur BAEC abaissé la production de NO en raison de eNOS découplage comme le montre la figure 2D. DMPO de spin piège a été utilisé pour O 2 • - la détection. DMPO seul ne donne pas un signal, comme indiqué dans les figure 3A confirmant que le spin est exempt d&#39;impuretés paramagnétiques. Figure 3B est le spectre de DMPO et les PMN seulement, et de même, il n&#39;ya pas de signal détectable suggérant que le DMPO ne provoque pas de l&#39;activation de la NADPH oxydase enzyme. figure 3C montre le signal observé EPR lors de la stimulation de PMN par le PMA. Les valeurs HFSC pour ce signal a été déterminé à être un N = 14,71 G, une &amp;beta;-H = 11,40 G et une γ-H = 1,25 G, et sont compatibles avec les valeurs de la littérature d&#39;un N = 14,3 G, un β-H = 11,7 G et une γ-H = 1,3 G 19 pour DMPO-O 2 H adduit. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="Figure 1" /> Figure 1. Diagramme pour la détection des radicaux de neutrophiles et de piégeage de spin utilisant BAEC EPR. (A) PMN ont été mélangés avec DMPO et de la PMA, et le mélange résultant transféré dans une cellule EPR plat pour l&#39;acquisition de données. (B) BAEC ont été cultivées sur une plaque, et on le lave avec du DPBS. Le spin Fe (MGD) 2 a été ajouté avec Cai. La solution a été soigneusement mélangé et incubé. Le mélange a été transféré dans une cellule EPR plat pour l&#39;EPR d&#39;acquisition de données. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="Figure 2" /> Figure 2. De détection EPR de NO de BAEC. (A) du spectre de Fe 2 +-MGD seulement (B) du spectre de Fe 2 +-MGD + CaI seulement. (C) spectre résultant de triplet NO piégeage par Fe 2 +-MGD utilisant CaI-cellules stimulées. (D) du spectre montrant la diminution de la production de NO en raison de 0,5 mM SIN-1 le traitement des cellules. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Figure 3" /> Figure 3. Détection EPR de DMPO-O 2 H à partir de neutrophiles activés. (A) du spectre de 10 mm seulement DMPO. (B) du spectre des PMN seul en présence de 10 mM DMPO. (C) du spectre des PMN activés par la PMA, dans la présence de 10 mM DMPO.

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Pas de conflits d&#39;intérêt déclarés.

Piégeage de spin EPR a été employé dans un large éventail d&#39;applications biomédicales pour la quantification et l&#39;identification des radicaux libres. Piégeage de spin est très sensible, capable de détecter les radicaux à des concentrations allant de nM à uM fait la rendant appropriée pour l&#39;application dans les systèmes biologiques. La formation de l&#39;adduit paramagnétique, NO-Fe 2 +-MGD, est à la base de non détection par l&#39;intermédiaire d&#39;EPR. Fe 2 +-MGD...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Phénol un milieu exempt de DMEM Glucose à haute 1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% de trypsine-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-glutamine </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM acides aminés non essentiels </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> De sérum foetal bovin </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Facteur de croissance endothélial </td><td> Millipore </td><td> 02-102 </td><td></td></tr><tr><td> CaI </td><td> Enzo Life Sciences </td><td> A-23187 </td><td> Dissoudre dans du DMSO </td></tr><tr><td> SIN-1 </td><td> Enzo Life Sciences </td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> Laboratoires Dojindo </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> Dissoudre dans du PBS avec Ca et Mg </td></tr><tr><td> MGD </td><td> Enzo Life Sciences </td><td> ALX-400-014-M050 </td><td> Dissoudre dans du PBS avec Ca 2 + et Mg 2 + </td></tr><tr><td> Cellules BAEC </td><td> Cell Systems </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 avec du CaCl 2 et MgCl 2 </td><td> Sigma Uneldrich </td><td> D8662 </td><td></td></tr><tr><td> Phorbol-myristate acétate (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

