这项工作是支持由国立卫生研究院（CA142664）。

1。 ROS的检测，使用羧基- H 2 DCFDA 羧基- H 2 DCFDA非荧光，但是，当这种试剂氧化的活性氧的存在，成为绿色荧光。 <ol><li>立即使用前，准备在无菌二甲基亚砜（DMSO）或100％的乙醇的新鲜原液的羧基- H 2 DCFDA 。避免你的染料多个解冻/冻结周期。 </li><li>洗净细胞，与HEPES缓冲盐溶液（HBSS）或磷酸盐缓冲液（PBS）删除原介质的痕迹。 </li><li>负载与染料的细胞（和控制染料，比照注意部分）。在这个实验中，我们使用的Jurkat，人白血病细胞株。使用常规培养基中的终浓度为1μm降低血清（2％），羧基- H 2 DCFDA。 </li><li>孵育30分钟，在黑暗中的文化，在传统的孵化器（37℃，5％CO2）。丢弃所有未使用的染料解决方案。 </li><li>删除羧基- H 2 DCFDA介质中的HBSS或PBS洗两次。从这一步，由轻保护您的细胞。 </li><li>加入新鲜的培养基中含有您的首选药物的羧基- H 2 DCFDA加载细胞和孵化需要。对于这个例子，我们用H 2 O 2（0.03％），1个小时。 </li><li>评估立即流量使用FL1通道（绿色荧光），或荧光酶标仪或荧光显微镜流式细胞仪分析细胞的活性氧。可检测到使用适合绿色荧光的激发和发射波长的荧光。 </li></ol>注:控制应包括羧基- H 2 DCFDA载入未经处理的细胞和未染色未经处理的细胞。羧基- H 2 DCFDA是众所周知的检测过氧化物，但也可以通过其他活性氧氧化。此试剂也可以通过其他方式进行修改，不敏感控制氧化染料，如5 - （和- 6） - 羧基- 2&#39;，7&#39; -二氯ofluorescein双乙酸钠（羧基DCFDA），因此，应包含在测试。 2。一氧化氮（NO）生产的测量您将需要磺胺和N - 1 - napthylethylenediamine盐酸盐（NED）解决方案，和亚硝酸盐标准。这个实验是所谓Griess检测。 NED的解决方案:请在无菌water.Sulfanilamide溶液稀释的N - 1 - napthylethylenediamine盐酸盐0.1％解决方案:1％溶液，稀释于5％磷酸的磺胺。亚硝酸盐标准:0.1M标准股票亚硝酸钠在无菌培养基稀释至100微米，在同一介质中做一个连续稀释。 储存条件:储存化学品在室温下制造商的指示。当重组，NED和磺胺解决方案存储立即使用后，在4 ° C，在黑暗中，最长不超过3个月。 <ol><li>在96孔板培养细胞，每个条件使用一式三份，并根据您的实验，包括适当的控制。 </li><li>治疗细胞诱导NO的产生。在我们的实验中，我们用脂多糖（LPS）（100毫微克/毫升）和重组IL - 4，对待我们的细胞。在这个协议中，我们使用的RAW 264.7，小鼠巨噬细胞株（小鼠白血病单核细胞巨噬细胞系）。 </li><li>在一天的检测，既带来试剂室温。 </li><li>旋转你的盘子，收集细胞上清50μL转移到一个新的96孔板。准备限制您的标准储备液稀释井，使标准曲线。 </li><li>加入50μL的磺胺解决方案以及每个样品和控制，并拌匀。 </li><li>在室温孵育10分钟在黑暗中。 </li><li>添加每个样品和控制以及N - 1个napthylethylenediamine盐酸盐溶液50μL，拌匀。 </li><li>在室温孵育10分钟在黑暗中。 </li><li>测量absorbaNCE立即用酶标仪波长520nm和550nm的过滤器之间的一个。 </li></ol>如果你使用不同的盘/碟大小，使用1/1/1每个解决方案的体积和样品上清。 紫罗兰的颜色会出现在正井。标准取得的成果将帮助您检查您的解决方案的稳定性。 3。检测ROS的行动:氧化蛋白质 ROS产生不同的方法来确定最终结果看，通过检测的蛋白质氧化。事实上，在大多数细胞中表达及抗氧化活性氧修改谷胱甘肽，对ROS的保护作用。以下氧化活性氧，谷胱甘肽（GSH）在其半胱氨酸的巯基（硫醇）通过二硫键连接到第二个谷胱甘肽组的结果修改。这会导致形成一个二聚体蛋白质（蛋白质氧化的GSSG）。 GSH可以通过修改酶谷胱甘肽还原酶的GSSG恢复。中的GSSG /谷胱甘肽比例的增加反映氧化应激。下面的实验是基于这些氧化蛋白的检测和定量。这种方法是没有选择性针对特定ROS的，而是检测NO的影响 ，H 2 O 2，O 2 -和其他活性氧。在这里，我们使用发光信号测量氧化（GSSG）和减少（GSH）的谷胱甘肽总量。 <ol><li>种子你的细胞在96孔板（白色/透明，平底）像往常一样。我们的实验中，我们用1 × 10，每孔壁RAW 264.7和A549细胞和悬浮Jurkat细胞的细胞。根据细胞的大小，可以调整这些数字。我们建议板块当天贴壁细胞在测试之前，让它们附加到该板块。足够的井应准备氧化和减少蛋白质检测，以及"中唯一的"未经处理的细胞作为对照。使用一式三份的所有条件。 </li><li>与您所需要的时间药物治疗你的细胞。在这里，我们处理Jurkat细胞和A549细胞1h后， 与 H 2 O 2（分别为5mm和2.5mm的）。 