Diese Arbeit wurde von den NIH National Heart, Lung, and Blood Institute Zuschuss RO1 HL81248 finanziert.

1. Kultur von Bovine Endothelzellen (BAEC) <ol><li> Die richtige aseptische Techniken befolgt wurden. </li><li> In einem Wasserbad, warme Medium ohne Antibiotika bei 37 ° C </li></ol> Hinweis: Das Medium besteht aus Phenol Dulbecco-modifiziertem Eagle-Medium (DMEM) mit 4,5 g / l D-Glucose, 4 mM L-Glutamin, 1% nicht essentielle Aminosäuren, mit 10% fötalem Rinderserum (FBS) und 2,5 ergänzt mg / L endothelial growth factor. <ol start="3"><li> Entfernen Sie die T75 Kolben mit Zellen aus dem Inkubator und reinigen Sie die Oberfläche der Flasche mit 70% Ethanol, bevor Sie es im Inneren der Kapuze. </li><li> Entfernen Sie die alte Medium mit einem Saug und Waschen zweimal mit 5 ml von Dulbeccos phosphatgepufferter Salzlösung (DPBS). </li><li> 2 ml Trypsin und warten Sie 4-5 min für Zellen zu trennen, während in regelmäßigen Abständen Inspektion unter dem Mikroskop. </li><li> 3 ml des Mediums und immer wieder mischen mit einer Pipette zu trennen ter Zellen und erzeugen eine gleichmäßige Suspension. </li><li> Übertragen 5 ml des Mediums mit Trypsin zu einer 15 ml-Röhrchen und zentrifugiert bei 121 g für 5 min. </li><li> Entfernen Sie den Überstand mit einem Aspirator. 5 ml DPBS und gründlich mischen. Zentrifugieren bei 121 g für 5 min. </li><li> Saugen Sie den Überstand und resuspendieren Sie das Zellpellet durch Zugabe von 6 ml des Mediums. </li><li> Auf einer 6-Well-Platte, 1 ml der Zellsuspension in jede Vertiefung dann 1 ml des Mediums. Mischen Sie die Suspension mit einer Pipette. </li><li> Beschriften Sie die Platte und Inkubation über Nacht bei 37 ° C und 5% CO 2. </li></ol> 2A. Detektion von NO mit BAEC Zelle <ol><li> Die richtige aseptische Techniken befolgt wurden. </li><li> Entfernen Sie die 6-Well-Platte aus dem Inkubator und Saugen Sie das Medium aus der ersten Bohrung. Die Zellen werden zweimal mit 1 ml DPBS. </li><li> Fügen Sie 210 ul von 1,9 mm Eisen (II) sulfat-Heptahydrat (FeSO 4 · 7H 2 O, bereiten frisch durch Lösen von 0,8 mg in1 ml DPBS mit CaCl 2 und MgCl 2) und 210 ul von Ammonium-N-Methyl-D-glucamin Dithiocarbamat (MGD, frisch zubereitet und durch Auflösen von 2,7 mg in 500 ul DPBS mit CaCl 2 und MgCl 2) mit einem Verhältnis von 1:7 . (Hinweis:. Dieses Verhältnis, wo überschüssiges MGD verwendet wird, wurde herkömmlicherweise für die Herstellung von Fe 2 +-MGD-Komplex durch die Tatsache, dass die Ausbeute für Fe 3 +-MGD in Gegenwart von überschüssigem MGD maximiert wird eingesetzt Die Zugabe von Ascorbat in Lösung, um die Fe 2 +-MGD Stabilisierung ist nicht notwendig, da die NO-Fe 3 +-MGD endogen gebildet wird, um den EPR-detektierbare NO-Fe 2 durch Ascorbat, Hydrochinon, oder Cystein mit Wirkungsgrad von bis +-MGD reduziert auf 99,9% .. Die Low-Spin-Zustand diamagnetisch von Fe 2 + ermöglicht den Nachweis von NO mit flachen Zelle oder Kapillarrohr, ohne die Notwendigkeit eines niedrigen Temperatur-Gerät) 15. </li><li> Schwenken Sie die resultierende Suspension gut und fügen 4,6 μl Calcium-Ionophor (CAI) (hergestellt aus einer Stammlösung von 1,9 mM durch Lösen von 1 mg in 1 ml DMSO). </li><li> Strudel die Lösung wieder und bei 37 ° C für 36 min, um eine weitere Reduktion von NO ermöglichen-Fe 3 zum EPR nachweisbare NO-Fe 2 +-MGD +-MGD. </li><li> Die überstehende Flüssigkeit (425 ul) in ein Eppendorf-Röhrchen und Überweisung auf ein EPR flache Zelle (oder einem 50 ul Kapillarrohr). </li><li> EPR-Aufnahme-Parameter sind: Mikrowellen-Frequenz: 9,8 GHz; Center-Bereich: 3427 G; Modulationsamplitude: 6,0 g; Sweep-Breite: 100 g; Empfänger Gewinn: 1 x 10 5; Mikrowelle: 10 mW; Gesamtzahl der Scans: 121; Ablaufzeit: 10 s; und die Zeitkonstante von 20 ms (Anmerkung: Da die Parameter von einem Instrument und die experimentellen Bedingungen zur anderen variieren, daher nur die mittlere Feld, Frequenz und der Modulationsamplitude die wichtigsten Parameter zu prüfen.). </li><li> Die Spektren bei Raumtemperatur und die 2-D-Spektren wurden integribewertet, um die Hintergrundgeräusche und Grundlinie korrigiert Bruker WinEPR Data Processing Software oder andere Software zur Datenverarbeitung zu reduzieren. Zur Quantifizierung der Adduktbildung, kann Standardkurven Konzentration gegen Signalintensität (oder Fläche) ausgebildet ist unter Verwendung eines NO-Donors SNAP werden. </li></ol> 2B. eNOS Entkopplung Experiment <ol><li> Entfernen Sie die 6-Well-Platte aus dem Inkubator und Saugen Sie das Medium aus der zweiten gut waschen und zweimal mit 1 ml DPBS. </li><li> Ein Donor Peroxynitrit, 5-Amino-3-(4-Morpholinyl) -1,2,3-oxadiazolium chlorid (SIN-1) wurde verwendet, um eNOS 16 zu entkoppeln. Fügen Sie 100 ul 0,5 mM SIN-1 (M r 206,6 g / mol, von 10 mM Stammlösung frisch durch Lösen von 1 mg SIN-1 in 500 ul PBS ohne Ca / Mg-Ionen vorbereitet) und verdünnte bis 2 ml mit DPBS und 10% FBS. </li><li> Inkubieren für 2 h bei 37 ° C und 5% CO 2. </li><li> Die Platte aus Inkubator und waschen zweimal mit DPBS. </li><li> Fügen Sie 210 ul2,8 mM FeSO 4 · 7H 2 O und 210 ul von 19,6 mm MGD frisch nach dem Verfahren des oben genannten vorbereitet. </li><li> Schwenken Sie die Lösung und fügen 4,6 ul von 1,9 mm Cai. </li><li> Schwenken Sie die Lösung wieder und Inkubation bei 37 ° C für 36 min. </li><li> Die überstehende Flüssigkeit (425 ul) in ein Eppendorf-Röhrchen und Überweisung auf ein EPR Flachzelle. </li><li> EPR-Aufnahme-Parameter sind: Mikrowellen-Frequenz: 9,8 GHz; Center-Bereich: 3427 G; Modulationsamplitude: 6 G; Sweep-Breite: 100 g; Empfänger Gewinn: 1 x 10 5; Mikrowelle: 