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面用70％乙醇。 </li><li>删除旧的介质，使用吸气和用5毫升杜尔贝科的磷酸盐缓冲液（DPBS）的两倍。 </li><li>加入2毫升的胰蛋白酶，等待4-5分钟为细胞分离，同时定期在显微镜下检查。 </li><li>加入3毫升的介质和反复混合使用吸管分开ţ他细胞，并创建一个甚至暂停。 </li><li>转让5毫升培养基，胰蛋白酶15毫升管和离心5分钟在121Ğ。 </li><li>使用抽吸去除上清。加入5毫升的DPBS调匀。在121克离心5分钟。 </li><li>吸上清液，再加入6毫升培养基暂停细胞沉淀。 </li><li>在6孔板，每个加1毫升细胞悬液，然后加入1毫升的媒介。混合暂停使用吸管。 </li><li>标示板孵育过夜37°C和5％CO 2。 </li></ol> 2A。 BAEC细胞细胞没有检测<ol><li>适当的无菌技术。 </li><li>从孵化器的6孔板和抽吸介质，从第一口井。洗细胞两次用1毫升DPBS。 </li><li>加入210微升1.9毫米铁（II）硫酸亚铁（硫酸亚铁4 .7 H 2 O，准备由溶解在0.8毫克新鲜CaCl 2和MgCl 2的 1毫升DPBS）和铵的N-甲基-D-葡萄糖胺二硫代氨基甲酸（MGD的，刚准备CaCl 2和MgCl 2的 2.7毫克，溶解于500μLDPBS）210μL，使用比例为1:7 （注：这多余的MGD的使用比例已编制的Fe 2 + MGD的复杂，由于对Fe 3 +-MGD的产量存在过剩MGD的最大化为传统雇用增补抗坏血酸溶液中稳定的Fe 2 + MGD的是没有必要的，因为铁的NO-3 + MGD的形成是内生减少的EPR检测无铁抗坏血酸，对苯二酚，或半胱氨酸与转换效率高达2 + MGD 99.9％..低自旋铁2抗磁状态+允许检测NO的低温设备的需求不使用扁平细胞或毛细管）15。 </li><li>涡流产生的悬浮增加4.6μ钙离子载体（蔡升）（从1.9毫米的储备溶液溶解在1 mL二甲基亚砜1毫克）。 </li><li>漩涡的解决方案和孵化，在37°C为36分钟，以进一步减少NO的铁3 + MGD的EPR检测NO铁2 + MGD的。 </li><li>收集上清（425μL）在一个Eppendorf管中，并转移到EPR扁平细胞（或50μl的毛细管）。 </li><li> EPR的收购参数：微波频率：9.8 GHz的中心领域：3427 G，调制幅度：6.0克;扫描宽度：100克;接收增益：1×10 5;微波功率：10毫瓦;扫描总数：121;扫描时间：10秒;时间常数：20毫秒（注：由于参数会有所不同，从一台仪器和实验条件，因此，仅在中心场，频率和调制幅度要考虑的最重要的参数。）。 </li><li>记录在室温下的光谱和二维光谱INTEG额定降低背景噪声和基线校正使用布鲁克WinEPR数据处理软件或其他数据处理软件。量化加合物的形成，浓度与信号强度（或地区）的标准地块可以建造使用NO供体SNAP。 </li></ol> 2B。 eNOS的解耦实验<ol><li>从孵化器的6孔板和吸第二口井从中期和1毫升DPBS洗两次。 </li><li>一个的过氧捐助，5 -氨基-3 - （4 -吗啉）-1,2,3 oxadiazolium的氯化物（单-1）是用来解耦eNOS的16。加入100μL0.5毫米单-1（M R206.6克/摩尔，从10毫米原液刚准备在500 PBS液无钙/镁离子溶解1毫克仲1）稀释至2毫升DPBS和10％FBS的。 </li><li>孵育2小时37°C和5％的CO 2。 </li><li>从孵化器中取出板和用DPBS两次。 </li><li>加入210μL2.8毫米硫酸亚铁4 .7 H 2 O和210微升19.6毫米MGD新鲜准备根据上述的程序。 </li><li>漩涡的解决方案，并加入4.6微升1.9毫米蔡。 </li><li>漩涡的解决方案，并在37°C孵育36分钟。 </li><li>在一个Eppendorf管中，并转移到一个EPR的扁平细胞收集上清（425μL）。 </li><li> EPR的采集参数为：微波频率：9.8 GHz的中心领域：3427Ğ;调制幅度：地下6;扫描宽度：100克;接收增益：1×10 5;微波功率：10毫瓦;扫描总数：121;扫描时间：10秒，时间常数：20毫秒。 </li><li>记录的光谱进行了整合，以减少背景噪声和基线纠正上述的2-D光谱。 </li></ol> 3。 -中性粒细胞（中性粒细胞检测的O 2） <ol><li>如先前所述17中性粒细胞从人体血液样本分离。 </li><li>使股票的1米DMPO *在PBS溶液中二乙烯三胺五乙酸（DTPA）的0.1毫米。 DMPO具有熔点25-29°C间，所以它是更方便吸取液体DMPO（密度〜1.02克/毫升在25°C）在一个玻璃小瓶（注：不使用塑料瓶称重自纯DMPO与塑料发生反应）。冷冻DMPO可以融化运行温水小瓶（注：不运行热水DMPO可以分解）。 </li><li>重要的是，使用高的DMPO纯度（&gt; 99％）以来的一些市售自旋陷阱包含顺磁杂质，因此，当务之急是只是DMPO解决方案单独运行的EPR谱（在这种情况下，10毫米）。这是至关重要的，没有任何背景信号是明显的（ 见图3A）。 </li><li>准备股票佛-12-肉豆蔻-13-醋酸酯（PMA），在DMSO溶液（1毫克/毫升）。稀释至10微克/毫升的PBS溶液等分。 </li><li>在1。5毫升的Eppendorf管，准备一个解决方案，除了序列与总体积为0.6毫升〜10 6个细胞，中性粒细胞每毫升，D-葡萄糖（1毫克/毫升）和白蛋白（1毫克/毫升），10毫米DMPO和PMA的0.2微克/毫升（注：PMA的是激进的激活和应补充的最后）。 </li><li>转让EPR的扁平细胞的解决方案。 </li><li> EPR的采集参数的条件是：微波频率：9.8 GHz的中心场：3486 G，调制幅度：0.5克;扫描宽度：100克;接收增益：5×10 5;扫描总数：10;扫描时间：30秒微波功率为20兆瓦;时间常数：81毫秒量化加合物的形成，使用，如速度或3羧酸PROXYL的，稳定的氮氧自由基浓度与信号强度（或地区）的标准地块可以建造。 </li></ol> *的DMPO使用的重要注意事项：通过扁平细胞，可以增加细胞密度，从而增加信号自旋加合物，但O 2的半衰期强度- DMPO加合物短（T 1/2〜1分钟）分解DMPO-OH。 DMPO可以使用相同浓度的EMPO，BMPO或DEPMPO的可供加合物的稳定增加，商业与T 1/2〜8和14分钟，分别取代。 4。代表结果 NO的自由基的自旋捕获使用铁2 + MGD的。 图2A和2B显示没有EPR信号由Fe 2 + MGD的或+ MGD蔡，表明这些试剂没有背景信号源于铁的混合物。后与蔡刺激的BAEC细胞释放NO的反应与Fe 2 + MGD形成的自旋加合物，铁2 + MGD的，显示了超精细分裂常数（HFSC）N = 12.66Ğ价值和特点的三重信号G = 2.040 g因子。 （ 图2C）。在HFSC值确定使用的WINSIM的模拟程序，可以从NIEHS的EPR软件数据库网站下载。实验HFSC 一个 N = 12.70 G和G = 2.041 18 NO铁2 + MGD的加合物的文学价值是一致的。同样，单仲偕-1的BAEC细胞的作用，降低NO的生产由于eNOS的解偶联图2D所示。 为O 2• -检测DMPO自旋陷阱。 DMPO单没有发出信号，确认是从顺磁杂质的自旋陷阱图3A所示图3B是DMPO频谱和中性粒细胞，同样，也没有检测到的信号表明，不会导致DMPO NADPH氧化酶的激活。 图3C显示由PMA刺激中性粒细胞后观察到的EPR信号。这个信号的HFSC值被确定为一个 N = 14.71 G，一个与β; - H = 11.40 G ​​和γ-H = 1.25克，是一个 N = 14.3Ğ，β - H = 11.7克和γ-H的文学价值= 1.3克19 DMPO - O 2Ĥ加合物。 <img src="/files/ftp_upload/2810/2810fig1.jpg" alt="图1" /> 图1。使用EPR的自旋捕获中性粒细胞和BAEC细胞的自由基的检测流程图。 （ 一 ）中性粒细胞与DMPO和PMA混合，和由此产生的混合物转移到一个EPR数据采集的扁平细胞。 （二）BAEC细胞生长盘子上，与DPBS洗涤。与蔡一起加入自旋陷阱铁（百万加仑）2。该解决方案是彻底混合培养。被转移到一个EPR EPR数据采集单位细胞的混合物。 <img src="/files/ftp_upload/2810/2810fig2.jpg" alt="图2" /> 图2。EPR检测从B NO的AEC。 （ 一 ）频谱的Fe 2 + MGD（二）对Fe 2 + MGD的+蔡谱。 （三）三重频谱从没有捕获导致铁2 + MGD的使用蔡刺激细胞。 （四）频谱显示NO产量下降至0.5毫米，SIN-1细胞治疗。 <img src="/files/ftp_upload/2810/2810fig3.jpg" alt="图3" /> 图3。EPR检测DMPO-O 2 H激活中性粒细胞。 （ 一 ）频谱DMPO只有10毫米。 （二）中性粒细胞在10毫米DMPO存在单独的频谱。 （三）由PMA激活中性粒细胞的频谱在10毫米DMPO存在。

