Questo lavoro è stato finanziato dal National NIH Heart, Lung, and Blood Institute concessione RO1 HL81248.

1. Coltura di cellule endoteliali dell&#39;aorta bovina (BAEC) <ol><li> Corrette tecniche asettiche sono stati seguiti. </li><li> In un bagno di acqua, terreno senza antibiotici calda a 37 ° C. </li></ol> Nota: Il mezzo è costituito da fenolo libero terreno Dulbecco modificato Eagle (DMEM) con 4,5 g / L D-glucosio, 4 mM L-glutammina, 1% di amminoacidi non essenziali, integrato con 10% siero bovino fetale (FBS) e 2,5 mg / L fattore di crescita endoteliale. <ol start="3"><li> Rimuovere le cellule T75 pallone contenente dall&#39;incubatore e pulire la superficie del pallone con il 70% di etanolo prima di inserirlo all&#39;interno della cappa. </li><li> Rimuovere il mezzo vecchio usando un aspiratore e lavare due volte con 5 ml di Dulbecco tampone fosfato salino (DPBS). </li><li> Aggiungere 2 ml di tripsina e attendere per 4-5 min per le cellule di staccare periodicamente, mentre l&#39;ispezione sotto il microscopio. </li><li> Aggiungere 3 ml del mezzo e ripetutamente miscelare con una pipetta a separato tegli cellule e creare una sospensione omogenea. </li><li> Trasferire 5 ml del mezzo con tripsina in un tubo da 15 ml e centrifugare a 121 g per 5 min. </li><li> Rimuovere il surnatante mediante un aspiratore. Aggiungere 5 ml di DPBS e mescolare accuratamente. Centrifugare a 121g per 5 min. </li><li> Aspirare il supernatante e risospendere il pellet aggiungendo 6 ml del mezzo. </li><li> Su una piastra da 6 pozzetti, aggiungere 1 ml della sospensione cellulare a ciascun pozzetto si aggiunge 1 ml di mezzo. Mescolare la sospensione con una pipetta. </li><li> Etichettare la piastra e incubare durante la notte a 37 ° C e 5% CO 2. </li></ol> 2A. Rilevamento di NO con Cell BAEC <ol><li> Corrette tecniche asettiche sono stati seguiti. </li><li> Rimuovere la piastra da 6 pozzetti dall&#39;incubatore e aspirare il mezzo dal primo pozzetto. Lavare le cellule due volte con 1 ml di DPBS. </li><li> Aggiungere 210 ul di 1,9 mM ferro (II) solfato eptaidrato (FeSO 4 .7 H 2 O, preparare fresco sciogliendo 0,8 mg in1 ml con DPBS CaCl 2 e MgCl 2) e 210 pl di ammonio N-metil-D-glucammina ditiocarbammato (MGD, preparare fresco sciogliendo 2,7 mg in 500 pl con DPBS CaCl 2 e MgCl 2) usando un rapporto di 01:07 . (Nota:. Questo rapporto dove viene utilizzato MGD eccesso è stato convenzionalmente impiegati per la preparazione di Fe 2 +-MGD complessa a causa del fatto che la resa di Fe 3 +-MGD è massimizzata in presenza di eccesso MGD L&#39;aggiunta di ascorbato in soluzione per stabilizzare il Fe 2 +-MGD non è necessaria poiché il NO-Fe 3 +-MGD formatosi viene endogenamente ridotto al rilevabile EPR NO-Fe 2 +-MGD da ascorbato, idrochinone, o cisteina con un&#39;efficienza di conversione fino al 99,9% .. Il basso stato di spin diamagnetico di Fe 2 + consente il rilevamento di NO con celle piane o tubo capillare senza la necessità di un dispositivo a bassa temperatura) 15. </li><li> Agitare la sospensione risultante bene e aggiungere 4,6 μl di calcio ionoforo (CAI) (preparato da una soluzione stock di 1,9 mM sciogliendo 1 mg in 1 ml di DMSO). </li><li> Agitare la soluzione nuovamente e incubare a 37 ° C per 36 min per permettere un&#39;ulteriore riduzione di NO-Fe 3 +-MGD al rilevabile EPR NO-Fe 2 +-MGD. </li><li> Raccogliere il supernatante (425 pl) in un tubo Eppendorf e il trasferimento di una cella EPR piatta (o ad un tubo capillare 50 pl). </li><li> Parametri di acquisizione EPR sono: frequenza delle microonde: 9,8 GHz; centro del campo: 3427 G; modulazione di ampiezza: 6,0 G, larghezza di scansione: 100 G; guadagno del ricevitore: 1 x 10 5, potenza microonde: 10 mW, il numero totale di scansioni: 121; tempo di scansione: 10 s, e la costante