Azote réactif / espèces oxygénées (ROS / RNS) à des concentrations faibles jouent un rôle important dans la régulation de la fonction des cellules, de signalisation, et la réponse immunitaire, mais à des concentrations non réglementés sont préjudiciables à la viabilité des cellules 1, 2. Alors que les systèmes vivants ont évolué avec endogènes et alimentaires mécanismes de défense antioxydants pour réglementer ROS génération, ROS sont produites en continu tant que sous-produits naturels du métabolisme normal de l&#39;oxygène et peut causer des dommages oxydatifs à des biomolécules entraînant la perte de la fonction des protéines, coupure de l&#39;ADN, ou des lipides peroxydation 3, et, finalement, à un stress oxydatif conduisant à des blessures ou la mort cellulaire 4. Anion superoxyde radical (O 2 • -) est le précurseur d&#39;majeure partie des espèces plus fortement oxydants connus dans des systèmes biologiques tels que le peroxynitrite radical hydroxyle et. La génération de O 2 • - signale le premier signe de la flambée oxydative, et, par conséquent, its de détection et / ou de séquestration dans les systèmes biologiques est important. Dans cette démonstration, O 2 • - a été générée à partir de polynucléaires neutrophiles (PMN). Grâce à la stimulation chimiotactique avec le phorbol-12-myristate-13-acétate (PMA), PMN génère O 2 • - via l&#39;activation de la nicotinamide adénine dinucléotide phosphate (NADPH) oxydase 5. D&#39;oxyde nitrique (NO) synthase qui se décline en trois isoformes, comme inductible, endothéliale et neuronale-NOS, ou iNOS, nNOS ou de eNOS, respectivement, catalyse la conversion de la L-arginine en L-citrulline, en utilisant le NADPH pour produire NO 6 . Ici, nous avons généré NO par les cellules endothéliales. Dans des conditions de stress oxydatif, eNOS par exemple, peut passer de la production de NO à O 2 • - dans un processus appelé découplage, qui est censé être causé par l&#39;oxydation de l&#39;hème 7 ou le co-facteur, tétrahydrobioptérine (BH 4) 8. Il ya seulement quelquesméthodes fiables pour la détection de radicaux libres dans les systèmes biologiques, mais sont limités par la spécificité et la sensibilité. Piégeage de spin est couramment utilisé pour l&#39;identification de radicaux libres et comprend la réaction d&#39;addition d&#39;un radical à un accepteur de spin formant un produit d&#39;addition de spin persistant qui peut être détectée par résonance paramagnétique électronique (RPE). Les adduits différents radicaux présentent spectre distinctif qui peut être utilisé pour identifier les radicaux étant générées et peuvent fournir une mine de renseignements sur la nature et la cinétique de la production de radicaux 9. Les nitrones cycliques, 5,5-diméthyl-pyrroline-N-oxyde, DMPO 10, l&#39;phosphoryle substitué DEPMPO 11, et l&#39;ester-substitué, EMPO 12 et 13, BMPO ont été largement utilisé comme pièges de spin - le spin-ci pièges présentant plus des demi-vies pour O 2 • - adduit. Fer (II)-N-méthyl-D-glucamine dithiocarbamate, Fe (MGD) 2 &lt;/ Sub&gt; est couramment utilisé pour piéger NO en raison de taux élevé de la formation d&#39;adduits et la grande stabilité de l&#39;adduit de spin 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

Résonance paramagnétique électronique (RPE) a été utilisé pour détecter l&#39;oxyde nitrique à partir de cellules endothéliales aortiques bovines et de l&#39;anion superoxyde radical de neutrophiles humains en utilisant le fer (II)-N-méthyl-D-glucamine dithiocarbamate, Fe (MGD)<sub&gt; 2</sub&gt; Et de 5,5-diméthyl-1-pyroroline-N-oxyde, DMPO, respectivement.

Détection de l&#39;oxyde nitrique et de l&#39;anion superoxyde radical par spectroscopie de résonance paramagnétique électronique à partir de cellules en utilisant des pièges de spin

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

18.8K vues.  The Ohio State University. Résonance paramagnétique électronique (RPE) a été utilisé pour détecter l'oxyde nitrique à partir de cellules endothéliales aortiques bovines et de l'anion superoxyde radical de neutrophiles humains en utilisant le fer (II)-N-méthyl-D-glucamine dithiocarbamate, Fe (MGD) 2 Et de 5,5-diméthyl-1-pyroroline-N-oxyde, DMPO, respectivement.

Vidéo: 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.

Résonance de spin électronique Micro-imagerie d&#39;espèces vivantes pour la cartographie d&#39;oxygène

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

Recherche

Biologie

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.

Expérience profil bioénergétique utilisant cellules C2C12 de myoblastes

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.

Concentration des métabolites issus de communautés planctoniques à faible densité de l&#39;environnement en utilisant métabolomique résonance magnétique nucléaire

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

Détection de Live<em&gt; Escherichia coli</em&gt; O157: H7 par les cellules 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. ...

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.

Deux photons imagerie intracellulaire de Ca<sup&gt; 2+</sup&gt; Manipulation et production d&#39;oxyde nitrique endothélial et en cellules de muscle lisse d&#39;un Rat aorte isolée

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 détection de visage de l&#39;oxyde nitrique et de superoxyde dans la couche endothéliale des artères intactes

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.

Développement et caractérisation de<em&gt; In vitro</em&gt; Microvaisseaux réseau et des mesures quantitatives de l&#39;endothélium [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Et production d&#39;oxyde nitrique

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.

Détection des formes modifiées de Cytosine Utilisation Sensible immunohistochimie

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.

Mesure de nitrites et de nitrates, Métabolites dans le Nitric Oxide Pathway, dans les matières biologiques en utilisant la méthode chimiluminescence

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

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Application de Codé génétiquement fluorescent oxyde nitrique (NO •) Sondes, les geNOps, pour l&#39;imagerie en temps réel de NO • Les signaux dans les cellules simples

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...