这项工作是由美国国家心脏，肺和血液研究所授予HL56237，糖尿病和消化研究所和肾脏疾病研究所DK97391-03，美国国家科学基金会（NSF-1227359和EPS-1003907）。

1.微流体设备制造<ol><li>一个SU-8 50母模标准光刻制造<ol><li>清洁前旋涂在硅晶片。冲洗，用丙酮进行15分钟，然后用异丙醇（IPA）15分钟的裸2英寸的硅晶片。通过在150℃下将其放置在热板上进行1小时脱水晶片。脱水后，冷却，在室温下的晶片。 </li><li>旋涂用的SU-8光致抗蚀剂的硅晶片。加2ml的SU-8光致抗蚀剂到晶片上。斜坡晶片在100转/秒的加速度，持续10秒以500rpm，接着在300转/秒的加速度以1000rpm进行30秒。 （见补充视频1） </li><li>预烘烤的热板上的晶片在65℃下进行10分钟，然后软烘烤在95℃下30分钟。 </li><li>用紫外线照射，以从薄膜掩模变换设计的图案，以旋涂光致抗蚀剂。上打印的薄透明膜作为膜掩模的图案。放置膜掩模上的顶旋涂光致抗蚀剂和暴露出的剂量为300mJ / cm 2的对紫外线。 （见补充视频2） 注:在这个协议中，微通道网络模式（159微米宽的母亲渠道分支成四个100微米宽的女儿通道）的设计与CAD软件。在胶片上的图案示出为透明字段和膜的其余部分被黑色油墨覆盖。因为SU-8是一种负光致抗蚀剂，暴露于紫外线光致抗蚀剂的部分将是交联和模具​​开发（步骤1.1.6）期间变得不溶于溶剂。开发后，该不溶部分将复制设计的网络模式，并作为一个模具师傅。 </li><li>后烘烤的热板上曝光晶片在65℃下1分钟，然后95℃10分钟。 </li><li>开发模具师傅。使用SU-8开发者为溶剂去除未交联光致抗蚀剂。沉浸在SU-8显影剂将晶片3分钟并用IPA冲洗1分钟。 </li><li>重复这个循环发展2-3次，直到有国际音标冲洗后无白纹形成（非固化光刻胶的残渣）。轻轻擦干​​用氮气晶片并在显微镜下检查该图案的小的特性。如有必要，重复额外开发周期。 </li><li>硬烤晶片上的热板上在150℃下进行15分钟。 </li></ol></li><li>软光刻<ol><li>在重量的两部分聚二甲基硅氧烷（PDMS）弹性体（基料和固化剂）手动混合。的10:1的比例。 </li><li>解气体与PDMS混合物与一所房子的真空15分钟连接的干燥器。投光致抗蚀剂母模的PDMS解决方案。与PDMS的厚度需要是超过3毫米到提供入口/出口管道的稳定保持。脱气15分钟，再与PDMS，并且如果需要除去残留的空气泡用氮气。固化在烘箱中铸造PDMS在65℃下3小时。 </li><li>清洁的表面玻璃盖玻片用透明胶带和等离子净化器（30秒的等离子体处理）。旋涂未固化的PDMS（0.5ml）中的玻璃盖玻片以500rpm /秒的加速率斜坡到4,000rpm下持续30秒。固化在烘箱中在PDMS旋涂盖玻片在65℃下至少30分钟。 </li><li>剪切并从主模释放固化的PDMS。然后创建的入口，并与在母亲的通道的远端的​​直径1毫米打孔器的出口。 </li><li>粘接装置向旋涂的PDMS玻璃盖玻片。氧化的PDMS的表面，并与1分钟的等离子体清洁器盖玻片。氧等离子体处理后，放置的去离子水（DI水）的液滴在接合表面上，并且两部分放在一起。烘烤设备在烘箱中于65℃下30分钟以实现永久的粘合。 </li></ol></li></ol><img alt="补充视频1" src="/files/ftp_upload/54014/54014movie1.jpg"/&gt; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/Supplemental_video_1.mp4" target="_blank">补充视频1（点击下载）。