RAW 264.7细胞经200ng/ml的LPS 16小时在此期间培养时间，带上所有的试剂室温（RT）。不超过30分钟，然后再执行测试的所有试剂。下表显示了每口井的试剂卷。在可能的情况下，重要的是要删除的媒体进行测试之前，含有还原剂。 </li></ol><table border="1"><tbody><tr><td rowspan="2"></td><td colspan="2"> 贴壁细胞 </td><td colspan="2"> 悬浮细胞 </td></tr><tr><td> 总谷氨酸裂解 </td><td> 氧化谷氨酸裂解 </td><td> 总谷氨酸裂解 </td><td> 氧化谷氨酸裂解 </s仲&gt; </td></tr><tr><td> NEM，25MM </td><td>没有</td><td> 0.5μL </td><td>没有</td><td> 0.5μL </td></tr><tr><td>荧光素- NT </td><td> 1μL </td><td> 1μL </td><td> 1μL </td><td> 1μL </td></tr><tr><td>被动裂解缓冲液，5X </td><td>加入10μl </td><td>加入10μl </td><td>加入10μl </td><td>加入10μl </td></tr><tr><td>蒸馏水</td><td> 39.0μl </td><td> 38.5μl </td><td> 14μl </td><td> 13.5μl </td></tr><tr><td> 每口井的最终体积</td><td> 50μL </td><td> 50μL </td><td> 25μl </td><td> 25μl </td></tr></tbody></table><ol start="3"><li>从贴壁细胞的水井中删除的媒介。不要删除介质悬浮细胞的水井。 </li><li>谷胱甘肽裂解试剂或氧化型谷胱甘肽裂解试剂添加到相应的井和动摇在室温下5分钟。 </li><li>添加荧光素代试剂和incubatE为30分钟，在室温下。 </li><li>加入荧光素检测试剂和孵育在室温下15分钟。 </li><li>阅读整合时间0.25-1第二，每使用一盘读者光度计的发光信号。 </li></ol><table border="1"><tbody><tr><td></td><td> 贴壁细胞 </td><td> 悬浮细胞 </td></tr><tr><td>每孔细胞数</td><td> 1 × 10 4 </td><td> 1 × 10 4 </td></tr><tr><td>每口井+药物的细胞悬液</td><td> 100μL，3.4之前删除</td><td> 25μl，不得被遣送</td></tr><tr><td>还原型谷胱甘肽裂解试剂</td><td> 50μL </td><td> 25μl </td></tr><tr><td>氧化型谷胱甘肽裂解试剂</td><td> 50μL </td><td> 25μl </td></tr><tr><td>荧光素代试剂</td><td> 50μL </td><td> 50μL </td></tr><tr><td>荧光素检测试剂</td><td> 100μL </td><td> 100μL </td></tr></tbody></table> 4。代表性的成果: <img alt="图1" src="/files/ftp_upload/3357/3357fig1.jpg" /> 图1的活性氧，用羧基- H2DCFDA染料的检测。非治疗细胞与H 2 O 2处理的Jurkat细胞（人白血病细胞株）进行了比较。活性氧诱导的羧基- H 2 DCFDA修改，通过流式细胞仪，荧光峰在H 2 O 2处理的细胞转移相比，在控制峰（H 2 O 2处理细胞氧化不敏感的染料和非染色检测荧光绿色治疗羧基- H 2 DCFDA染色细胞）。结果证实处理的细胞中ROS的。 <img alt="图2" src="/files/ftp_upload/3357/3357fig2.jpg" /> 图2检测Øf不使用Griess试剂。与LPS和IL - 4处理的RAW 264.7细胞（小鼠巨噬细胞）。检测处理的细胞相比，控制未经处理的细胞中的NO产量显着增加。 <table border="1"><tbody><tr><td> 细胞株 </td><td> 非治疗 </td><td> 治疗 </td></tr><tr><td> RAW 264.7 </td><td> 13.0 </td><td> 8.3 </td></tr><tr><td> A549 </td><td> 21.6 </td><td> 10.5 </td></tr><tr><td>的Jurkat </td><td> 5.2 </td><td> 2.8 </td></tr></tbody></table> 表1检测的ROS介导的蛋白质氧化。 RAW 264.7细胞治疗与LPS，Jurkat细胞和A549（人肺癌）细胞经与H 2 O 2。结果表示比例减少（GSH）/氧化谷胱甘肽（GSSG）。谷胱甘肽（GSH）/（GSSG）的检出率分别为处理的细胞相比，控制未经处理的细胞，揭示蛋白质氧化处理的细胞。

