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

Dit werk werd gefinancierd door de NIH National Heart, Lung, and Blood Institute subsidie ​​RO1 HL81248.

1. Cultuur van Bovine Aorta endotheelcellen (BAEC) <ol><li> De juiste aseptische technieken werden gevolgd. </li><li> In een waterbad, warme medium zonder antibiotica bij 37 ° C. </li></ol> Opmerking: Het medium bestaat fenolvrij Dulbecco&#39;s gemodificeerd Eagle&#39;s medium (DMEM) met 4,5 g / L D-glucose, 4 mM L-glutamine, 1% niet-essentiële aminozuren, aangevuld met 10% foetaal bovine serum (FBS) en 2,5 mg / L endotheliale groeifactor. <ol start="3"><li> Verwijder de ...

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EPR spin-trapping is werkzaam in een breed scala van biomedische toepassingen voor het kwantificeren en het identificeren van vrije radicalen. Spin trapping zeer gevoelig voor het opsporen van radicalen bij concentraties variërend van nM tot uM waardoor het geschikt is voor toepassing in biologische systemen. De vorming van het paramagnetische adduct, NO-Fe2 +-MGD, is de basis van NO detectie via EPR. Fe 2 +-MGD reageert met NO snel 18 met een snelheid van ~ 10 6 M <...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Opmerkingen (optioneel) </td></tr><tr><td> Fenol vrij DMEM medium Hoge glucose-1X </td><td> GIBCO </td><td> 31053 </td><td></td></tr><tr><td> 0,25% 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 Niet-essentiële aminozuren </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Foetaal runderserum </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Endotheliale groeifactor </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> Los op 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> 215.422-250G </td><td> Los in PBS met Ca en Mg </td></tr><tr><td> MGD </td><td> Enzo Life Sciences </td><td> ALX-400-014-M050 </td><td> Los in PBS met Ca2 + en Mg2 + </td></tr><tr><td> BAEC cellen </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 met CaCl2 en MgCl2 </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Forbol-myristaat acetaat (PMA) </td><td> Sigma Aldrich </td><td> 79346-1 mg </td><td></td></tr></tbody></table>

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

Reactief stikstof / oxygen species (ROS / RNS) bij lage concentraties spelen een belangrijke rol in het reguleren van cel-functie, signalering, en immuunrespons, maar in niet-gereguleerde concentraties schadelijk zijn voor de levensvatbaarheid van de cellen 1, 2. Terwijl levende systemen hebben zich ontwikkeld met endogene en voedingssupplementen antioxidant verdedigingsmechanismen om ROS generatie te reguleren, worden ROS continu geproduceerd als natuurlijke bijproducten van de normale stofwisseling van zuurstof en kan oxidatieve schade aan biomoleculen wat resulteert in verlies van de functies van eiwitten, DNA-splitsing, of lipide veroorzaken peroxidatie 3, en uiteindelijk tot oxidatieve stress leidt tot cel letsel of de dood 4. Superoxide radicaal-anion (O 2 • -) is de belangrijkste voorloper van enkele van de meest oxiderende soorten bekend zijn in biologische systemen zoals peroxynitriet en hydroxyl radicalen. De generatie van O 2 • - signaleert de eerste tekenen van oxidatieve burst, en daarom, its detectie en / of opslag in biologische systemen is belangrijk. In deze demonstratie, O 2 • - werd gegenereerd op basis van polymorfonucleaire neutrofielen (PMN). Door chemotactische stimulatie met forbol-12-myristaat-13-acetaat (PMA) PMN genereert O 2 • - activering van nicotinamide adenine dinucleotide fosfaat (NADPH) oxidase 5. Stikstofoxide (NO) synthase die wordt geleverd in drie isovormen, zoals induceerbare-, neuronale-en endotheel-NOS, of iNOS, nNOS of eNOS, respectievelijk, katalyseert de omzetting van L-arginine tot L-citrulline, met behulp van NADPH op NO 6 te produceren . Hier wordt gegenereerd NO uit endotheelcellen. Onder oxidatieve stress, kan eNOS bijvoorbeeld schakelen van de productie van NO tot O 2 • - in een proces dat ontkoppeling, waarvan wordt aangenomen veroorzaakt wordt door oxidatie van heem 7 of co-factor, tetrahydrobiopterinedeficiëntie (BH 4) 8. Er zijn weinigbetrouwbare werkwijzen voor de detectie van vrije radicalen in biologische systemen, maar beperkt door de specificiteit en gevoeligheid. Spin trapping wordt gebruikt voor de identificatie van vrije radicalen en omvat de additiereactie van een groep een spin trap die een aanhoudende rotatie adduct dat gedetecteerd kan worden met elektronen paramagnetische (EPR) spectroscopie. De verschillende radicale adducten vertonen opvallende spectrum dat kan worden gebruikt om de radicalen worden gegenereerd identificeren en kan een schat aan informatie over de aard en de kinetiek van de radicale productie 9 geven. De cyclische nitronen, 5,5-dimethyl-pyrroline-N-oxide, DMPO 10 de fosforylchloride gesubstitueerde DEPMPO 11 en de ester gesubstitueerde, Empo 12 en BMPO 13 zijn alom gebruikt als rotatie vallen - deze rotatie vallen vertonen langere halfwaardetijden voor O 2 • - adduct. IJzer (II)-N-methyl-D-glucamine dithiocarbamaat, Fe (MGD) 2 &lt;/ Sub&gt; wordt vaak gebruikt om NO te wijten aan hoge mate van adduct vorming en de hoge stabiliteit van de spin adduct 14 te vangen.

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

Elektronen paramagnetische resonantie (EPR) spectroscopie werd gebruikt om stikstofoxide te detecteren van runderen aorta endotheelcellen en superoxide radicaal anion van menselijke neutrofielen met behulp van ijzer (II)-N-methyl-D-glucamine dithiocarbamaat, Fe (MGD)<sub&gt; 2</sub&gt; En 5,5-dimethyl-1-pyroroline-N-oxide, DMPO respectievelijk.

Detectie van stikstofoxide en superoxide radicaal anion door Electron Paramagnetische Resonantie Spectroscopie van cellen met behulp van 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 ...

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