</a> <img alt="补充视频2" src="/files/ftp_upload/54014/54014movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/Supplemental_video_2.mp4" target="_blank">补充视频2（点击下载）。</a> 2.内皮细胞播种和文化<ol><li>灭菌设备和纤维连接蛋白涂层<ol><li>保留周围的氧化入口/出口与PDMS表面的疏水性。盖周围的入口PDMS表面和透明胶带的小条纹防止氧化插座。这个程序可以防止DI水从在填充步骤（2.1.3）遍布器件的顶表面。 </li><li>对待PDMS设备用氧等离子体FOR 5分钟，以改变它的表面是亲水性的，从而有利于通过所述微通道的流体流动，并防止泡沫在填充过程（2.1.3）中捕集。 </li><li>使用一个注射器附有针头或移液管从入口填充用DI水的设备。继续填充，直到有在出口累计DI水（30-50微升）的液滴。在显微镜下检查装置，以检查是否有截留在微通道内的气泡。在入口和出口用薄纸除去过量的水滴。 </li><li>消毒装置在培养皿用UV一种用于在层流生物安全罩最少3小时。为了防止蒸发，盖上一块薄薄的PDMS的入口/出口，把湿纸组织培养皿内，并包装用封口膜的菜。 </li><li>涂上纤连蛋白的设备。用磷酸盐缓冲盐水（PBS），100微克/毫升纤连蛋白过夜第一冲洗设备，然后涂层它在冰箱（4℃）。放置的在入口溶液的液滴，然后创建在用室内真空，用针或吸移管的注射器的出口负压。 </li><li>填充细胞培养介质的设备。加载细胞培养基之前手动用PBS冲洗设备3次。在37℃预热设备的孵化器。 </li></ol></li><li>内皮细胞播种和长期灌注 注意:与传统静态2D EC培养比较，以连续灌注体外微血管网络模型所取得的细胞培养环境接近体内条件。在这个协议中表现出的体外微血管网络可以维持长达两周。该井控灌注将持续补充肌肤养分，清除废弃物，防止过度汇合EC单层的。因此，它可以扩展到一个较长期的研究，不同的生理学下模仿细胞和血液动力学环境升和病理状况。 <ol><li>在混合HUVEC培养基用8％的葡聚糖（分子量为70,000）的主要血管内皮细胞（MCDB 131）。葡聚糖是用来增加装载介质的粘度和达到更好的细胞接种的结果。推荐的细胞密度为约2-4×10 6个细胞/ ml。 </li><li>种子的细胞进入设备。将细胞在进口的10-20微升滴。引进由任一倾斜的装置中，或者使用在出口处的玻璃巴斯德吸管毛细管作用平缓流动。对于一个成功的小区负载的关键是控制流速小于1毫米/秒的最小信道之内。 </li><li>检查细胞种植。最初的接种后，检查在显微镜下播种状态之前孵育设备15-20分钟（在37℃的5％CO 2的加湿气氛）。如果需要的话，执行额外的小区负载来实现所需的细胞密度。 </li><li>轻轻漂洗用细胞培养基的装置（37℃）以除去葡聚糖。培养（37℃，5％CO 2）的设备在没有流动的用于在培养箱的前6小时。 </li><li>设置为长期灌注管连接。用胶带将设备插入培养皿以防止移动。上创建培养皿为入口和出口管道插入的盖小孔。入口管道连接到一个注射器与用于细胞培养基灌注针。出口管路连接到一个废物收集。 注:将设备与在细胞培养培养箱废液收集器，同时将一个灌注泵的保育箱的外面，并通过长进口管道它与设备连接。灌注速率由实验设计来确定。在本装置中，使用0.35微升/分的灌注速率和1-2达因/厘米2之间的剪切应力范围内。在这种灌注条件下，内皮细胞达到约3-4天汇合。 </li></ol></li></ol> 3.我mmunofluorescent染色<ol><li>在4℃下30分钟，接着通过1×PBS中于室温下漂洗15分钟，和1％的BSA封闭30分钟固定的内皮细胞在装置2％多聚甲醛灌注。使用灌注率是一样的用于细胞培养。 </li><li>透固定内皮抗体染色用0.1％的Triton X-100。灌流用Triton设备5-7分钟，以标签VE-cadherin的，3分钟到标签F-肌动蛋白。 </li><li>加载抗体到器件中。灌注用1×PBS的设备15分钟洗出以前的解决方案。手动加载针对VE-钙粘蛋白（抗山羊）一级抗体进入设备以2微克/毫升（总体积为20-50微升）的浓度。器件保持在冰箱中在4℃下过夜。灌注通过注射器泵的第二抗体（驴抗山羊）以7微克/毫升45-60分钟的浓度。 </li><li>标签EC F-肌动蛋白与鬼笔。灌注用1×PBS FO设备为7至10分钟手动装载鬼笔环肽（10微克/毫升）之前R 15分钟。 </li><li>标签EC核（见材料清单）。 </li><li>冲洗用1X PBS的设备，其保持在4℃，直到成像。 </li></ol> 4.内皮细胞的荧光成像[ 钙 离子浓度 <ol><li>灌注与白蛋白的装置（10毫克/毫升）-Ringer的在37℃下15分钟的溶液。使用相同的灌注速率作为用于细胞培养。的白蛋白林格氏溶液中所有组分的浓度在材料清单中描述。 </li><li>建立了共聚焦系统EC 内钙 我和荧光- 4点成像。使用共焦显微系统， 钙成像。使用氩激光（488纳米）激发并设置发射带为510-530纳米。在512×512扫描格式:用25X客观收集的图像（0.95水浸泡，NA）。用2微米对于i的每个堆叠的z步骤和扫描间隔20秒的法师。 </li><li>灌注与FLUO-4上午（5微米），在37℃下40分钟的设备，然后洗出流明FLUO-4AM白蛋白林格氏溶液。 </li><li>收购荧光 - 4加载微血管的图像。收集基线图像10分钟。灌注与ATP（10μM）的设备和20分钟记录的荧光强度（FI）的变化。 </li></ol> 5.一氧化氮产生荧光成像<ol><li>灌注与白蛋白林格氏15分钟溶液的设备。使用灌注率是一样的用于细胞培养。 </li><li>设置了荧光成像系统来测量ATP诱导的一氧化氮产生。使用配有12位数字CCD照相机和计算机控制的快门一氧化氮测量的显微镜。使用一个75瓦的氙灯作为光源。 注意:通过一个干涉滤光器（四十零分之四百八十零毫微米）和分色镜选择DAF-2 DA的激发和发射波长（505纳米）与带通屏障（五十零分之五百三十五纳米）。使用20X物镜（NA:0.75）收集图像。为了最大限度地减少漂白，定位在干涉滤光器前的中性密度滤光器（0.5 N）和限制曝光时间以0.12秒。 </li><li>负载DAF-2 DA（5微米）的细胞。灌注用DAF-2 DA设备约35-40分钟，在37℃。初始加载后记录DAF-2的荧光强度（FI DAF）的基线。 注意:DAF-2 DA是连续地存在于该灌洗液整个实验7。 </li><li>收集DAF-2图像。记录FI DAF的基线5分钟。灌注与ATP（10μM）的设备，并收集图像与1分钟的间隔30分钟。收集所有来自同一组的细胞的图像中的同一焦平面上。 </li></ol> 6.数据分析<ol><li>手动选择从个别细胞水平上采集到的图像利益（投资回报）​​的区域。每ROI覆盖可以由荧光轮廓来指示一个个体单元的面积。 </li><li>减去背景荧光和计算每个投资回报率的平均FI。 </li><li>测量EC ATP引起的变化[ 钙 离子浓度和NO的产生。量化ATP诱导的变化EC [ 钙 离子浓度通过计算荧光- 4 FI（FI / FI 0 * 100，其中FI 0是白蛋白林格氏液灌注过程中的平均基线FI加入ATP之前）的变化减去测背景。通过进行经时的FI DAF的第一差分转换量化NO产生率。 注意:FI DAF的时间过程表示累积的NO产生与时间。因此，NO的产生速率是基于FI DAF的基础和ATP的刺激条件下，轮廓计算。数据分析和实验程序的细节已在先前的出版物7进行了描述。 </li></ol>

