作者要感谢在制定血液中的亚硝酸盐测量使用亚硝酸盐保存液A. Dejam博士和MM佩尔蒂埃的重要贡献。

所有协议，包括使用动物都是由NIDDK动物护理和使用委员会批准使用，并从美国国立卫生研究院血库健康供者获得人血。 1.样品制备<ol><li> 亚硝酸盐保存溶液的制备 <ol><li>制备含890毫铁氰化钾溶液（K 3的Fe（CN）6）和118毫NEM（N-乙基马来酰亚胺）在蒸馏水。充分溶解，直到很明显的黄色，没有晶体存在。 9比例（V / V，NP-40 /溶液）:在一个1添加的NP-40（辛基phenoxylpolyethoxylethanol）。轻轻混匀，避免起泡，并储存在4℃下一个星期左右）。 </li></ol></li><li> 收集和制备的血液样品的 <ol><li>使用至少20G的针，以避免与肝素溶血防止凝血（5U / ml）的收集血液。用于全血样品，与亚硝酸盐在1立即保存液混合血液:4的比例（ⅴ/ v溶液/血）。 </li><li>对于血浆和红血细胞的样品，离心在4℃下将收集的血液在4000 xg离心5分钟以分离红细胞（RBC）和血浆。取上清（血浆），并将其与上述用于测定血浆中的亚硝酸盐水平的所述亚硝酸盐保存液混合。 </li><li>小心除去残留从管的底部的样本和吸移管红细胞样品的血浆和血沉棕黄层，以避免由其它类型的使用的截止枪头血细胞的污染，并转移到一个管含有在同一亚硝酸保存液比如上述。 </li><li>混合各样品（血浆和红细胞）用冷甲醇在一个比为1:1或1:2（V / V样品/甲醇）和以13,000 xg离心离心他们在4℃15分钟以沉淀蛋白质。取上清，并使用它用于亚硝酸盐测量或冻结制备的样品，如果需要的话，在干冰上并储存在-80℃。 注:亚硝酸盐快速REA克拉与血液形成硝酸盐氧合血红蛋白（oxyHb）。因为oxyHb总是存在于大过量的亚硝酸盐，该反应导致具有的〜10分钟的时间帧天然血液亚硝酸盐的几乎完全湮没。为了保存最内源性血液亚硝酸盐，亚硝酸盐，防腐剂溶液在抽血后立即加入到全血样品。该解决方案将快速溶解红血细胞和氧化oxyHb成高铁血红蛋白（metHb），血红蛋白的无活性形式，不发生化学亚硝酸盐反应。 </li></ol></li><li> 样品从其它类型的体液的制备 <ol><li>样品收集到适当的容器后，用亚硝酸盐保存液，deproteinate拌匀，即可测量或在-80℃冷冻和储存。 </li></ol></li><li> 收集和制备的组织样本 <ol><li>从收集与肝素生理盐水（10U HEPA灌注动物组织RIN / ml）中。消费约1g所需的组织的，均质或者使用手动匀浆器或自动均化器。 </li><li>添加亚硝酸盐根据需要实现平滑匀浆保存液的已知量。 </li><li>如上所述（:1或1 2比V / V样品/甲醇1），以13,000 xg离心然后离心样品在4℃，15分钟一次组织匀浆化，用冷甲醇沉淀蛋白质。 </li><li>收集上清和测量亚硝酸盐的含量。如果需要的话，在制备干冰和存储的任何阶段冷冻样品在-80℃下。 </li></ol></li></ol> 2.减少溶液的制备<ol><li> 三碘（I 3）减少亚硝酸盐和R-（X）-NO种决心1,3,4解决方案 <ol><li>用138在水中毫I 2溶液一起制备301毫碘化钾的溶液。 7比上约30分钟，直到所有的磁力搅拌器（体积/体积溶液/乙酸）:在2冰醋酸混合晶体溶解。最好是保持在黑暗瓶，碘化物对光敏感，并准备在一周内使用。 注意:此解决方案将减少亚硝酸盐成NO，它也将自R-SNO，R-NNO和Fe-NO官能团（R-（X）-NO）释放NO，但硝酸也不会受到影响。从上面的基于无官能基的信号可以从真亚硝酸信号通过用酸化的磺胺（AS）和进行比较的AS-处理和未处理的信号的样本的一半隔开。 AS不可逆地形成重氮阳离子与亚硝酸并且不能由我3溶液可以减少这种复杂。对于AS处理，制备磺胺290毫溶液在1M HCl和它在比1添加到样品的等分试样:9（V / V AS /样品）。测量在样品的AS-处理和未处理部分CL信号。计算真实的亚硝酸盐的信号作为样本的AS-处理的和未处理部分的差。亚硝酸盐可以通过使用选择性尼特可以还测定RITE还原为部分2.2描述抗坏血酸/乙酸混合物溶液。但是，由于R-（X）-NO品种目前的通常少量使用AS-未经处理的样品测量是在大多数情况下，总的亚硝酸盐含量的一个很好的近似。 </li></ol></li><li> 抗坏血酸/乙酸（4A）为亚硝酸盐2的选择性测定溶液 <ol><li>制备在水中的500mM抗坏血酸。混合用冰醋酸该溶液在一个比为1:7（体积/体积，抗坏血酸/乙酸）中以制备反应混合物。 注意:此溶液是具体的亚硝酸盐，不会从任何R-（X）-NO物种或硝酸盐释放NO。然而，抗坏血酸和醋酸的溶液必须被新鲜的测量之前每天制备，如抗坏血酸容易在溶液中氧化。 