10 mW; Gesamtzahl der Scans: 121; Sweep-Zeit: 10 s; und Zeitkonstante: 20 ms. </li><li> Die Spektren und die 2-D-Spektren wurden integriert, um das Hintergrundrauschen und Baseline-korrigierte wie oben erwähnt zu reduzieren. </li></ol> 3. Nachweis von O 2 • - von neutrophilen Granulozyten (PMN) <ol><li> Neutrophile wurden aus menschlichen Blutprobe isoliert, wie zuvor 17 beschriebenen. </li><li> Machen Sie eine Stammlösung von 1 M * DMPO in PBS mit 0,1 mM Diethylentriaminpentaessigsäure (DTPA). DMPO hat einen Schmelzpunkt von 25-29 ° C, so ist es bequemer, um Flüssigkeit DMPO (Dichte ~ 1,02 g / ml bei 25 ° C) in einem Glasfläschchen (Anmerkung pipettieren: Verwenden Sie keine Kunststoff-Fläschchen zum Wiegen da reines DMPO reagiert mit Kunststoff). Gefrorene DMPO kann durch Ausführen von lauwarmem Wasser auf dem Fläschchen (: Führen Sie nicht heißes Wasser als DMPO kann sich zersetzen Hinweis) geschmolzen werden. </li><li> Es ist wichtig, um die hohe Reinheit DMPO (&gt; 99%) zu verwenden, da einige der im Handel erhältlichen Spinfallen paramagnetische Verunreinigungen enthalten, und daher ist es unerlässlich, EPR-Spektrum nur die DMPO Lösung allein (10 mM in diesem Fall) ausgeführt. Entscheidend ist, dass kein Hintergrund-Signal erkennbar ist (siehe Abbildung 3A). </li><li> Planen-Stammlösung (1 mg / ml) Phorbol-12-myristat-13-acetat (PMA) in DMSO. Machen Aliquots durch Verdünnen der Lösung auf 10 pg / ml in PBS. </li><li> In einem 1.5 ml Eppendorf-Röhrchen, Herstellung einer Lösung mit einem Gesamtvolumen von 0,6 ml in folgender Reihenfolge der Zugabe: ~ 10 6 Zellen pro ml PMN, D-Glucose (1 mg / ml) und Albumin (1 mg / ml), 10 DMPO mM und 0,2 ug / ml PMA. (Hinweis: PMA ist die radikale Aktivator und sollte als letztes zugegeben werden). </li><li> Die Lösung wird in einem EPR Flachzelle. </li><li> EPR-Aufnahme-Parameter sind: Mikrowellen-Frequenz: 9,8 GHz; Center-Bereich: 3486 G; Modulationsamplitude: 0,5 g; Sweep-Breite: 100 g; Empfänger Gewinn: 5 x 10 5; Gesamtzahl der Scans: 10; Sweep-Zeit: 30 s ; Mikrowellenleistung: 20 mW; und Zeitkonstante: 81 ms. Zur Quantifizierung der Adduktbildung, kann Standardkurven Konzentration gegen Signalintensität (oder Fläche) unter Verwendung der stabilen Nitroxide, wie TEMPO oder 3-carbonsäure-PROXYL werden. </li></ol> * Wichtiger Hinweis zur Verwendung von DMPO: Durch die Verwendung eines flachen Zellen, kann man erhöhen die Zelldichte und erhöht damit die SignalqualitätIntensität des Spinadduktes, aber die Halbwertszeit von O 2 • - Addukt von DMPO kurz ist (t 1/2 ~ 1 min), welche zersetzt DMPO-OH ist. DMPO können substituiert mit den gleichen Konzentrationen von EMPO, BMPO oder DEPMPO im Handel erhältlich sind für eine erhöhte Stabilität Addukt mit t 1/2 ~ 8 und 14 min, jeweils werden. 4. Repräsentative Ergebnisse Spintrapping der NO-Radikal wurde mit Fe 2 +-MGD. 2A und 2B zeigen kein EPR-Signal von Fe 2 +-MGD oder eine Mischung von Fe 2 + mit Cai-MGD, was darauf hinweist, dass kein Signal von NO Hintergrund dieser Reagenzien stammt. BAEC nach Stimulation mit CaI NO freisetzt, die mit Fe 2 +-MGD reagiert, um die Spinadduktes, NO-Fe 2 +-MGD zu bilden, und zeigt ein charakteristisches Signal mit Triplett Hyperfeinaufspaltung Konstante (HFSC) Wert eines N = 12,66 g und g-Faktor von g = 2,040. (Abbildung 2C). Die HF-SC-Wert wurde mit Hilfe des WinSim Simulationsprogramm, das aus NIEHS EPR-Software Datenbank-Website heruntergeladen werden kann. Die experimentelle HFSC steht im Einklang mit der Literatur Wert einer N = 12,70 G und g = 2,041 18 für NO-Fe 2 +-Addukt MGD. In ähnlicher Weise abgesenkt die Wirkung von SIN-1 auf die NO-Produktion BAEC durch eNOS Entkopplung, wie in 2D gezeigt ist. Erkennung - DMPO wurde für O 2 • Einsatz. DMPO allein nicht ein Signal geben, wie in den 3A bestätigt, dass der Spin-frei von paramagnetischen Verunreinigungen gezeigt. 3B ist das Spektrum der DMPO und PMN nur, und in ähnlicher Weise gibt es kein nachweisbares Signal ab, dass die nicht zu DMPO Aktivierung des Enzyms NADPH-Oxidase. 3C zeigt die beobachteten EPR-Signals nach der Stimulierung von PMN durch PMA. Die HFSC Werte für dieses Signal waren entschlossen, ein N = 14,71 g, a &amp; seinbeta;-H = 11,40 g γ und-H = 1,25 G und stehen im Einklang mit den Literaturwerten von einer N = 14,3 g, einer β-H = 11,7 G und einer γ-H = 1,3 G 19 für-O DMPO 2 H-Addukt. <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="1" /> 1. Ablaufdiagramm für den Nachweis von Resten von Neutrophilen und BAEC mit EPR Spin trapping. (A) PMNs wurden gemischt mit DMPO und PMA, und die resultierende Mischung eine EPR Flachzelle zur Datenerfassung übertragen. (B) BAEC wurden auf eine Platte gezüchtet, gewaschen und mit DPBS. Der Spin-Trap-Fe (MGD) 2 wurde zusammen mit Cai hinzu. Die Lösung wurde gründlich gemischt und inkubiert. Das Gemisch wurde auf einem EPR Flachzelle für EPR Datenerfassung übertragen. <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="2" /> Abbildung 2. EPR Detektion von NO aus BAEC. (A) Spektrum des Fe 2 +-MGD nur (B) Spektrum von Fe 2 +-MGD + CaI nur. (C) Triplettspektrum aufgrund fehlender Trapping von Fe 2 +-MGD mit CAI-Zellen stimuliert. (D) Spectrum zeigt Rückgang der NO-Produktion durch 0,5 mM SIN-1 Behandlung von Zellen. <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="Abbildung 3" /> Abbildung 3. EPR Detektion von DMPO-O 2 H aus aktivierten neutrophilen Granulozyten. (A)-Spektrum von 10 mM DMPO nur. (B) Spektrum von PMN allein in der Gegenwart von 10 mM DMPO. (C) Spektrum von PMN durch PMA in Gegenwart von 10 mM DMPO aktiviert.