<ol>
	<li>Guzik, T. J., Korbut, R., Adamek-Guzik, T. <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+and+superoxide+in+inflammation+and+immune+regulation.">Nitric oxide and superoxide in inflammation and immune regulation.</a> J Physiol Pharmacol. 54, 469-487 (2003).</li><li>Perl, A., Gergely, P., Banki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+T+cells+of+patients+with+systemic+lupus+erythematosus.">Mitochondrial dysfunction in T cells of patients with systemic lupus erythematosus.</a> Int Rev Immunol. 23, 293-313 (2004).</li><li>Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., Telser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+antioxidants+in+normal+physiological+functions+and+human+disease.">Free radicals and antioxidants in normal physiological functions and human disease.</a> Int J Biochem Cell Biol. 39, 44-84 (2007).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol Rev. 82, 47-95 (2002).</li><li>Nakamura, K., Yube, K., Miyatake, A., Cambier, J. C., Hirashima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+CD4+D3-D4+membrane+proximal+extracellular+domain+for+the+inhibitory+effect+of+oxidative+stress+on+activation-induced+CD4+down-regulation+and+its+possible+role+for+T+cell+activation.">Involvement of CD4 D3-D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation.</a> Mol Immunol. 39, 909-921 (2003).</li><li>Los, M., Droge, W., Stricker, K., Baeuerle, P. A., Schulze-Osthoff, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+as+a+potent+activator+of+T+lymphocyte+functions.">Hydrogen peroxide as a potent activator of T lymphocyte functions.</a> Eur J Immunol. 25, 159-165 (1995).</li><li>Deem, T. L., Cook-Mills, 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=Vascular+cell+adhesion+molecule+1+(VCAM-1)+activation+of+endothelial+cell+matrix+metalloproteinases:+role+of+reactive+oxygen+species.">Vascular cell adhesion molecule 1 (VCAM-1) activation of endothelial cell matrix metalloproteinases: role of reactive oxygen species.</a> Blood. 104, 2385-2393 (2004).</li><li>Pacher, P., Beckman, J. S., Liaudet, L. <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+and+peroxynitrite+in+health+and+disease.">Nitric oxide and peroxynitrite in health and disease.</a> Physiol Rev. 87, 315-424 (2007).</li><li>Griendling, K. K., Sorescu, D., Lassegue, B., Ushio-Fukai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+kinase+activity+and+gene+expression+by+reactive+oxygen+species+and+their+role+in+vascular+physiology+and+pathophysiology.">Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology.</a> Arterioscler Thromb Vasc Biol. 20, 2175-2183 (2000).</li><li>Loh, K., Deng, H., Fukushima, A., Cai, X., Boivin, B., Galic, S., Bruce, C., Shields, B. J., Skiba, B., Ooms, L. M., Stepto, N., Wu, B., Mitchell, C. A., Tonks, N. K., Watt, M. J., Febbraio, M. A., Crack, P. J., Andrikopoulos, S., Tiganis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+enhance+insulin+sensitivity.">Reactive oxygen species enhance insulin sensitivity.</a> Cell Metab. 10, 260-272 (2009).</li><li>Goldstein, B. J., Mahadev, K., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+paradox:+insulin+action+is+facilitated+by+insulin-stimulated+reactive+oxygen+species+with+multiple+potential+signaling+targets.">Redox paradox: insulin action is facilitated by insulin-stimulated reactive oxygen species with multiple potential signaling targets.</a> Diabetes. 54, 311-321 (2005).</li></ol>