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作者没有利益冲突或利益冲突的披露。

<html>在这篇文章中，我们提出详细的协议为培养微血管网络的发展，EC路口和细胞骨架F-肌动蛋白分布特征，以及EC的定量测量钙离子浓度 且使用微流体装置NO的产生。灌注的微流体装置提供的体外模型，其允许在体内微血管几何形状和剪切流动状况的密切模拟。由于培养的微血管网络可以通过初级人内皮细胞来形成，这种方法可以作为一种有用的工具，调查从患者样品改变的血液成分如何病理影响由患者的血液样本的直接灌注人类内皮到培养的微血管和提供深入临床的问题。开发微血管人类内皮细胞也可以用于药物筛选，以避免在人类和动物的组织之间药物反应的潜在差异。 ve_content"&gt;在这个协议中所示的微流体装置的基板是玻璃盖玻片（150-180微米）旋涂的PDMS薄层。这种薄的PDMS层是用于高分辨率和活组织实时必不可少成像，因为较大的数值孔径物镜通常具有短工作距离。所述微通道（亚毫米范围）内的层流具有非常低的雷诺数，与压力差线性相关的流量，从而使剪切应力分布是完全依赖所述信道的几何形状，因此，它可以通过高精度的灌注泵20良好控制的，在我们目前的研究中，施加到HUVEC培养微血管网络的剪切应力是10达因/厘米2，这是内1之间范围内静脉系统21,22测量的剪切应力，宏观流体系统如平行流室缺乏复杂的流动模式。在大规模流室（毫米厘米范围），内皮细胞通常只生长在底表面（2D单层），缺乏几何相似体内微血管。当流室的宽度大于2毫米，均匀流动被限制在，通常是从出口/入口几毫米远的区域内，和几百微米从侧壁23的路程。当腔室的宽度增大到厘米的范围内，该流程将分化成各种流线和流速将在整个区域24变化。此外，较大规模的流动室消耗至少100倍以上的灌注液以达到相同的剪切应力，在微通道。所述微通道网络内的剪切应力分布可以使用数值建模工具来模拟，并在固定的不可压缩Navier-Stokes方程可以被大多数有限元软件来解决。 微流体装置可与SINGL制成Ë面膜微细加工。培养微血管发展的成功，但是，需要特别关注的细节。加载溶液放入微通道之后，每个装置需要EC播种前，在显微镜下进行检查气泡俘获。如果发生气泡，它仍然是可能通过在一个封闭的出口的入口施加液压以迫使它在这个阶段，由于PDMS是具有透气性的材料。以保持所需的灌注速率，而不泄漏紧身是必要的所有的针/管路连接的。匹配的打孔机和泵管尺寸都实现了紧身的关键。为了保证流速的精确输送，在灌注泵和装置的注射器应放置在同一水平。要改变灌流液，插入管应轻轻地从设备断开连接，以避免任何伸展。为了增加剪切应力，提级（如10步增长在18小时）的recommenDED避免突发流量变化而引起脱皮EC。 单独灌注完好微血管进行先前的研究表明，在欧共体激动剂诱导的增加的[Ca 2+] i和后续的内皮型一氧化氮合酶（eNOS）活化，NO产生是炎症性疾病6,7-下微血管通透性增加必要的信令，9-13,25。在这个协议中，我们选择的ATP为代表激动剂，因为它通常是由红血细胞，聚集的血小板和受损组织或体内炎症条件下释放。的荧光钙指示剂，荧光-凌晨四点，是用来衡量暴露后ATP内皮的变化[ 钙 离子浓度 。欧盟委员会[ 钙 离子浓度在培养的微血管的反应是相似的在完整的微血管7,9找到。测量EC NO生产已具有技术挑战性的生物学家。迄今为止，还没有荧光指示剂，使得在NO浓度以类似的方式，以钙指标的动态变化的探测。 DAF-2 DA已被广泛用于评估NO产生。然而，正确使用，数据解释和数据分析需要吸引用户的注意。因为DAF-2在NO存在下化学转化来的DAF-2T是不可逆的（单程转化率）26中 ，检测到的DAF荧光强度分布并不代表NO浓度的变化。相反，它表示所积累的DAF-2T的生产用的时间。在此基础上累积DAF-2荧光（FI DAF）曲线，NO生产速率（DF / DT）可以由FI DAF 7,11的第一差分转换得到。即，FI曲线的斜率指示NO生产速率和平台期表示没有进一步增加NO的产生。转换后，从该协议的公关产生的数据esented两个基底NO产生速率和微血管网络内的个别EC水平响应的变化的NO产生对ATP与时间和空间分辨率。 目前的PDMS已广泛用于微流体的应用，因为它是一种光学透明的，无毒的，并且可透气的软的弹性体27-30。然而，由于PDMS不透水性，我们无法直接测量EC单层的通透性。为了克服此限制，某些应用程序修改了通道壁的结构，例如一个渗透膜整合到信道31，或制造可渗透"窗孔"与微柱或微孔32,33，而另一些被集中在设备材料，例如使用水凝胶的装置或水凝胶/ PDMS混合设备17,34-36的修饰。这种装置的缺点是它们的脆弱性质脱水和他们的相对较弱的米器的机械性能站立压力或压力。与机械强度渗透性材料的未来发展将进一步完善设备和更广泛的生物应用潜力。