di tempo: 20 ms (Nota: poiché i parametri possono variare da uno strumento e le condizioni sperimentali per un altro, quindi, solo il campo centrale, la frequenza e modulazione di ampiezza sono i parametri più importanti da considerare.). </li><li> Registrare il spettri a temperatura ambiente e il 2-D spettri sono stati integritànominale per ridurre il rumore di fondo e di base corretta con Bruker WinEPR software di elaborazione dati o di altri software di elaborazione. Per la quantificazione della formazione di addotti, trame standard di concentrazione in funzione del intensità del segnale (o area) può essere costruito usando un donatore di NO SNAP. </li></ol> 2B. eNOS Uncoupling Experiment <ol><li> Rimuovere la piastra da 6 pozzetti dall&#39;incubatore e aspirare il terreno dal secondo pozzo e lavare due volte con 1 ml DPBS. </li><li> Un donatore perossinitrito, 5-ammino-3-(4-morfolinil) -1,2,3-oxadiazolium cloruro (SIN-1) è stato usato per disaccoppiare eNOS 16. Aggiungere 100 pl di 0,5 mM SIN-1 (M r 206,6 g / mol, da 10 mM soluzione fotografia preparata sciogliendo 1 mg SIN-1 in 500 pl di PBS senza Ca / Mg ioni) e diluire a 2 ml con DPBS e il 10% FBS. </li><li> Incubare per 2 ore a 37 ° C e 5% CO 2. </li><li> Rimuovere la piastra dalla camera umida e lavare due volte con DPBS. </li><li> Aggiungere 210 pl di2,8 mM FeSO 4 .7 H 2 O e 210 microlitri di 19,6 mM MGD preparata secondo la procedura di cui sopra. </li><li> Agitare la soluzione e aggiungere 4,6 microlitri di 1,9 mM CAI. </li><li> Agitare la soluzione nuovamente e incubare a 37 ° C per 36 min. </li><li> Raccogliere il supernatante (425 pl) in un tubo Eppendorf e il trasferimento di una cella EPR piatta. </li><li> Parametri di acquisizione EPR sono: frequenza delle microonde: 9,8 GHz; centro del campo: 3427 G; modulazione di ampiezza: 6 G, larghezza di scansione: 100 G; guadagno del ricevitore: 1 x 10 5, potenza microonde: 10 mW, il numero totale di scansioni: 121; Tempo di scansione: 10 s, e la costante di tempo: 20 ms. </li><li> Registrare il spettri e il 2-D spettri sono stati integrati per ridurre il rumore di fondo e basale corretta come menzionato sopra. </li></ol> 3. Rilevamento di O 2 • - da polimorfonucleati neutrofili (PMN) <ol><li> I neutrofili sono stati isolati dal campione di sangue umano come descritto in precedenza 17. </li><li> Ottenere una soluzione stock di 1 M * DMPO in PBS contenente 0,1 mM dietilentriammina-pentaacetico acido (DTPA). DMPO ha un punto di fusione di 25-29 ° C ed è quindi più conveniente per pipettare liquidi DMPO (densità ~ 1,02 g / ml a 25 ° C) per una fiala di vetro (Nota: non usare i flaconi di plastica per la pesatura in quanto pura DMPO reagisce con plastica). DMPO congelato può essere sciolto da acqua corrente tiepida al flacone (Nota: non eseguire l&#39;acqua calda come DMPO può decomporsi). </li><li> È importante utilizzare DMPO elevata purezza (&gt; 99%) poiché alcune delle trappole di spin disponibili in commercio contengono impurità paramagnetici, e pertanto, è indispensabile eseguire spettro EPR della sola soluzione DMPO sola (10 mM in questo caso). È fondamentale che non segnale di fondo è evidente (vedi Figura 3A). </li><li> Preparare la soluzione madre (1 mg / ml) di forbol-12-miristato-13-acetato (PMA) in DMSO. Effettuare aliquote diluendo la soluzione a 10 ug / ml in PBS. </li><li> In un 1.5 ml provetta Eppendorf, preparare una soluzione con un volume totale di 0,6 ml seguendo la sequenza di aggiunta: ~ 10 6 cellule per ml di PMN, D-glucosio (1 mg / ml) e albumina (1 mg / ml), 10 DMPO mM e 0,2 mg / ml di PMA. (Nota: PMA è il radicale attivatore e dovrebbe essere aggiunto per ultimo). </li><li> Trasferire la soluzione in una cella EPR piatto. </li><li> EPR parametri di acquisizione condizioni sono: frequenza