This work was funded by the NIH National Heart, Lung, and Blood Institute grant RO1 HL81248.

1. Culture of Bovine Aortic Endothelial Cells (BAEC)
<ol>
 <li>Proper aseptic techniques were followed.</li>
 <li>In a water bath, warm medium without antibiotics at 37 &deg;C.</li>
</ol>
Note: The medium consists of phenol free Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L D-glucose, 4 mM L-glutamine, 1% non-essential amino acids, supplemented with 10% fetal bovine serum (FBS) and 2.5 mg/L endothelial growth factor.
<ol start="3">
 <li>Remove the T75 flask conta...

<ol>
	<li>Winterbourn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconciling+the+chemistry+and+biology+of+reactive+oxygen+species.">Reconciling the chemistry and biology of reactive oxygen species.</a> Nat. Chem. Biol. 4, 278-286 (2008).</li><li>Winterbourn, C. C., Hampton, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiol+chemistry+and+specificity+in+redox+signaling.">Thiol chemistry and specificity in redox signaling.</a> Free Radic. Biol. Med. 45, 549-561 (2008).</li><li>Clerch, L. B., Massaro, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oxygen, Gene Expression, and Cellular Function. , (1997).</li><li>Gutteridge, J. M. C., Halliwell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants:+Molecules,+medicines,+and+myths.">Antioxidants: Molecules, medicines, and myths.</a> Biochem. Biophys. Res. Commun. 393, 561-564 (2010).</li><li>Sumimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+regulation+and+evolution+of+Nox-family+NADPH+oxidases+that+produce+reactive+oxygen+species.">Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species.</a> FEBS J. 275, 3249-3277 (2008).</li><li>Ignarro, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Editor Nitric Oxide: Biology and Pathobiology. , (2009).</li><li>Moreau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+alkyl-+and+arylguanidines+on+the+stability+and+reactivity+of+inducible+NOS+heme-dioxygen+complexes.">Differential effects of alkyl- and arylguanidines on the stability and reactivity of inducible NOS heme-dioxygen complexes.</a> Biochemistry. 45, 3988-3999 (2006).</li><li>Vasquez-Vivar, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+generation+by+endothelial+nitric+oxide+synthase:+the+influence+of+cofactors.">Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 9220-9225 (1998).</li><li>Villamena, F. A., Zweier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+and+nitrogen+species+by+EPR+spin+trapping.">Detection of reactive oxygen and nitrogen species by EPR spin trapping.</a> Antioxid. Redox Signal. 6, 619-629 (2004).</li><li>Finkelstein, E., Rosen, G. M., Rauckman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+trapping+of+superoxide+and+hydroxyl+radical:+practical+aspects.">Spin trapping of superoxide and hydroxyl radical: practical aspects.</a> Arch. Biochem. Biophys. 200, 1-16 (1980).</li><li>Frejaville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Diethoxyphosphoryl-5-methyl-1-pyrroline+N-oxide+(DEPMPO):+a+new+phosphorylated+nitrone+for+the+efficient+in+vitro+and+in+vivo+spin+trapping+of+oxygen-centered+radicals.">5-Diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO): a new phosphorylated nitrone for the efficient in vitro and in vivo spin trapping of oxygen-centered radicals.</a> J. Chem. Soc., Chem. Commun. , 1793-1794 (1994).</li><li>Olive, G., Mercier, A., Le Moigne, F., Rockenbauer, A., Tordo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide:+Evaluation+of+the+spin+trapping+properties.">2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide: Evaluation of the spin trapping properties.</a> Free Radical. Biol. Med. 28, 403-408 (2000).</li><li>Villamena, F. A., Zweier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+radical+trapping+and+spin+adduct+decay+of+5-tert-butoxycarbonyl-5-methyl-1-pyrroline+N-oxide+(BocMPO):+kinetics+and+theoretical+analysis.">Superoxide radical trapping and spin adduct decay of 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BocMPO): kinetics and theoretical analysis.</a> J. Chem. Soc., Perkin Trans. 2, 1340-1344 (2002).</li><li>Tsuchiya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide-forming+reactions+of+the+water-soluble+nitric+oxide+spin-trapping+agent,+MGD.">Nitric oxide-forming reactions of the water-soluble nitric oxide spin-trapping agent, MGD.</a> Free Radical Biol. Med. 27, 347-355 (1999).</li><li>Vanin, A. F., Poltorakov, A. P., Mikoyan, V. D., Kubrina, L. N., van Faassen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+iron-dithiocarbamates+ensure+detection+of+nitric+oxide+in+cells+and+tissues.">Why iron-dithiocarbamates ensure detection of nitric oxide in cells and tissues.</a> Nitric Oxide. 15, 295-311 (2006).</li><li>RojasWahl, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decomposition+mechanism+of+3-N-morpholinosydnonimine+(SIN-1):+A+density+functional+study+on+intrinsic+structures+and+reactivities.">Decomposition mechanism of 3-N-morpholinosydnonimine (SIN-1): A density functional study on intrinsic structures and reactivities.</a> J. Mol. Model. 10, 121-129 (2004).</li><li>Klempner, M. S., Gallin, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+functional+characterization+of+human+neutrophil+subpopulations.">Separation and functional characterization of human neutrophil subpopulations.</a> Blood. 51, 659-669 (1978).</li><li>Pou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+trapping+of+nitric+oxide+by+ferro-chelates:+kinetic+and+in+vivo+pharmacokinetic+studies.">Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies.</a> Biochim. Biophys. Acta. 1427, 216-226 (1999).</li><li>Finkelstein, E., Rosen, G. M., Rauckman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+trapping.+Kinetics+of+the+reaction+of+superoxide+and+hydroxyl+radicals+with+nitrones.">Spin trapping. Kinetics of the reaction of superoxide and hydroxyl radicals with nitrones.</a> J. Am. Chem. Soc. 102, 4994-4999 (1980).</li><li>Britigan, B. E., Rosen, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin-trapping+and+human+neutrophils.+Limits+of+detection+of+hydroxyl+radical.">Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical.</a> J. Biol. Chem. 264, 12299-12302 (1989).</li><li>Frejaville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline+N-oxide:+A+new+efficient+phosphorylated+nitrone+for+the+in+vitro+and+in+vivo+spin+trapping+of+oxygen-centered+radicals.">5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen-centered radicals.</a> J. Med. Chem. 38, 258-265 (1995).</li><li>Snyrychova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+the+sensitivity+of+EPR+spin+trapping+in+biological+systems+by+cyclodextrins:+A+model+study+with+thylakoids+and+photosystem+II+particles.">Improvement of the sensitivity of EPR spin trapping in biological systems by cyclodextrins: A model study with thylakoids and photosystem II particles.</a> Free Radical Biol. Med. 48, 264-274 (2010).</li><li>Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipophilic+beta-cyclodextrin+cyclic-nitrone+conjugate:+Synthesis+and+spin+trapping+studies.">Lipophilic beta-cyclodextrin cyclic-nitrone conjugate: Synthesis and spin trapping studies.</a> J. Org. Chem. 74, 5369-5380 (2009).</li><li>Han, Y., Tuccio, B., Lauricella, R., Villamena, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+spin+trapping+properties+by+beta-cyclodextrin-cyclic+nitrone+conjugate.">Improved spin trapping properties by beta-cyclodextrin-cyclic nitrone conjugate.</a> J. Org. Chem. 73, 7108-7117 (2008).</li><li>Hardy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+characterization,+and+decay+kinetics+of+ROS+and+thiyl+adducts+of+mito-DEPMPO+spin+trap.">Detection, characterization, and decay kinetics of ROS and thiyl adducts of mito-DEPMPO spin trap.</a> Chem. Res. Toxicol. 20, 1053-1060 (2007).</li><li>Kim, S. -. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+reactivity+of+a+cyclic+nitrone-calix[4]pyrrole+conjugate+with+superoxide+radical+anion:+Theoretical+and+experimental+studies.">Fast reactivity of a cyclic nitrone-calix[4]pyrrole conjugate with superoxide radical anion: Theoretical and experimental studies.</a> J. Am. Chem. Soc. 132, 17157-17173 (2010).</li></ol>