内皮细胞（EC）衬血管壁体内经常暴露于流动，但培养的内皮细胞通常在静态条件下生长并表现出促炎性表型1,2。微流体技术使得精确地控制流体通过几何约束微尺度（亚毫米）通道3，其提供了用于培养细胞的机会，特别是对血管内皮细胞，所需的流量的条件下生长。这些特点使得比常规的，静态的2D细胞培养更接近体内细胞培养条件。它们是当微流体装置用于模拟不同类型的脉管的，并研究EC响应机械和/或化学的刺激非常重要的。 尽管微通道网络在一个静态的细胞培养，生物医学F中的适应和微流体的应用表现出的优势ield仍然是有限的。通过最近的一篇综述报道，大部分该领域（85％）的出版物仍然是工程类期刊4。微流体装置的性能尚未足够的说服力大多数生物学家从当前技术切换诸如Transwell小测定和宏观尺度培养皿/载玻片该小型化的设备。微流控是一个多学科领域，需要跨学科的合作，以推动这一领域。本技术本文的目的是减少知识缺口学科之间，使制造程序可理解的生物学家，同时提供生物应用和微流体微血管功能验证。可视化实验方案包括微流体装置及其生物实用工具，它代表的工程师和生物学家之间的密切合作制造。 我们最近的一些报道使用体外微血管网络与微流体装置5生物学应用。为了适当地设计所述微通道网络的尺寸和应用所需的剪切应力，数值模型用计算流体动态软件密切估算流动轮廓建造。原代人脐静脉内皮细胞（HUVECs），该接种到达到汇合的微通道， 即覆盖所述微通道的整个内表面上，在3-4天连续灌注。适当的阻挡层形成通过VE-钙粘蛋白染色显示，并与静态细胞培养条件下，并在完整的微血管形成的比较。通过施加在单独灌注完好微血管6-8开发的实验方案，我们定量测量在EC的变化的[Ca 2+] i和一氧化氮（NO）产生的荧光响应于三磷酸腺苷（ATP）的修复作用和共聚焦和常规荧光显微镜。在欧共体激动剂诱导增加[ 钙 离子浓度和NO据报道，作为在微血管通透性6-15炎性介质引起的必要增加细胞内的信号。虽然以前的一些研究表明DAF-2 DA加载微流体装置16,17的图像，合适的分辨率和数据分析还没有达到18。据我们所知，这项研究表明内皮[ 钙 离子浓度和NO利用微流体为基础的系统激动剂诱导动态变化的第一个定量测量。 微细加工技术，灵活地制造微下降到几微米和使复杂的图案的发展，以模仿体内微血管的几何形状。在这里，我们提出了一个典型的微通道网络，三级分支。这个网络是由在一个微细洁净室和软光刻进行光刻的组合制成。

