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

这项工作是由NIH的国家心脏，肺和血液研究所授予RO1的HL81248。

1。培养的牛主动脉内皮细胞（BAEC） <ol><li>适当的无菌技术。 </li><li>在水洗澡，温暖的介质无抗生素在37°C。 </li></ol> 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。 <ol start="3"><li>从孵化器的T75瓶中含有细胞和前罩内放置清洁瓶表...

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	<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>

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?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>苯酚免费DMEM培养基 高血糖1X </td><td> GIBCO公司</td><td> 31053 </td><td></td></tr><tr><td> 0.25％胰蛋白酶EDTA </td><td> GIBCO公司</td><td> 25200 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Fisher Scientific则</td><td> BP379-100 </td><td></td></tr><tr><td>的MEM非必需氨基酸</td><td> GIBCO公司</td><td> 11140 </td><td></td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td><td></td></tr><tr><td>内皮生长因子</td><td> Millipore公司</td><td> 02-102 </td><td></td></tr><tr><td>蔡</td><td>恩佐生命科学</td><td> A-13187 </td><td>溶于DMSO中</td></tr><tr><td> SIN-1 </td><td>恩佐生命科学</td><td> BML-CN245-0020 </td><td></td></tr><tr><td> DMPO </td><td> dojindo实验室</td><td> D048-10 </td><td></td></tr><tr><td>硫酸亚铁4 .7 H 2 O </td><td> Sigma Aldrich公司</td><td> 215422-250G </td><td>溶解在PBS中钙和镁</td></tr><tr><td> MGD的</td><td>恩佐生命科学</td><td> ALX-400-014-M050 </td><td>在PBS溶解的Ca 2 +和Mg 2 + </td></tr><tr><td> BAEC细胞的细胞</td><td>电池系统</td><td> 2B2-C75 </td><td></td></tr><tr><td>二甲基亚砜</td><td> Fisher Scientific则</td><td> BP231-100 </td><td></td></tr><tr><td> DPBS </td><td> Sigma Aldrich公司</td><td> D8537 </td><td></td></tr><tr><td> DPBS与CaCl 2和MgCl 2的 </td><td>西格玛一个ldrich </td><td> D8662 </td><td></td></tr><tr><td>佛波醇酯（PMA） </td><td> Sigma Aldrich公司</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

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 &lt;/ SUB&gt;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。

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

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（I​​I）-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&gt; 2</sub&gt;和5,5 - 二甲基-1-pyroroline - N-氧化物，DMPO分别。

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

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Research

JoVE Journal

Biology

Electron Spin Resonance Micro-imaging of Live Species for Oxygen Mapping

电子自旋共振氧测绘活体物种显微成像

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

科研

生物学

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

使用C2C12成肌细胞的生物能简介实验

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

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

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

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

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

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

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

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

一氧化氮和超氧化物歧化酶恩的人脸检测在完整的动脉内皮细胞层

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

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

发展与表征<em&gt;体外</em&gt;微血管网络和内皮的定量测量的[Ca<sup&gt; 2+</sup&gt;]<sub&gt;我</sub&gt;和一氧化氮含量

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

胞嘧啶修饰形式使用敏感免疫组化检测

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

用化学发光法测定硝酸盐和亚硝酸盐，代谢产物中的一氧化氮途径，在生物材料

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

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

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