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EPR Spin trapping wurde in einem weiten Bereich von biomedizinische Anwendungen für die Quantifizierung und Identifizierung von freien Radikalen verwendet. Spin trapping ist hochempfindlich, zum Aufspüren von Resten in Konzentrationen von nM bis uM eignet sich daher für die Anwendung in biologischen Systemen. Die Bildung des paramagnetischen Addukt, NO-Fe 2 +-MGD, ist die Basis der NO-Erkennung durch EPR. Fe 2 +-MGD reagiert mit NO schnell 18 an einer Rate von ~ 10 6</su...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Phenol freien DMEM-Medium Hohe Glukose-1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% Trypsin-EDTA </td><td> GIBCO </td><td> 25200 </td><td></td></tr><tr><td> L-Glutamin </td><td> Fisher Scientific </td><td> BP379-100 </td><td></td></tr><tr><td> MEM nicht-essentiellen Aminosäuren </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Rinderfötenserum </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Endothelial growth factor </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> Man löst 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 · 7H 2 O </td><td> Sigma Aldrich </td><td> 215422-250G </td><td> Lösen Sie in PBS mit Ca und Mg </td></tr><tr><td> MGD </td><td> Enzo Life Sciences </td><td> ALX-400-014-M050 </td><td> Lösen Sie in PBS mit Ca 2 + und Mg 2 + </td></tr><tr><td> BAEC Zellen </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 mit CaCl 2 und MgCl 2 </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Phorbol-Myristat-Acetat (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