No conflicts of interest declared.

EPR spin trapping has been employed in a wide range of biomedical applications for quantifying and identifying free radicals. Spin trapping is highly sensitive, capable of detecting radicals at concentrations ranging from nM to &mu;M thus making it suitable for application in biological systems. The formation of the paramagnetic adduct, NO-Fe2+-MGD, is the basis of NO detection via EPR. Fe2+-MGD reacts with NO rapidly18 at a rate of ~ 106 M-1 s-1....

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td> Phenol free DMEM medium 
High 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-Glutamine</td>
 <td>Fisher Scientific</td>
 <td>BP379-100</td>
 <td></td>
 </tr>
 <tr>
 <td>MEM Non Essential Amino acids</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal Bovine serum</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>Dissolve 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>FeSO4.7H2O</td>
 <td>Sigma Aldrich</td>
 <td>215422-250G</td>
 <td>Dissolve in PBS with Ca and Mg</td>
 </tr>
 <tr>
 <td>MGD</td>
 <td>Enzo Life Sciences</td>
 <td>ALX-400-014-M050</td>
 <td>Dissolve in PBS with Ca2+ and Mg2+</td>
 </tr>
 <tr>
 <td>BAEC cells</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 with CaCl2 and MgCl2</td>
 <td>Sigma Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Phorbol-myristate acetate (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

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

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

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

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

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Biology

18.8K Görüntüleme.  The Ohio State University. Elektron paramanyetik rezonans (EPR) spektroskopisi demir (II)-N-metil-D-glukamin ditiokarbamat, Fe (MGD) kullanılarak, insan nötrofil gelen sığır aort endotelyal hücreler ve süperoksit anyon radikal nitrik oksit tespit etmek için kullanılmıştır 2 Ve 5,5-dimetil-1-pyroroline-N-oksit, sırasıyla, DMPO,.