delle microonde: 9,8 GHz; centro del campo: 3486 G; modulazione di ampiezza: 0,5 G, larghezza di scansione: 100 G; guadagno del ricevitore: 5 x 10 5, il numero totale di scansioni: 10; tempo di scansione: 30 s ; microonde potenza: 20 mW, e costante nel tempo: 81 ms. Per la quantificazione della formazione di addotti, trame standard di concentrazione in funzione del intensità del segnale (o area) può essere costruito utilizzando i nitrossidi stabili come TEMPO o 3-carbossilico-PROXYL. </li></ol> * Attenzione sull&#39;uso di DMPO: Utilizzando una cella piatta, si può aumentare la densità cellulare e aumentando il segnaleintensità dell&#39;addotto rotazione ma l&#39;emivita di O 2 • - addotto di DMPO è breve (t 1/2 ~ 1 min) che si decompone a DMPO-OH. DMPO può essere sostituito con le stesse concentrazioni di Empo, BMPO o DEPMPO che sono disponibili commercialmente per una maggiore stabilità addotto con t 1/2 ~ 8 e 14 minuti, rispettivamente. 4. Risultati rappresentativi Spin intrappolamento di NO radicale è stata effettuata utilizzando Fe 2 +-MGD. Figure 2A e 2B mostrano alcun segnale EPR da Fe 2 +-MGD o miscela di Fe 2 +-MGD con CAI, indicando che non NO segnale di fondo proviene da questi reagenti. BAEC dopo stimolazione con CAI rilascia NO che reagisce con Fe 2 +-MGD per formare l&#39;addotto di spin, NO-Fe 2 +-MGD, e mostra un segnale di caratteristica tripletto con divisione iperfine costante (hfsc) valore di N = 12,66 G e g-fattore g = 2,040. (Figura 2C). Il hfsc valore è stato determinato utilizzando il programma di simulazione WINSIM che può essere scaricato dal sito NIEHS Database Software EPR. Il hfsc sperimentale è coerente con il valore di una letteratura N = 12,70 e G = 2,041 g per 18 NO-Fe 2 +-MGD addotto. Analogamente, l&#39;effetto di SIN-1 sul BAEC abbassa la produzione di NO per eNOS disaccoppiamento come mostrato nella Figura 2D. DMPO spin trap è stato utilizzato per O 2 • - rilevamento. DMPO sola non ha dato un segnale, come mostrato nella Figura 3A confermano che la trappola rotazione è esente da impurità paramagnetiche. Figura 3B è lo spettro di DMPO e PMN solo, e analogamente, non c&#39;è segnale rilevabile suggerendo che il DMPO non provoca attivazione del ossidasi NADPH enzima. figura 3C mostra il segnale osservato EPR ​​alla stimolazione di PMN da PMA. I valori hfsc per questo segnale sono stati determinati per essere un N = 14,71 G, a &amp;beta;-H = 11,40 G e γ-H = 1,25 G, e sono coerenti con i valori di letteratura di un N = 14,3 G, una β-H = 11,7 G e γ-H = 1,3 G 19 per DMPO-O 2 H addotto. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="Figura 1" /> Figura 1. Diagramma di flusso per la rilevazione di radicali da neutrofili e BAEC utilizzando trapping EPR rotazione. (A) sono stati mescolati con PMN DMPO e PMA, e la miscela risultante trasferita in una cella EPR piatta per l&#39;acquisizione dei dati. (B) BAEC sono state fatte crescere su una piastra, e lavata con DPBS. Lo spin trap Fe (MGD) 2 è stato aggiunto insieme con il CAI. La soluzione è stata accuratamente miscelati e incubati. La miscela è stata trasferita ad una cella EPR appartamento EPR acquisizione dati. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="Figura 2" /> Figura 2. Rilevazione EPR di NO da BAEC. (A) Spettro di Fe 2 +-MGD solo (B) Spettro di Fe 2 +-MGD + CAI solo. (C) Triplet spettro derivante da NO trapping da Fe 2 +-MGD con CAI-cellule stimolate. (D) Spettro che mostra diminuzione della produzione di NO per 0,5 mM SIN-1 trattamento di cellule. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Figura 3" /> Figura 3. Rilevazione EPR di DMPO-O 2 H dai neutrofili attivati. (A) Spettro di 10 DMPO soli mm. (B) Spettro di PMN solo in presenza di 10 mM DMPO. (C) Spettro di PMN attivato dal PMA in presenza di 10 mM DMPO.