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">ATP</td>
			<td style="width:247px;">Sigma-Aldrich</td>
			<td style="width:327px;">A2383</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Fisher Scientific</td>
			<td>A929</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biopsy punch</td>
			<td>Miltex</td>
			<td>33-31 AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Albumin</td>
			<td>MP Biomedicals</td>
			<td>810014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Brain Extract (BBE)</td>
			<td>Lonza</td>
			<td>CC-4098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover-slip</td>
			<td>Fisher Scientific</td>
			<td>12-542C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAF-2 DA</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-Goat IgG (H+L) Secondary Antibody</td>
			<td>Life technologies</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DRAQ5 (nuclei staining)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>4084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td>Sigma-Aldrich</td>
			<td>E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluo-4 AM</td>
			<td>Life technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750-078</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-548B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Pasteur pippette</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-10KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human umbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl alcohol (IPA)</td>
			<td>VWR</td>
			<td>89125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mammalian Ringer Solution Ingredient</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl (132 mM)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL (4.6 mM)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 &#183; 2H2O (2.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4 &#183;7H2O (1.2 mM)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose(5.5 mM)</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3 (5.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Salt (9.1 mM)</td>
			<td>Research Organics</td>
			<td>6007H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Acid (10.9 mM)</td>
			<td>Research Organics</td>
			<td>6003H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB 131 Culture Medium</td>
			<td>Life technologies</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin (F-actin staining)</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline&#160;</td>
			<td>Life technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>Exel Int</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scotch tape</td>
			<td>3M</td>
			<td>34-8711-3070-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicon wafer</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2050 Y111072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tissue culture flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z707503-100EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Chemical Book</td>
			<td>T6878</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin Neutralizer solution</td>
			<td>Gibco</td>
			<td>R-002-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin/EDTA Solution (TE)</td>
			<td>Gibco</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Cole-Parmer</td>
			<td>PTFE microbore tubing, 0.012&#34; ID x 0.030&#34; OD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VE-cadherin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Laminar hood</td>
			<td>NuAire</td>
			<td>NU-425 Class II, Type A2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD camera</td>
			<td>Hamamatsu</td>
			<td>ORCA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art</td>
			<td>F42022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>Wenesco</td>
			<td>HP-1212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp oven</td>
			<td>Barnstead</td>
			<td>3608-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithography bench</td>
			<td>Karl Suss</td>
			<td>MA6 Contact Lithography</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical microscope</td>
			<td>Nikon</td>
			<td>L200 ND &#38; Diaphod 300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shutter for the CCD camera</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>PVA TePla/Harrick plasma</td>
			<td>M4L/PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Brewer Science</td>
			<td>Cee 200X</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump system</td>
			<td>Harvard Apparatus</td>
			<td>703005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Software</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD 2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFD simulation software</td>
			<td>COMSOL</td>
			<td>COMSOL Multiphysics 4.0.0.982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Images acquire and analyse for NO production&#160;</td>
			<td>Universal Imaging</td>
			<td>Metafluor</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production

