Name: detection of nitric oxide and superoxide radical anion by electron paramagnetic resonance spectroscopy from cells using spin traps
Uploaded: 2012-08-18T14:00:00
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

Dette arbejde blev finansieret af NIH National Heart, Lung, og Blood Institute tilskud RO1 HL81248.

1. Dyrkning af bovine aortiske endotheliale celler (BAEC) <ol><li> Korrekt aseptiske teknikker blev fulgt. </li><li> I et vandbad, varmt medium uden antibiotika ved 37 ° C. </li></ol> Bemærk: medium består af phenol fri Dulbeccos modificerede Eagles medium (DMEM) med 4,5 g / L D-glucose, 4 mM L-glutamin, 1% non-essentielle aminosyrer, suppleret med 10% føtalt bovint serum (FBS) og 2,5 mg / L endotelvækstfaktor. <ol start="3"><li> Fjerne T75-kolbe, der indeholder celler...

<ol>
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EPR spin-trapping har været ansat i en bred vifte af biomedicinske anvendelser til kvantificering og identifikation af frie radikaler. Spin trapping er meget følsom, er i stand til at detektere radikaler ved koncentrationer i området fra nM til uM, hvilket således gør den egnet til anvendelse i biologiske systemer. Dannelsen af den paramagnetiske addukt, NO-Fe 2 +-MGD, er grundlaget for NO detektion via EPJ. Fe2 +-MGD reagerer med NO hurtigt 18 med en hastighed på ~ 10 <su...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer (valgfri) </td></tr><tr><td> Phenol DMEM medium Høj glucose 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 ikke-essentielle aminosyrer </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Føtalt bovint serum </td><td> Atlanta Biologicals </td><td> S11550 </td><td></td></tr><tr><td> Endotelvækstfaktor </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> Opløses i 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> FeSO4 0,7 H2O </td><td> Sigma Aldrich </td><td> 215.422-250G </td><td> Opløses i PBS med Ca og Mg </td></tr><tr><td> MGD </td><td> Enzo Life Sciences </td><td> ALX-400-014-M050 </td><td> Opløses i PBS med Ca2 + og Mg2 + </td></tr><tr><td> Baec celler </td><td> Cellesystemer </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 med CaCl2 og MgCl2 </td><td> Sigma Aldrich </td><td> D8662 </td><td></td></tr><tr><td> Phorbol-myristat-acetat (PMA) </td><td> Sigma Aldrich </td><td> 79.346-1mg </td><td></td></tr></tbody></table>

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

Reaktive nitrogen / oxygenspecies (ROS / RNS) ved lave koncentrationer spiller en vigtig rolle i reguleringen af celle-funktion, signalering, og immunrespons, men i regulerede koncentrationer er skadelige for cellelevedygtighed 1, 2. Mens levende systemer har udviklet med endogene og diætmæssige antioxidant-forsvarssystemer mekanismer til regulering ROS generation, ROS produceres kontinuerligt som naturligt biprodukter fra normale metabolisme af oxygen og kan forårsage oxidativ beskadigelse biomolekyler resulterer i tab af protein-funktion, DNA-spaltning eller lipid peroxidering 3, og i sidste ende at oxidativt stress, der fører til celleskade eller død 4. Superoxidradikal anion (O 2 • -) er større precursor for nogle af de mest stærkt oxiderende arter vides at eksistere i biologiske systemer, såsom peroxynitrit og hydroxylradikal. Generering af O2 • - signalerer første tegn på oxidativ burst, og derfor, its påvisning og / eller binding i biologiske systemer er vigtig. I denne demonstration, O2 • - blev genereret fra polymorfnukleære neutrofiler (PMN&#39;er). Gennem kemotaxisk stimulering med phorbol-12-myristat-13-acetat (PMA), genererer PMN O2 • - via aktivering af nicotinamid-adenin-dinucleotid-phosphat (NADPH) oxidase 5. Nitrogenoxid (NO)-synthase, der findes i tre isoformer, som inducerbare-, neuronal-og endothel-NOS, eller iNOS, nNOS eller eNOS henholdsvis katalyserer omdannelsen af L-arginin til L-citrullin, under anvendelse NADPH at producere NO 6 . Her har vi frembragt NO fra endotelceller. Under oxidative stress-tilstande, kan eNOS for eksempel gå fra fremstilling NO til O2 • - en proces, der kaldes frakobling, som menes at være forårsaget af oxidering af hæm 7 eller co-faktoren, tetrahydrobiopterin (BH 4) 8. Der er kun fåpålidelige metoder til påvisning af frie radikaler i biologiske systemer, men er begrænset af specificitet og følsomhed. Spin trapping er almindeligt anvendt til identifikation af frie radikaler og omfatter additionsreaktion af en radikal med en spin trap danner en vedvarende centrifugering addukt, som kan detekteres ved elektronmikroskopi paramagnetisk resonans (EPR) spektroskopi. De forskellige radikale Addukterne udstille karakteristisk spektrum, som kan bruges til at identificere de radikaler, der produceres og kan give et væld af oplysninger om arten og kinetikken af radikal produktionen 9. De cykliske nitroner, 5,5-dimethyl-pyrrolin-N-oxid, DMPO 10, phosphoryl-substituerede DEPMPO 11, og den ester-substituerede, EMPO 12 og BMPO 13, er blevet udbredt anvendt som spin-fælder - sidstnævnte centrifugering fælder udstiller længere halveringstider for O 2 • - addukt. Jern (II)-N-methyl-D-glucamin dithiocarbamat, Fe (MGD) 2 &lt;/ Sub&gt; er almindeligt anvendt til at indfange NO på grund af høj hastighed for adduktdannelse og den høje stabilitet af spin adduktet 14.

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

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

Elektron paramagnetisk resonans (EPR) spektroskopi blev anvendt til at detektere nitrogenoxid fra bovine aortiske endotheliale celler og superoxidradikal anion fra humane neutrofiler med jern (II)-N-methyl-D-glucamin dithiocarbamat, Fe (MGD)<sub&gt; 2</sub&gt; Og 5,5-dimethyl-1-pyroroline-N-oxid, DMPO hhv.

Påvisning af nitrogenoxid og superoxid Radical anion ved elektron paramagnetisk resonans-spektroskopi fra celler under anvendelse Spin Fælder

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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