资金由美国国家科学基金会通过NUE工程和领导的联盟程序表示感谢。

1。胶体银奈米粒子的合成克莱顿法（略作修改，廉价的）22的反应机制中很详细的描述，在辅助信息的参考帕维尔et.al一起的不需要的水解侧的NaBH 4，在室温或更高温度下反应23 <ol><li> 12-24小时，在10％HNO 3的浴，然后清洁所有的玻璃器皿为4-12小时，在1.25 M的NaOH在40％的乙醇浴中，最后的高压釜。玻璃器皿应彻底漂洗最低五次后，用超纯水（17MΩ或更高）的酸和碱浴步骤。 </li><li>准备2 mM的NaBH 4溶液的300毫升和100毫升1mM的AgNO 3溶液，使用灭菌水冷却在10℃下的较低的温度下，将防止侧的NaBH 4反应。 </li><li>加入300毫升2mM的NaBH 4溶液到500毫升锥形反应烧瓶中续癌宁搅拌棒和烧瓶用铝箔包裹，以防止银氧化。将烧瓶放置在冰浴上搅拌盘和搅拌溶液10分钟，在325 rpm的转速下。 </li><li>总理25毫升滴定管具有列满的超纯水漂洗。浸水后，填写滴定管AgNO 3溶液和包装用铝箔。 </li><li>在一个黑暗的房间中，添加1mM的AgNO 3溶液50毫升，在连续搅拌下（ 图1A）的NaBH 4溶液1滴秒-1的速率。量的装置与一个“箔帐篷”的中间部分，以尽量减少在AgNO 3的除了曝光。 硝酸银除了将需要30-40分钟。定期补货冰浴。 </li><li> AgNO 3的除了完成后，补充冰浴，并继续搅拌该胶体溶液中的一个额外的45-50分钟。信号形成的胶体银奈米粒子颜色的变化由无色金黄色，这是表面等离子体共振最多AGNPS（ 图1B）的特征。 </li><li>一旦反应完成后，冷藏胶体。胶体AgNP批次可以合并在一个星期后的胶体保持一致， 即胶体溶液不会合并，和批量使用紫外-可见吸收光谱法和拉曼光谱的特点，以确定可能的聚集或污染物。 </li></ol> 2。胶体银奈米粒子的特性在Cary 50 UV-VIS-NIR分光光度计（Varian公司）和一个LabRamHR的800拉曼系统（HORIBA Jobin Yvon公司，公司），配有奥林巴斯BX41共焦拉曼显微镜，用于AgNP特性。 ，LabSpec的卡里WinUV软件第5节地8.0软件的数据收集和分析。 注：采集参数将必须进行优化FOR其他仪器模型。 通过紫外 - 可见分光光度法测定胶体银奈米粒子的表面等离子体共振<ol><li>填写1厘米3的一次性反应杯与克莱顿胶体和超纯水在1:10的体积比。填写另1 cm 3的比色皿，用超纯水为空白的基线校正。擦拭与Kimwipe两个比色皿的外侧。 </li><li>设置的分光光度计吸光度模式从-0.5 Y最小的Y的最大值为1.0。设置X到200-800纳米扫描窗口，并选择较快的扫描速度为4800 nm的基线校正分钟-1。 </li><li>将装满水的试管中，放入仪器和运行基准扫描。如有必要，重复上述步骤，直到一个非零的基线控制的实现。 </li><li>更换空比色皿与样品比色皿和启动的吸光度扫描收集的胶体样品的UV-Vis吸收光谱（ 图1C）。 </lI&gt; </ol>通过拉曼光谱纯度的胶体银奈米粒子由于视频演示（10-15分钟的视频）的时间限制和空间限制的协议文本（最多3页），这个实验的部分将不会进行录像。 <ol start="5"><li>设定仪器参数设置如下：激励源（632.8 nm波长的He-Ne），过滤器（没有过滤器，样品〜17毫瓦的激光功率），共焦孔（300微米），光谱仪（730厘米-1），全息光栅（600园/毫米），物镜（50倍长的工作距离空气物镜），曝光时间（30秒），和积累周期（5）。 </li><li>使用干净的移液管，以填补与胶体2毫升的石英比色皿，并轻轻地插入插头。使用Kimwipe的比色皿的表面，清洁过的指纹，污迹或胶体。显着降低，在显微镜阶段。选择50倍的物镜和搬上舞台，将试管。 </li><li>聚焦拉斯维加斯呃束上​​的AgNP的胶体直接的比色皿使用仪器和奥林巴斯相机的视频模式下的内壁。关闭室内灯光，并获得拉曼光谱（ 图1D）。 </li></ol> 3。胶体银奈米粒子的大小选择和集中，通过切向流超滤（TFU） 甲KrosFlo II研究过滤系统（Rancho Dominguez的频谱实验室，CA）用于限制的AgNP的多分散性和浓缩（ 图2）。 TFU过程的三个步骤：（1）尺寸银奈米粒子和AgNP-聚集体的50-nm的直径和较大的使用50纳米MidiKros聚砜模块（460厘米2），2）大小的选择和浓度的选择银奈米粒子1-20纳米的直径，使用一个100 kD的MicroKros的聚砜过滤器（20厘米2）（ 图3），使用100 kD的MidiKros过滤器（200厘米2），和（3）进一步减容。 <ol><li>连接尺寸为17 MASTERFLEX进料管的蠕动泵，根据图2A。一个Y字路口和管交界处，将需要的设置。连接管到的50纳米MidiKros的模块。一定要确保管道过滤器采用拉链关系。选择管道尺寸17 SIZE按钮。 </li><li>选择使用DIR“按钮逆时针泵方向。确保MODE按钮是INT。 </li><li>前启动泵，泵的速度降低到低于300毫升分钟-1。根据所使用的管材的尺寸，应调节的泵速率。它应该是一个小的设置，以允许操作者及时反应潜在的泄漏，但足够大的起动系统的效果仍然有。为了创建需要绘制胶体从水库进入管件和过滤器的真空，剪切，导致从过滤器的底部部分的Y结的顶部部分中的中间的油管油管。 </li><li&gt;放置的管道部分，导致从储液瓶过滤器进入和夹紧用卡子或手的底部附近的Y结油管。打开在泵上。开始创建的真空虹吸胶体。 </li><li>一旦自由流动的液体通过管子，关闭泵，加入破碎的部分管与管接头和保护带拉链的关系。再次开启泵，继续过滤。 </li><li>检查油管回路的泄漏。如果发现泄漏，，调整装修或重新用扎带确保，修复泄漏。一旦管道系统是无泄漏，泵的流速可以增加，以不大于700毫升分钟-1。该泵的速度值，根据管道大小，以避免管道发生故障，应进行优化。继续过​​滤在储液瓶，直到液体被耗尽到几乎为零。 </li><li>一旦完成过滤，收集滤液，其中包含银奈米粒子的直径为50纳米以及小型LER。滞留物可被保存根据具体AgNP应用程序以供进一步分析。 </li></ol>第2步<ol start="8"><li>冲洗管与2％HNO 3和超纯水之前安装100 kD的MidiKros的过滤器使用相同的设置为50纳米的模块。 </li><li>重复步骤3.3使用100 kD的MidiKros的模块。 </li><li>一旦过滤完成后，收集的油管和过滤器（100-kD的滞留物）的内容。的体积应为约50毫升。 </li></ol>第3步<ol start="11"><li>连接大小14 MASTERFLEX管和100 kD的MicroKros的过滤器根据图2B蠕动泵。所有路口固定带拉链的关系。选择油管尺寸14对泵使用的大小按钮，降低泵的速度分-1〜30毫升。 </li><li>开始过滤过程。检查油管回路的泄漏。如果发现泄漏，修复泄漏，通过调整合适婷安全与扎带。 </li><li>一旦管道系统是无泄漏，泵的流速可以增加，以不大于90毫升分钟-1。继续过​​滤直至剩余的液体中的储液瓶包含最小量的浓缩物。 </li><li>通过除去从瓶泵仍在运行时的进料管，油管和过滤器的剩余的内容可以被收集到储液瓶。一旦管和过滤器的内容是在储液瓶，泵可被关闭。 </li></ol> 4。银量的定量胶体银奈米粒子的电感耦合等离子体发射光谱仪（ICP-OES） 每个胶体样品化学消化和使用A 710E光谱仪（Varian公司）用ICP-OES的银的量进行定量。银（ 图4）的线性回归的校准曲线是使用八个银标准（0，3构造，7，10，15，25，50，和100微克L-1），制备了10000微克毫升-1银痕量金属分析标准，（超低科学）。 <ol><li>化学消解样品用HNO 3。的有代表性的样品是原来的胶体（步骤1），50-nm的滤液（步骤1），100-kD的滞留物（步骤2），和最后的100-kD的滞留物（步骤3）（ 图3）。 </li><li>应是稀释后的样品用2％的HNO 3使用下面的体积比：1:1000原始胶体的1:1000的50-nm的滤液，1:25,000头100-kD的滞留物，和1:250,000为最后的100 kD的滞留物。为了防止银浸出，所有的样品应当存放在低密度的聚丙烯容器中。 </li><li> ICP-OES仪器参数设置如下：对Ag（328.068 nm）的波长，功率（1.20千瓦），血浆流量（最小15.0 L -1），辅助流量（1.50 L最小-1），雾化器压力（200千帕）。 </li><li>每个SA复制时间为10秒，mple应在一式三份。之间的测量的稳定化时间为15秒和30秒的样品的吸收延迟应该被使用。每个样品之​​间应采用的方法空白，以减少潜在的交叉污染。 </li></ol> 5。胶体银奈米粒子的粒径分布，通过透射电子显微镜（TEM） 十字EM 208S TEM用于可视化的胶体银奈米粒子。电子显微照片，使用高解析度的加坦BIOSCAN相机捕获和分析在ImageJ软件24 <ol><li>用超纯水（1:100的体积比）稀释100 kD的滞留样本。存款原始胶体和稀释的100-kD的滞留物（步骤3）对20μl的300目福尔瓦被覆金网格（电子显微学）。允许电网在干燥器中干燥。在一天之内。 </li><li>设置在70千伏可视化农业非点源瞬变电磁仪的加速潜力。 Çapture电子显微照片（ 图5），使用高清晰度的摄像头，并保存为标签图像文件格式（TIFF）。 </li></ol> 6。代表性的成果胶体银奈米粒子的合成与表征使用设置显示在图1A，成功地合成了4升的克赖顿胶体银奈米粒子。最终的胶体的特性的金黄色的颜色（ 图1B）22，23在此胶体的UV-Vis吸收光谱有一个典型的尖锐，对称的表面等离子体振子峰（SPR），在394纳米（ 图1C）。原始克莱顿胶体和最后的100-kD的滞留物的拉曼光谱中提出只有三个振动模式，即在弯曲（1640 cm -1）的对称和非对称伸缩模式的H 2 O（3245厘米-1和3390厘米-1 ，分别）（ 图1D）。/ P&gt; TFU胶体AGNPS 的TFU安装和3步TFU过程的示意图在图2和图3所示，分别50纳米的过滤器（460厘米2）在步骤1中，利用尺寸选择，以除去银奈米粒子和AgNP-聚集体的50-nm的直径和较大的从原来的胶体（约100毫升的50-nm的滞留物）。这一步也从原来的胶体4 L，3.9 L的50纳米滤液的体积小，减少陪同。无反洗或流量中断步骤使用。量最大的降低（ 即 ，除去水），得到在步骤2中，当通过一个100-kD的过滤器（200厘米2）的50-nm的滤液，随后运行。将所得的100-kD的滞留物的总体积为50ml。大部分的合成副产物和过量的试剂，在此步骤中，通过水溶剂（3.850毫升100 kD的滤液的）销。另外，AgNP浓度由ADDIT实现离子先前报道的方法的第三过滤工序19在该步骤3中，一个100-kD的滤波器的一个较小的表面区域（20厘米2）100-kD的滞留体积减少至4.0毫升。 TEM测量表明，这最后的100 kD的滞留物主要是由直径为1-20纳米的的弱旅汇总AGNPS的。 ICP-OES和TEM的胶体AGNPS 从八个标准（0，3，7，10，15，25，50，和100μgL-1）的线性回归校准曲线（ 图4）构建银。在每一个四个有代表性的胶体样品的银的量，然后确定从ICP-OES校准曲线通过外推法：原始胶体（15.2 ppm的， 图3A），50-nm的滤液（14.1 ppm的， 图3B），第一个100 - kD的滞留物（683.1 ppm的， 图3C）和最后的100-kD的滞留物（586.5 ppm的， 图3D）。15.2 ppm的实际产量是非常接近的克莱顿反应的典型的理论产率的15.4 ppm的。极端的银奈米粒子浓度（4毫升586.5 PPM），反映了一个戏剧性的变化，颜色从原来的胶体金黄色到深褐色的最后100 kD的滞留物（ 图3，插图的小瓶的图片）。被发现的过滤器的质量是至关重要的的TFU过程，特别是步骤1。最终阻滞浓度为446.5 ppm的9,333.3百万分之根据过滤器的条件（大量使用与全新）。