亚硝酸盐还原的完成取决于抗坏血酸的浓度;并建议完全r至少为50 mm抗坏血酸血浆中的亚硝酸盐排出。然而，建议与最终样品测量之前对不同浓度的抗坏血酸和亚硝酸盐的标准在预期亚硝酸浓度范围执行一些预试验。请记住，抗坏血酸在测量过程中略有消耗，建议在玻璃反应室的反应混合物如此频繁的变化。 </li></ol></li><li> 钒（III）硝酸盐测定氯9减少解决方案 <ol><li>制备的VCl 3的1 M盐酸51毫溶液。通过200微米的过滤器和储存在一个黑暗的瓶过滤器解决方案，在两个星期内使用。 注意:此解决方案将减少硝酸盐，亚硝酸盐和所有R-（X）-NO物种，因此，如果比较的量的亚硝酸盐或其它R-（X）的-NO物种存在与硝酸一起，最终硝酸盐内容来计算与的VCl 3和I中得到的CL信号之间的差3降低了解决方案。 </li></ol></li></ol> 3.化学发光（CL）分析仪的安装和测量<ol><li> 标准的解决方案 <ol><li>制备的亚硝酸钠1μM的溶液（ 亚硝酸钠）。相同的溶液可用于亚硝酸盐和硝酸盐测定。 </li></ol></li><li> 不使用分析仪 注意:目前有两种市售是用于生物研究的目的不够敏感NO分析仪-西弗斯NOA和Ecophysics CLD 88Y。他们都同样的原则进行操作;的主要区别在于，CLD 88Y使用氧气从室内空气，使臭氧（O 3），而NOA要求用于此目的的外部氧气罐。下面的过程说明了设立西弗斯NOA。 <ol><li> 仪器设置 <ol><li>根据制造商的建议进行NO分析仪的初始设置如看到的图1。 </li><li>打开O 2罐连接到仪器。选择"分析"，并从面板上的主菜单中的"输入"。在下一屏幕上选择"开始"，然后按"Enter"键。 （含有1 M氢氧化钠）酸阱连接到仪器， 如图1中所见。 </li><li>等到光电倍增冷却至温度低于-12℃，将反应室抽真空至6乇。大约需要30分钟才能达到稳定平稳的基线。基线需要1内稳定 - 2毫伏，其标称值比其稳定性不太重要。 注意:如果硝酸盐测量，打开冷却水浴（设定为4℃，作为冷阱为酸和水蒸汽）和加热水浴（主反应室中，95℃）。的硝酸盐还原成否的VCl 3溶液的速率是温度相关的，并且它在室温坦佩非常缓慢叉涂抹，需要温度，所以大幅度提高，观察反应。 </li><li>打开他坦克和玻璃反应室连接到酸陷阱在图1中所看到。填充适当还原溶液的反应室，减少鼓泡到最小和反应室连接到酸陷阱。采用试错，调整他冒泡率匹配仪器吸率（对西瑞尔的NOA通常〜200毫升/分钟）。 </li><li>打开电脑控制仪器并启动基于LabVIEW的Liquid软件。要打开仪器和采集软件，按之间的通信"进入"中的"数据菜单"，直到屏幕上显示"输出使能"的时候，然后按"清除"要返回数据的屏幕。 </li><li>等待屏幕上的一个稳定的基线跟踪并开始注入样本和亚硝酸盐标准纳入通过隔膜还原溶液。总是等到达到稳定的基线船尾呃高峰。这最多需要2分钟。液体的软件有一个选项为"标记"时代注入和注释注入，它将这些意见导出到一个单独的文件（filename.info）作为一个有益的补充数据文件（filename.data）。 </li><li>一旦样品和标准衡量，选择"停止"在Liquid软件菜单选项 - 这会从临时文件中的数据写入到被称为"filename.data"永久的文件。按"退出"选项，将终止测量没有获得的数据写入到一个永久文件。 </li><li>终止实验，通过在反应容器的顶部关闭旋塞从反应容器断开机器并断开酸阱和反应容器之间的管（ 见图1）。现在停止NOA分析 - 按"清除"，进入"分析"，把机器上的"待机"和"确认"待命。如果经常，使用的仪器可在待机模式下放置数周。关闭氧气供应。然后冲洗从反应室，断开的还原性溶液，并关闭从玻璃反应容器中，他罐和完成清洁反应容器中。 </li></ol></li><li> 标准和样品进样 <ol><li>注入50微升的1μM的亚硝酸盐溶液成使用公洗涤Hamilton注射器反应混合物。等待至少1 - 注射之间2分钟达到高峰的良好分离。 50微升重复注射以获得复制到每个数据点。每次注射后要用去离子水的注射器。 </li><li>为了获得全套的标准曲线数据，继续与100，150微升重复注射1微米的亚硝酸盐溶液（如果预期的高浓度的亚硝酸盐或硝酸盐可能需要额外的200微升）。在每种情况下，照顾等到信号回落至基线，然后获得1 - 2英里基准的n个达到峰值的良好分离。 注意:亚硝酸盐的标准可在实验过程中的任何时间进行测量。然而，优选的是采集数据测量前的标准曲线，因为它也可作为仪器的功能的独立的检查。 当收集数据时，样品的注入量应导致良好明显的峰值。然而，应当记住的是，所述光电倍增管是线性的仅达〜800毫伏，所以没有峰应比〜700毫伏以上。这可以通过注入量样品的降低或用去离子水稀释的样品来实现任一。每个点应该至少一式两份进行测定。 </li></ol></li></ol></li></ol>