我们收到这本刊物的Promega公司的支持。

一些病理情况，如炎症性疾病，癌症，缺血/再灌注，如放疗或化疗治疗（即顺铂）诱导ROS的生产过剩。因此，检测和测量ROS水平是很重要的，在很多的基础，临床前和临床研究。但是，活性氧有很短的半衰期，可能是复杂的检测。在这里，我们建议经常使用，并免费在哺乳动物细胞中的激进生产检测被广泛接受的简单测试。 使用染料的检测协议允许实验者使用各种广泛使用?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> 5 - （和6） - 羧基-2&#39;，7&#39; -二氯荧光素二乙酸酯（羧基DCFDA） </td><td>分子探针</td><td> C369 </td><td>控制</td></tr><tr><td>羧基- H 2 DCFDA </td><td>分子探针</td><td> C400 </td><td></td></tr><tr><td>磺胺</td><td>西格玛</td><td> S9251 - 100G </td><td></td></tr><tr><td> N - 1 - napthylethylenediamine盐酸盐</td><td>西格玛</td><td> N9125 - 10G </td><td></td></tr><tr><td>亚硝酸盐标准</td><td>西格玛</td><td> 237213 - 100G </td><td></td></tr><tr><td> GSH / GSSG - GLO含量</td><td> Promega公司</td><td> V6612 </td><td>为了量化氧化，减少或氧化/还原型谷胱甘肽</td></tr></tbody>
</table>

production and detection of reactive oxygen species (ros) in cancers

活性氧分子，破坏DNA和RNA和氧化的蛋白质和脂质（脂质peroxydation）。这些反应的分子中含有氧和包括H 2 O 2（双氧水），NO（一氧化氮），O 2 - （氧化物阴离子），过氧亚硝基阴离子（ONOO - ），hydrochlorous酸（次氯酸）和羟自由基（OH - ） 。 氧化品种生产不仅病理情况下（癌症，缺血/再灌注，神经系统及心血管病症，传染性疾病，炎症性疾病，自身免疫性疾病等... ...），而且在非生理（病理）的情况下， 如细 ​​胞的新陈代谢3 4。事实上，活性氧在许多细胞信号通路中发挥重要作用（增殖，细胞活化5，6，7迁移等。） 。 ROS可以是有害的（然后被称为"氧化和nitrosative强调"）时，在细胞内的车厢和细胞产生高量一般回应ROS通过上调抗氧化剂，如超氧化物歧化酶（SOD）和过氧化氢酶（CAT），谷胱甘肽过氧化物酶（GPX）和谷胱甘肽（GSH），保护这些危险的自由基转换成无害的分子（即水）。维生素C和E也被形容为ROS的拾荒者（抗氧化剂）。 自由基是在3低量是有益的。巨噬细胞和中性粒细胞介导的免疫反应涉及NO的产生和释放，从而抑制病毒，病原体和肿瘤增殖8。 NO还与其他ROS反应，因此，也有作为解毒剂（活性氧清除剂）的作用。最后船只上的任何行为，调节血液流量，这是非常重要的肌肉适应长时间运动 9 ，10。一些出版物也表明，活性氧参与胰岛素SENSitivity 11，12。 许多方法来评价ROS产生。在这篇文章中，我们提出了几个简单，快速，经济实惠的的检测;这些实验已经验证了许多出版物，通常用来检测在哺乳动物细胞中ROS或其影响。虽然这些化验检测多种活性氧，其他检测只有一个单一的活性氧。

Watch this Scientific Journal Video about Production and Detection of Reactive Oxygen Species ROS in Cancers at JoVE.com

Production and Detection of Reactive Oxygen Species ROS in Cancers

在这里，我们提出简单的方法来测试和评估细胞活性氧的存在。

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

production-and-detection-of-reactive-oxygen-species-ros-in-cancers

Research

JoVE Journal

Medicine

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

69.9K 次观看.  Baylor College of Medicine. 在这里，我们提出简单的方法来测试和评估细胞活性氧的存在。

视频: Production and Detection of Reactive Oxygen Species ROS in Cancers

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

可视化和肝脏微循环血流量和耗氧量分析:急性肝炎模型中的应用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

科研

医学

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

隔离和使用的牛肠系膜动脉和静脉部分，作为生物测定在小肠测试的Vasoactivity

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host&#8217;s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host&#8217;s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

评估传染性海绵状脑病物种界限与<em&gt;在体外</em&gt;朊蛋白转化测定法

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they ...