内皮细胞（EC）衬血管壁体内经常暴露于流动，但培养的内皮细胞通常在静态条件下生长并表现出促炎性表型。虽然微流体装置的发展已被工程师超过二十年被接受，其生物学应用仍然有限。通过生物 ​​应用验证更生理有关体外模型微血管重要的是推动该领域和桥体之间在体外研究的空白。这里，我们提出的详细程序为培养的微血管网络的使用微流体设备与长期灌注能力的发展。我们还使用共聚焦和常规荧光显微镜表明实时其在EC的[Ca 2+] i和一氧化氮（NO）产生的激动剂诱导的变化的定量测量的应用。所形成的微血管网连续灌注工作表明内皮之间发达结。 VE-钙粘蛋白分布更接近在完整微血管比静置培养乳油单层观察。 ATP诱导的EC瞬时增加钙离子浓度 i和NO的产生进行了定量的单个细胞的水平，这验证了培养微血管的功能进行测量。此微流体装置允许内皮细胞到良好控制，生理学相关的流，这使得细胞培养环境比更接近于体内在以往的，静态的2D培养下生长。所述微通道网络的设计是高度通用的，并且制造工艺简单，重复性好。该装置可以方便地集成到共焦的或常规的显微系统，使高分辨率成像。最重要的是，因为培养的微血管网络可以通过初级人内皮细胞来形成，这种方法将作为一个有用的工具来调查如何从病理患者样本改变血液成分影响人类内皮细胞，并提供深入了解临床的问题。它还可以被开发作为用于药物筛选的平台。