Reaktivem Stickstoff / Sauerstoff-Spezies (ROS / RNS) in geringen Konzentrationen spielen eine wichtige Rolle bei der Regulierung der Zellfunktion, Signal-, und Immunantwort aber in unregulierten Konzentrationen sind schädlich für die Lebensfähigkeit der Zellen 1, 2. Während lebenden Systemen mit endogenen und diätetische antioxidativen Mechanismen entwickelt, um ROS zu regulieren, werden ROS kontinuierlich natürliche Nebenprodukte der normalen Stoffwechsel von Sauerstoff erzeugt und kann oxidativen Schäden an Biomolekülen was zu einem Verlust der Funktion des Proteins, DNA-Spaltung, oder Lipid zu Peroxidation 3, und schließlich gegen oxidativen Stress, der zu Verletzung oder Tod Zelle 4. Superoxid-Radikal-Anion (O 2 • -) ist das wichtigste Vorläufer einige der stark oxidierenden Spezies, auf die in biologischen Systemen, wie Peroxynitrit und Hydroxylradikale abgegeben. Die Erzeugung von O 2 • - signalisiert den ersten Anzeichen von oxidativem Burst, und deshalb habe ichts Nachweis und / oder Speicherung in biologischen Systemen wichtig ist. In dieser Demonstration O 2 • - wurde von neutrophilen Granulozyten (PMN) generiert. Durch chemotaktische Stimulation mit Phorbol-12-myristat-13-acetat (PMA), erzeugt PMN O 2 • - über die Aktivierung von Nicotinamid-Adenin-Dinucleotid-Phosphat (NADPH) Oxidase 5. Stickoxid (NO)-Synthase, die in drei Isoformen kommt, induzierbare-, neuronale und Endothel-NOS oder iNOS, nNOS oder eNOS, jeweils, katalysiert die Umwandlung von L-Arginin, L-Citrullin, mit NADPH, um NO 6 zu erzeugen . Hier erzielten wir NO aus Endothelzellen. Unter oxidativem Stress kann zum Beispiel von eNOS Herstellung NO zu O 2 • wechseln - in einem Prozess namens Entkopplung, die vermutlich durch Oxidation von Häm-7 oder der Co-Faktor, Tetrahydrobiopterin (BH 4) 8 verursacht wird. Es gibt nur wenigezuverlässiger Verfahren zur Detektion von freien Radikalen in biologischen Systemen, aber von Spezifität und Sensitivität begrenzt. Spin trapping wird allgemein für die Identifizierung von freien Radikalen verwendet und umfasst die Addition von einem Rest einer Spin-Trap Bilden einer persistenten Spinadduktes, die durch paramagnetische Elektronenresonanz (EPR)-Spektroskopie nachgewiesen werden kann. Die verschiedenen Radikaladdukte zeigen markante Spektrum, mit dem die Radikale erzeugt identifizieren und können eine Fülle von Informationen über die Art und Kinetik der Radikal-Produktion 9 bieten kann. Die cyclischen Nitrone, 5,5-Dimethyl-pyrrolin-N-oxid, DMPO 10, die Phosphoryl-substituiert DEPMPO 11 und die Ester-substituierte, EMPO 12 und BMPO 13, werden weithin als Spinfallen verwendet - letztere Spin Fallen ausstellenden längere Halbwertszeiten für O 2 • - Addukt. Eisen (II)-N-Methyl-D-glucamin Dithiocarbamat, Fe (MGD) 2 &lt;/ Sub&gt; wird häufig verwendet, um die NO aufgrund der hohen Rate von Adduktbildung und der hohen Stabilität des Spin-Addukt 14 abfangen.