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Trapping centrifuga EPR è stato impiegato in una vasta gamma di applicazioni biomediche per quantificare e identificare i radicali liberi. Trapping Spin è altamente sensibile, in grado di rilevare radicali a concentrazioni comprese tra nM a pM rendendo così adatto per applicazioni in sistemi biologici. La formazione dell&#39;addotto paramagnetico, NO-Fe 2 +-MGD, è la base di NO rilevamento via EPR. Fe 2 +-MGD reagisce rapidamente con NO 18 ad una velocità di ~ 10 6</su...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Fenolo DMEM libero Alta glucosio 1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% tripsina-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-Glutammina </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM non amminoacidi essenziali </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Siero bovino fetale </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Fattore di crescita endoteliale </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> Sciogliere in 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> 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> Sciogliere in PBS con Ca e Mg </td></tr><tr><td> MGD </td><td> Enzo Life Sciences </td><td> ALX-400-014-M050 </td><td> Sciogliere in PBS con Ca 2 + e Mg 2 + </td></tr><tr><td> Cellule BAEC </td><td> Sistemi di celle </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 e MgCl 2 </td><td> A Sigmaldrich </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

Azoto reattivo / specie di ossigeno (ROS / RNS) a basse concentrazioni gioca un ruolo importante nella regolazione della funzione delle cellule, di segnalazione, e la risposta immunitaria, ma in concentrazioni non regolamentati sono dannose per la vitalità cellulare 1, 2. Mentre i sistemi viventi si sono evoluti con meccanismi antiossidanti di difesa endogeni e dietetici per regolare ROS generazione, ROS vengono prodotti continuamente come naturali sottoprodotti del normale metabolismo di ossigeno e può causare danni ossidativi alle biomolecole con conseguente perdita di funzione della proteina, taglio del DNA, o lipidi perossidazione 3, e, infine, allo stress ossidativo che porta a lesioni o morte delle cellule 4. Radicale superossido (O 2 • -) è il principale precursore di alcune delle specie più altamente ossidanti noti presenti in sistemi biologici, come perossinitrito e radicale idrossile. La generazione di O 2 • - segnala il primo segnale di burst ossidativo, e pertanto, its rilevamento e / o il sequestro nei sistemi biologici è importante. In questa dimostrazione, O 2 • - è stato generato da neutrofili polimorfonucleati (PMN). Attraverso la stimolazione chemiotattica con forbol-12-miristato-13-acetato (PMA), PMN genera O 2 • - attraverso l&#39;attivazione di nicotinammide adenin dinucleotide fosfato (NADPH) ossidasi 5. Ossido nitrico (NO) sintasi che viene in tre isoforme, come inducibile-,-neuronale e endoteliale-NOS, o iNOS, eNOS nNOS o, rispettivamente, catalizza la conversione di L-arginina a L-citrullina, usando NADPH per produrre NO 6 . Qui, abbiamo generato NO da cellule endoteliali. In condizioni di stress ossidativo, eNOS ad esempio possibile passare dalla produzione di NO a O 2 • - in un processo chiamato disaccoppiamento, che è ritenuto essere causato da ossidazione di eme 7 o la co-fattore, tetraidrobiopterina (BH 4) 8. Ci sono solo pochemetodi affidabili per la rilevazione di radicali liberi in sistemi biologici, ma sono limitati dalla specificità e sensibilità. Trapping Spin è comunemente utilizzato per l&#39;identificazione di radicali liberi e comporta la reazione di addizione di un radicale di una trappola di spin formando un addotto persistente centrifuga che può essere rilevato dal risonanza paramagnetica elettronica (EPR) spettroscopia. Gli addotti vari radicali presentano spettro distintivo che può essere utilizzato per identificare i radicali che vengono generati e può fornire una grande quantità di informazioni sulla natura e la cinetica della produzione di radicali 9. Le nitroni ciclici, 5,5-dimetil-pirrolina-N-ossido, DMPO 10, il fosforil-sostituito DEPMPO 11, e l&#39;estere-sostituito, Empo BMPO 12 e 13, sono stati ampiamente impiegati come trappole di spin - spin quest&#39;ultimo trappole esibendo emivita più lunga per O 2 • - addotto. Ferro (II)-N-metil-D-glucammina ditiocarbammato, Fe (MGD) 2 &lt;/&gt; Sub è comunemente utilizzato per intercettare NO dovuto alla elevata di formazione di addotti e l&#39;elevata stabilità dell&#39;addotto rotazione 14.