本节展示了一些与此协议开发的培养微血管网络所取得的成果。所述微通道图案是一个三级分支网络（ 图1A）。在此设计中，一个159微米宽的母信道分支成两个126微米宽的通道，和分支再次分成四个100微米宽女儿频道。进行三维数值模拟来估计的0.35微升/分钟的流速（ 图1B），这表明这微通道网络内的三个不同级别的剪切应力下的剪切应力分布。在微通道内的层流被预测为不干扰或二次流的存在下流体动力学稳定。一个SU-8母模用标准光刻工艺（ 图1C）制成后，一个PDMS设备通过软光刻制造以复制所述微通道网元twork设计成透明的，透气的支架（ 图1D - E）。 EC播种（ 图1F - G）后和连续灌注的3-4天，内皮细胞达到汇合，并成功地覆盖微通道的内表面。 EC F-肌动蛋白和核网络内被荧光标记，并用共焦图象中所示。下1.0达因/厘米2的剪切应力，所述内皮细胞的约30％沿着具有增加的中央应力纤维的流动方向上拉长的，而其余的70％保持其鹅卵石图案与主导外围F-肌动蛋白5。剪切诱导的细胞形态变化似乎比2D静态细胞培养中观察到不那么突出。实际上在体内条件下，内皮细胞在不同类型的船只不同细胞的形状，但在响应不会轻易改变细胞形状与流动茶nges。需要更多的研究，以确定该差值是否是信道的几何形状和灌注的环境是接近体内条件的结果。 我们还成功地测量在欧共体的[Ca 2+] i和NO的产生（ 图4和5）ATP诱导的变化。 ATP引起的短暂增加EC [ 钙 离子浓度和NO的产生。的平均峰的FI FLUO-4的基线，这在35±10秒发生的ATP灌流开始后的187±22％。 NO生产速率从每分钟0.15±0.05 AU的基础水平增加至每分钟1.18±0.37 AU ATP曝光的第一个五分钟内。所有的结果被报告为平均值±标准误差（SE）。他们媲美单独从灌注完好静脉6,9,14,19得出的结果。 图1:微流体器件制造和EC装载 （A）的所述微通道网络的示意图。微通道的宽度和分叉角在每个分支的水平表示。 （B）剪应力分布的数值模拟。流速为0.35微升/分钟。显影后的微通道的高度为80微米。这两个数字已经从以前的出版物与改装更多详细信息5。 （C）主模具的发展。所述微通道网络的主模具被制造硅晶片上的SU-8光致抗蚀剂。 （D）的软光刻的方法。 PDMS的混合物浇铸到主模和在烘箱中固化。 （E）的粘合的PDMS通道到基底上。用于该设备的基板是盖玻片的spiN涂层用的PDMS薄层。入口和出口与一个空穴的冲床创建。 （F和G）EC加载。大约10微升的细胞溶液被放置在进气口。一个温柔的流然后通过经毛细流动的出口放置一个巴斯德吸管诱导。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/54014fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54014/54014fig2.jpg" /> 图2:在培养的微网EC F-肌动蛋白的共聚焦图像 （A）网络设计的示意图。在B中所示的选定的区域-中D图形表示。 （B - D）的 在体外微血管网络的EC F-肌动蛋白（红色）和细胞核（蓝色）的共聚焦图像b 1分配 。 </strong&gt;，C 1和D 1是从微通道图像栈的上半部分，和B 2，C 2和D 2中的投影图像从图像栈的下半部分中的图像预测。 d 1的 ，比例尺= 100微米- E的剖视图B中1表示为1-1&#39;，2-2&#39;和3-3&#39;。这些图像是改编自先前公布5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/54014fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54014/54014fig3.jpg" /> 图3:比较VE-钙粘蛋白在培养的微血管网络与静置培养EC单层和完整的大鼠肠系膜微静脉分布（。 <stroNG&gt; A）网络设计与B中所示选定区域的示意图- ð。 （B - D）的 VE-钙粘着蛋白（绿色）和细胞核的体外微血管网络d 1的 （蓝色）染色和D 2分别是从同一分叉区域收集，投影图像的顶部和底部。 （E）VE-cadherin的静态培养的内皮细胞的染色。 （F）VE-cadherin的单独灌注完整的大鼠小静脉的染色。这个数字之前5日公布。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/54014fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54014/54014fig4.jpg" /> 图4:ATP诱导EC中 Ca2 +浓度 增加 </suP&gt;] 我 。实验从4设备，每个设备7至12的ROI（个人内皮细胞）进行被选定为数据分析。 （ 一 ）ATP的时间过程（10μM）从一个代表性的实验诱导的EC [ 钙 离子浓度 （4的Fluo FI / FI 0×100％±SE）的变化。 （B）中的代表性实验的图像（数据是示出）。这是先前公布的数字5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/54014fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54014/54014fig5.jpg" /> 图5:ATP诱导的NO产生实验在3个设备进行，并且被选择用于数据分析每个设备11至12的ROI（个体内皮）。 （A </st荣&gt;）（FI DAF±SE，左Y轴）基本条件下和暴露在ATP后DAF-2荧光强度的时程变化的内皮细胞。 NO生产速率（DF / dt的，点线），从累积FI DAF（右边的Y轴）的第一差分转换而得。 （B）中从一个实验代表性图像。将FI DAF NO供体，硝普钠（SNP），加入到灌注液后进一步增加，表明细胞内DAF-2的足够量，没有反应。这是先前公布的数字5。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54014/54014fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

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 ...

development-characterization-vitro-microvessel-network-quantitative

Nanyang Technological University

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JoVE Journal

Biology

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

8.5K 次观看.  College of Medicine, Penn State University. 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.

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

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

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 ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

赖氨酸去甲基化酶活性的定量测量中的应用的MassSQUIRM

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

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 ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

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 ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和"剪切环"中的应用:一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

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 ...