<ol>
	<li>Piknova, B., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Nitrite+in+Blood+Samples+Using+the+Ferricyanide-Based+Hemoglobin+Oxidation+Assay.">Measurement of Nitrite in Blood Samples Using the Ferricyanide-Based Hemoglobin Oxidation Assay.</a> Methods Mol Biol. 704, 39-56 (2011).</li><li>Nagababu, E., Rifkind, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+plasma+nitrite+by+chemiluminescence+without+interference+of+S-,+N-nitroso+and+nitrated+species.">Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species.</a> Free Radic Biol Med. 42, 1146-1154 (2007).</li><li>Pinder, A. G., Rogers, S. C., Khalatbari, A., Ingram, T. E., James, P. E., Hancock, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+nitric+oxide+and+its+metabolites+in+biological+samples+by+ozone-based+chemiluminescence.">The measurement of nitric oxide and its metabolites in biological samples by ozone-based chemiluminescence.</a> Methods in Molecular Biology, Redox-Mediated Signal Transduction. 476, 11-28 (2008).</li><li>Pelletier, M. M., Kleinbongard, P., Ring-wood, L., Hito, R., Hunter, C. J., Schechter, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+measurement+of+blood+and+plasma+nitrite+by+chemiluminescence:+pitfalls+and+solutions.">The measurement of blood and plasma nitrite by chemiluminescence: pitfalls and solutions.</a> Free Radic Biol Med. 41, 541-548 (2006).</li><li>Mac Arthur, P. H., Shiva, S., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+circulating+nitrite+and+S-nitrosothiols+by+reductive+chemiluminescence.">Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence.</a> J Chromatogr B. 851, 93-105 (2007).</li><li>Bryan, N. S., Grisham, 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=Methods+to+detect+nitric+oxide+and+its+metabolites+in+biological+samples.">Methods to detect nitric oxide and its metabolites in biological samples.</a> Free Radic Biol Med. 43, 645-657 (2007).</li><li>Hendgen-Cotta, U., Grau, M., Rasaaf, T., Gharinin, P., Kelm, M., Kleinbongard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductive+gas-phase+chemiluminescence+and+flow+injection+analysis+for+measurement+of+nitric+oxide+pool+in+biological+matrices.">Reductive gas-phase chemiluminescence and flow injection analysis for measurement of nitric oxide pool in biological matrices.</a> Method Enzymol. 441, 295-315 (2008).</li><li>Yang, B. K., Vivas, E. X., Reiter, C. D., Gladwin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+for+the+sensitive+and+specific+measurement+of+S-nitrosothiols,+iron-nitrosyls+and+Nitrite+in+biological+samples.">Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls and Nitrite in biological samples.</a> Free Radic Res. 37, 1-10 (2003).</li><li>Sm&#225;rason, A. K. 1., Allman, K. G., Young, D., Redman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevated+levels+of+serum+nitrate,+a+stable+end+product+of+nitric+oxide,+in+women+with+pre-eclampsia.">Elevated levels of serum nitrate, a stable end product of nitric oxide, in women with pre-eclampsia.</a> Br J Obstet Gynaecol. 104 (5), 538-543 (1997).</li><li>Beckman, J. S., Congert, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Measurement+of+Dilute+Nitric+Oxide+in+Solution+with+an+Ozone+Chemiluminescent+Detector.">Direct Measurement of Dilute Nitric Oxide in Solution with an Ozone Chemiluminescent Detector.</a> Methods: A companion to Methods in Enzymology. 7, 35-39 (1995).</li><li>Bates, J. N. <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+measurements+by+chemiluminescence+detection.">Nitric oxide measurements by chemiluminescence detection.</a> Neuroprotocols: A companion to Methods in Neuroscience. 1, 141-149 (1992).</li></ol>