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

Elektronenspin-Resonanz (EPR)-Spektroskopie wurde eingesetzt, um Stickoxid aus Rinder-Endothelzellen und Superoxidradikalanion von menschlichen Neutrophilen unter Verwendung von Eisen (II)-N-Methyl-D-glucamin Dithiocarbamat, Fe (MGD) erkennen<sub&gt; 2</sub&gt; Und 5,5-Dimethyl-1-pyroroline-N-oxid, DMPO, jeweils.

Nachweis von Stickstoffmonoxid und Superoxid-Radikalanion von paramagnetischer Resonanzspektroskopie von Zellen mit Spinfallen

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 Aufrufe.  The Ohio State University. Elektronenspin-Resonanz (EPR)-Spektroskopie wurde eingesetzt, um Stickoxid aus Rinder-Endothelzellen und Superoxidradikalanion von menschlichen Neutrophilen unter Verwendung von Eisen (II)-N-Methyl-D-glucamin Dithiocarbamat, Fe (MGD) erkennen 2 Und 5,5-Dimethyl-1-pyroroline-N-oxid, DMPO, jeweils.

Serie: 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.

Elektronen-Spin-Resonanz-Micro-Imaging an lebenden Spezies, bei Oxygen Mapping

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

Forschung

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.

Bioenergetische Profil Experiment mit C2C12 Myoblasten Cells

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.

Konzentration der Metabolite von Low-Density-Planktonic Gemeinschaften für Umweltforschung Metabolomics mit Kernresonanzspektroskopie

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

Erkennung von Live-<em&gt; Escherichia coli</em&gt; O157: H7-Zellen durch 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.

Zwei-Photonen-Imaging von intrazellulären Ca<sup&gt; 2+</sup&gt; Transport- und Stickoxid-Produktion in Endothelzellen und glatte Muskelzellen eines isolierten Rattenaorta

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 Gesichtserkennung von Stickstoffmonoxid und Superoxid in Endothelschicht Intact Arterien

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.

Entwicklung und Charakterisierung von<em&gt; In Vitro</em&gt; Mikrogefäßnetz und quantitative Messungen von Endothelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Und die Produktion von Stickoxid

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.

Der Nachweis von modifizierte Formen von Cytosin Mit Sensitive Immunhistochemie

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.

Mess Nitrit und Nitrat, Metabolite in der Stickoxid-Bahn, in biologischen Materialien der Chemilumineszenz-Methode unter Verwendung von

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.

Die Anwendung von genetisch kodierter Fluorescent Stickstoffmonoxid (NO) • Sonden, die geNOps, für Echtzeit-Imaging von NO • Signale in Einzelzellen

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