从细胞中一氧化氮和超氧负离子自由基的电子顺磁共振光谱的检测，使用自旋陷阱
Summary
采用电子顺磁共振（EPR）光谱检测牛主动脉内皮细胞和人类使用铁（II）-N-甲基-D葡萄糖胺二硫代氨基甲酸铁（百万加仑）中性粒细胞超氧阴离子自由基一氧化氮<sub> 2</sub>和5,5 – 二甲基-1-pyroroline – N-氧化物，DMPO分别。
Abstract
活性氮/氧（ROS / RNS的）在低浓度时发挥了重要作用，在调节细胞功能的信号，以及免疫反应，但不稳定的浓度是有害的细胞活力1，2。虽然生活系统演变与内源性和膳食抗氧化防御机制来调节活性氧的产生，活性氧产生自然正常的氧代谢的产品不断，并可能导致氧化损伤生物大分子蛋白质功能丧失，DNA断裂，或脂质过氧化反应，并最终以氧化应激导致细胞损伤或死亡4。
超氧阴离子自由基（O2• – ）是主要易制毒化学一些如过氧亚硝基阴离子和羟自由基的生物系统中存在已知的最高度氧化的物种。一代的O 2• -标志着氧化爆发的第一个迹象，因此，我TS检测和/或封存在生物系统是重要的。在这个演示中，O 2的 • -中性粒细胞（中性粒细胞）的产生。通过与佛-12-肉豆蔻-13-醋酸酯（PMA）的趋化刺激，中性粒细胞产生O 2的 • -通过激活烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶5。
一氧化氮（NO）合酶三种亚型，作为诱导，神经元和内皮一氧化氮合酶，或iNOS，nNOS或eNOS的，分别催化L-精氨酸转化为L-瓜氨酸，用NADPH的产生NO 6 。在这里，我们从血管内皮细胞NO生成。 eNOS的，例如可以在氧化应激条件下，不产生切换到O 2• -在一个名为解偶联过程中，这被认为是造成氧化血红素7或共同因素，呤（波黑）8。
有只有少数自由基在生物系统中检测的可靠方法，但仅限于由特异性和敏感性。常用的自旋捕获自由基的鉴定，并涉及到自旋陷阱，形成一个持久的自旋加合物，可通过电子顺磁共振（EPR）光谱检测自由基的加成反应。各种激进的加合物，表现出鲜明的频谱可以用来确定所产生的自由基，并能提供丰富的信息的性质和动力学激进生产9。
循环硝，5,5 -二甲基吡咯氮氧化物，DMPO 10，磷取代DEPMPO 11，酯取代，EMPO 12和13 BMPO，已广泛采用自旋陷阱-后者的自旋陷阱参展较长的半衰期为O 2• -加合物。铁（II）-N-甲基D-葡萄糖胺二硫代氨基甲酸铁（百万加仑）2 </ SUB>常用捕获没有因加合物的形成和稳定性高自旋加合物14率高。
Protocol
1。培养的牛主动脉内皮细胞（BAEC） 适当的无菌技术。 在水洗澡，温暖的介质无抗生素在37°C。 注：中等苯酚免费Dulbecco的改良鹰的介质，4.5 g / L的Ð-葡萄糖（DMEM培养基），4毫米L-谷氨酰胺，1％非必需氨基酸氨基酸，辅以10％胎牛血清（FBS），和2.5 mg / L的内皮细胞生长因子。 从孵化器的T75瓶中含有细胞和前罩内放置清洁瓶表…
Discussion
在医学领域的应用范围广泛，量化，并确定自由基的EPR自旋捕获已受聘。是高度敏感，能够检测从纳米从而使其适合在生物系统中的应用到μM的浓度自由基的自旋捕获。形成顺加合物，无铁2 + MGD的，是没有通过的EPR检测的基础上。的Fe 2 +-MGD的反应与NO迅速率10 6米-1 S -1的〜18。无铁2 + MGD的加合物，具有半衰期长，高度斯塔比尔。事实上，人?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由NIH的国家心脏，肺和血液研究所授予RO1的HL81248。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Phenol free DMEM medium High glucose 1X |
GIBCO | 31053 | |
| 0.25% Trypsin- EDTA | GIBCO | 25200 | |
| L-Glutamine | Fisher Scientific | BP379-100 | |
| MEM Non Essential Amino acids | GIBCO | 11140 | |
| Fetal Bovine serum | Atlanta Biologicals | S11550 | |
| Endothelial Growth factor | Millipore | 02-102 | |
| CaI | Enzo Life Sciences | A-23187 | Dissolve in DMSO |
| SIN-1 | Enzo Life Sciences | BML-CN245-0020 | |
| DMPO | Dojindo Laboratories | D048-10 | |
| FeSO4.7H2O | Sigma Aldrich | 215422-250G | Dissolve in PBS with Ca and Mg |
| MGD | Enzo Life Sciences | ALX-400-014-M050 | Dissolve in PBS with Ca2+ and Mg2+ |
| BAEC cells | Cell Systems | 2B2-C75 | |
| DMSO | Fisher Scientific | BP231-100 | |
| DPBS | Sigma Aldrich | D8537 | |
| DPBS with CaCl2 and MgCl2 | Sigma Aldrich | D8662 | |
| Phorbol-myristate acetate (PMA) | Sigma Aldrich | 79346-1MG |
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Tags
DetectionNitric OxideSuperoxide Radical AnionElectron Paramagnetic Resonance SpectroscopyCellsSpin TrapsReactive Nitrogen SpeciesReactive Oxygen SpeciesCell FunctionImmune ResponseAntioxidant Defense MechanismsOxidative DamageProtein FunctionDNA CleavageLipid PeroxidationOxidative StressCell InjuryCell DeathSuperoxide Radical Anion GenerationPeroxynitriteHydroxyl RadicalPolymorphonuclear NeutrophilsChemotactic StimulationPhorbol-12-myristate-13-acetateNicotinamide Adenine Dinucleotide Phosphate OxidaseNitric Oxide Synthase
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Gopalakrishnan, B., Nash, K. M., Velayutham, M., Villamena, F. A. Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps. J. Vis. Exp. (66), e2810, doi:10.3791/2810 (2012).