艾伦·谢克特博士被列为在发给美国国立卫生研究院的使用亚硝酸盐对心血管疾病的治疗几项专利的共同发明人。他接收基于这些专利用于临床开发，但没有其他补偿NIH许可费。

该议定书中的关键步骤用于制备，稀释或以其他方式处理原始样品的所有解决方案（包括水）的等分试样必须保存并检查可能的亚硝酸盐或（更通常）硝酸盐的污染。我们发现大多数污染来自水和用于治疗样品（铁氰化物，特别）许多化学物质也包含在一些地段，与内源性的硝酸盐判定干扰硝酸盐污染的显著量。因此，我们检查所有化学品我们对亚硝酸盐和硝酸盐污染之前，我...

亚硝酸盐，并不太延长硝酸盐，血中水平反映机体的整体状态NO代谢。在血液亚硝酸盐的浓度和最器官和组织是仅在高纳摩尔或低微摩尔范围，硝酸盐通常存在高得多的量 - 在微摩尔范围内。在亚硝酸盐水平由于疾病进展变化或饮食习惯的改变是相当小的，并且可以使用一个非常敏感的方法只测定。由于它们非常不同等级和不同的代谢过程，硝酸盐和亚硝酸盐水平的单独的测定是必不可少的。所谓哪里硝酸盐和亚硝酸盐是在一起测量具有非常小的值"NO x的决心"。 关于各种生物样品中定量亚硝酸盐的几种方法已经被开发 - 最常见的是最老的，基于已在1879年即使现代改性中最初描述的格里斯反应NS，亚硝酸盐达到由格里斯&#39;方法的灵敏度极限是低微摩尔范围内。化学发光（CL）与三碘化物还原溶液相结合，被认为是当前最敏感的方法，允许量化在亚硝酸盐的浓度1-8,10,11的低纳摩尔范围内。相同的CL的方法，用三氯化钒还原溶液相结合，可以用于硝酸盐敏感测量，精确在纳摩尔范围9。 CL检测游离气体NO。因此，亚硝酸盐，硝酸盐，R-亚硝基硫醇（R-SNO），R-亚硝胺（R-NNO），或金属-NO化合物（以后在手稿称作"R-（X）-NO"），必须将其转换成免费无气，以通过CL量化其原始金额。转化为NO是使用几种不同的减少解决方案来实现，这取决于NO代谢物的性质。转换后，游离NO的气体从反应容器中由载体气体（他，N-吹扫2或Ar）到CL分析仪的反应室，其中臭氧（O 3）与NO组合以形成二氧化氮（NO 2）在它的激活状态。与返回到基态，NO 2 *发射在红外线区域和发射的光子由CL仪的光电倍增管（PMT）来检测。发出的光的强度成正比的反应室的NO浓度，这允许使用适当的校准曲线原种的浓度的计算。 在我们的协议，我们硝酸盐和亚硝酸盐的第一本基于CL-测定中最常用的临床设置 - 在血液和血浆，然后我们将讨论如何确定组织样本中的这些离子。我们还详细解释如何保持在亚硝酸盐反应的环境中，如血液及其隔间，血浆和红血细胞的原始生理亚硝酸盐浓度。