Detection of Disease-associated &#945;-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated &#945;-synuclein

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) in experimental models of synucleopathies. A transgenic mouse line (M83) over-expressing the human A53T &#945;S and spontaneously developing a dramatic clinical phenotype between eight and 22 months of age, characterized by symptoms including weight loss, prostration, and severe motor impairment, was used in this study. For molecular analyses of &#945;SD (disease-associated &#945;S) in these mice, an ELISA was designed to specifically quantify &#945;SD in sick mice. Analysis of the central nervous system in this mouse model showed the presence of &#945;SD mainly in the caudal brain regions and the spinal cord. There were no differences in &#945;SD distribution between different experimental conditions leading to clinical disease, i.e., in uninoculated and normally aging transgenic mice and in mice inoculated with brain extracts from sick mice. The specific detection of &#945;SD immunoreactivity using an antibody against Ser129 phosphorylated &#945;S by ELISA essentially correlated with that obtained by Western blot and immunohistochemistry. Unexpectedly, similar results were observed with several other antibodies against the C-terminal part of &#945;S. The propagation of &#945;SD, suggesting the involvement of a &#8220;prion-like&#8221; mechanism, can thus be easily monitored and quantified in this mouse model using an ELISA approach.

An ELISA offering a novel quantitative approach is described. It specifically detects disease-associated &#945;-synuclein (&#945;SD) in a transgenic mouse model (M83) of synucleinopathy using several antibodies against either the Ser129 phosphorylated &#945;S form or the C-terminal part of the protein.

发现疾病相关的α突触核蛋白通过增强ELISA在转基因小鼠表达人A53T的突变脑α突触核蛋白

In addition to established methods like Western blot, new methods are needed to quickly and easily quantify disease-associated &#945;-synuclein (&#945;SD) ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

触发反应胶质细胞增生<em&gt;在体内</em&gt;由前脑锐器伤

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

以质子磁共振成像和氧为造影剂对人肺特异性通气进行定量定位

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Characterization of Sickling During Controlled Automated Deoxygenation with Oxygen Gradient Ektacytometry

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When deoxygenated, HbS can polymerize, forming rigid rods of hemoglobin, resulting in the sickling of red blood cells (RBCs). These sickled RBCs have significantly reduced deformability, causing vaso-occlusion, which leads to numerous SCD-related clinical complications, including pain, stroke, and organ damage. RBC deformability is also reduced by RBC dehydration, resulting in dense red blood cells that are more likely to sickle. To date, there is not a single widely available, rapid, and reproducible laboratory assay capable of predicting the disease severity or directly monitoring the treatment effects for novel, non-fetal hemoglobin inducing therapies. In this study, we describe a protocol to measure RBC deformability as a function of pO2 that allows for the quantitation of sickling behavior in SCD patients. Oxygen gradient ektacytometry measures RBC deformability, expressed as the elongation index (EI), as a function of pO2. RBCs are exposed to a fixed shear stress of 30 Pa during one round of deoxygenation and reoxygenation. Six readout parameters are produced. Of these, the point of sickling (PoS), defined as the pO2 at which maximum EI (EImax) shows a 5% decrease, and minimum EI during deoxygenation (EImin) are the most informative, reflecting an individual patient&#8217;s pO2 at which sickling starts and the minimal deformability of a patient&#8217;s red blood cells, respectively. PoS is associated with an individual patient&#8217;s hemoglobin affinity for oxygen, whereas EImin shows a strong correlation with fetal hemoglobin levels. We conclude that oxygen gradient ektacytometry is a promising technique to monitor the treatment of patients with SCD, as a biomarker for anti-sickling agents in clinical and preclinical trials, and an important tool to study sickling behavior of RBCs from individuals with SCD and sickle cell traits.

Here, we present oxygen gradient ektacytometry, a rapid and reproducible method to measure red blood cell deformability in samples from patients with sickle cell disease under controlled deoxygenation and reoxygenation. This technique provides a way to study red blood cell sickling and to monitor sickle cell disease treatment efficacy.

受控自动脱氧过程中与氧梯度压质测定的病带特征

In sickle cell disease (SCD), a single point mutation in the gene coding for beta-globin causes the production of abnormal hemoglobin S (HbS). When ...