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

La risonanza paramagnetica elettronica (EPR) spettroscopia è stato impiegato per rilevare ossido nitrico da cellule endoteliali aortiche bovine e radicale superossido da neutrofili umani utilizzando ferro (II)-N-metil-D-glucammina ditiocarbammato, Fe (MGD)<sub&gt; 2</sub&gt; E 5,5-dimetil-1-pyroroline-N-ossido, DMPO, rispettivamente.

Rilevamento di ossido nitrico e anione superossido radicale da spettroscopia di risonanza paramagnetica elettronica da cellule con Spin Traps

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 Views.  The Ohio State University. La risonanza paramagnetica elettronica (EPR) spettroscopia è stato impiegato per rilevare ossido nitrico da cellule endoteliali aortiche bovine e radicale superossido da neutrofili umani utilizzando ferro (II)-N-metil-D-glucammina ditiocarbammato, Fe (MGD) 2 E 5,5-dimetil-1-pyroroline-N-ossido, DMPO, rispettivamente.

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.

Electron Spin Resonance micro-imaging delle specie dal vivo per la mappatura di ossigeno

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

Ricerca

Biologia

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.

Profilo Esperimento bioenergetica utilizzando cellule dei mioblasti C2C12

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.

Concentrazione dei metaboliti a bassa densità Comunità planctoniche per Metabolomica ambientali utilizzando la spettroscopia a risonanza magnetica nucleare

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

Rilevamento dal vivo<em&gt; Escherichia coli</em&gt; O157: H7 Celle di 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.

A due fotoni Imaging di Intracellulare Ca<sup&gt; 2+</sup&gt; Manipolazione e l&#39;ossido nitrico Produzione in endoteliale e della muscolatura liscia cellule di un isolato Rat Aorta

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 Face Detection di ossido nitrico e superossido in strato endoteliale delle arterie Intact

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.

Sviluppo e caratterizzazione di<em&gt; In Vitro</em&gt; Microvasi Rete e misurazioni quantitative di endoteliale [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E ossido nitrico Produzione

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.

Il rilevamento di forme modificate di Citosina Uso Sensitive immunoistochimica

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.

Misurazione nitriti e nitrati, metaboliti in ossido nitrico Pathway, nei materiali biologici utilizzando il metodo chemiluminescenza

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.

L&#39;applicazione di geneticamente codificato fluorescente ossido nitrico (NO •) sonde, i geNOps, in tempo reale Imaging di NO • I segnali in singole cellule

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