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			<td height="26" style="height:26px;width:351px;">potassium ferricyanide; K3Fe(CN)6</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">702587</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEM; N-ethylmaleimide</td>
			<td style="width:135px;">Sigma</td>
			<td>4260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40; 4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">74385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sulfanilamide; AS&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td>S9251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HCl</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">H1758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">acetic acid, glacial</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A9967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ascorbic acid&#160;</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">A7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">potassium iodide; KI</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">60399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">iodine; I2</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">207772</td>
			<td>light sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">sodium nitrite; NaNO2</td>
			<td style="width:135px;">Sigma</td>
			<td>563218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">vanadium(III) chloride; VCl3</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">208272</td>
			<td>ligt sensitive, toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GentleMac</td>
			<td style="width:135px;">Miltenyi</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sievers NOA 280i</td>
			<td style="width:135px;">GE</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLD 88Y</td>
			<td style="width:135px;">&#160;Ecophysics&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring nitrite and nitrate, metabolites in the nitric oxide pathway, in biological materials using the chemiluminescence method

一氧化氮（NO）是在血管内平衡，并且也是神经递质主要调节分子之一。酶产生的NO是由各种含氧血红素蛋白等仍然不为人所熟知的途径相互作用氧化成亚硝酸盐和硝酸盐。相反的过程，还原硝酸盐和亚硝酸盐的成NO已经在哺乳动物中发现了在过去十年中，它是受到关注的可能的途径，以防止或减轻，被认为是心血管，代谢和肌肉疾病的整个范围中的一个与NO的水平降低有关。血液，体液及各种组织 - 它来估计在不同的身体区室NO及其代谢物的量是重要的。血，因为它容易获得，是用于NO代谢物的估计优选隔室。由于它的使用寿命短（几毫秒）和低次纳摩尔浓度，血液没有直接可靠的测量&lt;EM&gt;在体内存在很大的技术困难。因而NO可用性是根据其氧化产物，亚硝酸盐和硝酸盐的用量通常估计。这两种代谢物始终单独测量。有几种公认的方法来确定在生物体液和组织中的浓度。在这里，我们提出了一个协议，用于化学发光法（CL）的基础上，分别由三碘或三氯化钒的解决方案，亚硝酸盐或硝酸盐还原后分光光度检测NO的。对亚硝酸盐和硝酸盐的检测灵敏度低纳摩尔范围，这台CL作为目前最敏感的方法来确定NO代谢途径的变化。我们详细讲解了如何才能在收集，以及如何的时间来确定样品中相应的金额保留原始金额硝酸盐和亚硝酸盐存在的准备从生物体液和组织样本。在CL技术的局限性也是EXPlained。

图2示出了从标准和五种不同样品收集代表性结果。如该图所示，光电倍增电压增加溶液（标准或样品）含亚硝酸注入还原溶液后立即返回到基线值的所有出现在注射一次亚硝酸盐（注射时间由曲线下方红色箭头表示）溶液降低。它也是从该图准确体积的测量是必要的，以获得高重现性的数据清楚。我们建议采用精密汉密尔顿注射器来衡量的注射量。减少I <sub...

Watch this Scientific Journal Video about Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method at JoVE.com

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

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 ...

measuring-nitrite-nitrate-metabolites-nitric-oxide-pathway-biological

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Signaling Networks of G Protein-coupled Receptors

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22.4K 次观看.  NIDDK, NIH. 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.

视频: Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

科研

生物学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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.

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.

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞:隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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

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此视频中的章节

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

利用磁调制生物传感系统的生物检测的快速均质检测

测量线虫寿命在96孔板

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

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

测量渗透水渗透系数（P

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

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

核酸的分离过滤:一种简单快速的DNA提取方法