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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>

没有利益冲突的声明。

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 ...

detection-nitric-oxide-superoxide-radical-anion-electron-paramagnetic

Research

JoVE Journal

Biology

18.8K 次观看.  The Ohio State University. 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 2和5,5 - 二甲基-1-pyroroline - N-氧化物，DMPO分别。

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

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

This protocol describes an electron spin resonance (ESR) micro-imaging method for three-dimensional mapping of oxygen levels in the immediate environment of live cells with micron-scale resolution1. Oxygen is one of the most important molecules in the cycle of life. It serves as the terminal electron acceptor of oxidative phosphorylation in the mitochondria and is used in the production of reactive oxygen species. Measurements of oxygen are important for the study of mitochondrial and metabolic functions, signaling pathways, effects of various stimuli, membrane permeability, and disease differentiation. Oxygen consumption is therefore an informative marker of cellular metabolism, which is broadly applicable to various biological systems from mitochondria to cells to whole organisms. Due to its importance, many methods have been developed for the measurements of oxygen in live systems. Current attempts to provide high-resolution oxygen imaging are based mainly on optical fluorescence and phosphorescence methods that fail to provide satisfactory results as they employ probes with high photo-toxicity and low oxygen sensitivity. ESR, which measures the signal from exogenous paramagnetic probes in the sample, is known to provide very accurate measurements of oxygen concentration. In a typical case, ESR measurements map the probe's lineshape broadening and/or relaxation-time shortening that are linked directly to the local oxygen concentration. (Oxygen is paramagnetic; therefore, when colliding with the exogenous paramagnetic probe, it shortness its relaxation times.) Traditionally, these types of experiments are carried out with low resolution, millimeter-scale ESR for small animals imaging. Here we show how ESR imaging can also be carried out in the micron-scale for the examination of small live samples. ESR micro-imaging is a relatively new methodology that enables the acquisition of spatially-resolved ESR signals with a resolution approaching 1 micron at room temperature2. The main aim of this protocol-paper is to show how this new method, along with newly developed oxygen-sensitive probes, can be applied to the mapping of oxygen levels in small live samples. A spatial resolution of ~30 x 30 x 100 &mu;m is demonstrated, with near-micromolar oxygen concentration sensitivity and sub-femtomole absolute oxygen sensitivity per voxel. The use of ESR micro-imaging for oxygen mapping near cells complements the currently available techniques based on micro-electrodes or fluorescence/phosphorescence. Furthermore, with the proper paramagnetic probe, it will also be readily applicable for intracellular oxygen micro-imaging, a capability which other methods find very difficult to achieve.

This protocol describes a method for micron-scale three-dimensional imaging of oxygen concentration in the immediate environment of live cells by electron spin resonance microscopy.

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

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

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

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

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

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. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

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

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

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

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

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

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated 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

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

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

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

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

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 exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

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

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

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

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

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

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

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

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

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 ...