如果膜毛孔成为损害，还可以具有的直径小于50纳米的银奈米粒子将保留，并随后将降低整体收集在滤液中的银奈米粒子量。过滤过程的优化，包括压力监测和适当的清洁可以提高过滤器的寿命。 代表的TEM照片<html>原始克莱顿胶体和最后的100-kD的滞留物（步骤3）在图5A和图5C所示，分别。在他们的非聚集状态下，AGNPS的浅灰色背景显示为黑色的圆形区域。大约有800银奈米粒子中确定的每一个，两个样品的TEM显微照片，使用Image J软件进行分析。一个粒子定义的一个完整的和封闭的周边。一个面积阈值定为1.0纳米2的TEM照片的分辨率。的的AgNP数和面积数据，然后导出到Microsoft Excel的AgNP直径推算。在原来的胶体和最后的100-kD的滞留物的的平均AgNP直径测定，分别为9.3 nm和11.1纳米。的银奈米粒子的直径测量，然后将其导出到来源地8.0软件对每个样品和大小的TEM直方图构建（ 图5B和图5D）。 “SRC =”/ files/ftp_upload/4167/4167fig1.jpg“/&gt; 图1：A）合成的设置，B）的典型色泽，C），紫外-可见吸收光谱，和D）的克赖顿胶体银奈米粒子的拉曼光谱。 <img alt="图2" src="/files/ftp_upload/4167/4167fig2.jpg" /> 图2。TFU实验装置A）步骤1和步骤2：I）水库克赖顿胶体银奈米粒子。 II）水库滤液收集。 III）Y-交界处的管道。 IV）蠕动泵头。 V）为50纳米或100 kD的南部库洛过滤器。B）步骤3：I）水库的克赖顿胶体银奈米粒子。 II）水库滤液收集。 III）100 kD的微：库洛过滤器。 <img alt="图3" src="/files/ftp_upload/4167/4167fig3.jpg" /> 图3的流程图描绘的TFU过程。蓝色阴影框马克银奈米粒子的胶体悬浮液中以供进一步分析收集。瓶photographs表明甲）原本的胶体批处理，B）的 50-nm的滤液收集处理后的原始胶体通过50纳米的过滤器（460厘米2），C）的头100-kD的滞留物后得到的体积减少使用100-kD的南比利库洛过滤器（200厘米2）， 和D）最后100-kD的滞留物从使用100 kD的微：库洛过滤器（20厘米2）的体积减小所导致。 100 kD的滤液看起来像水。 <img alt="图4" src="/files/ftp_upload/4167/4167fig4.jpg" /> 图4。ICP-OES线性的校准采用八银标准：0，3，7，10，15，25，50，和100μg·L-1。 <img alt="图5" src="/files/ftp_upload/4167/4167fig5.jpg" /> A）原克赖顿农业非点源污染和 C的TEM照片如图5所示。）最后的100 kD的滞留物（比例尺为为100nm）。 TEM建造的规模直方图分析约800 AGNPS B）原克赖顿AGNPS，D）最后100 kD的截留的。 图5B中的插图显示了展开的的41-75纳米尺寸范围内作比较。 <a href="https://www.jove.com/files/ftp_upload/4167/4167fig5large.jpg" target="_blank">点击此处查看大图</a> 。

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没有利益冲突的声明。

胶体银奈米粒子的紫外 - 可见吸收光谱法和拉曼光谱这是众所周知的，数量的表面等离子体共振一种胶体的吸收光谱的峰的对称性的AGNPS增加而减小。此外，AgNP聚合导致的外观更广泛或红移的峰。25,26的存在下，在394 nm处的一个单一的，尖锐的和对称的SPR峰值表示中度聚集和粒度分布的小的，球形的银奈米粒子。 前和超滤后的胶体样品的纯度，证明...

<table border="1"><tr><td>硝酸银（AgNO 3的 ） </td><td> Acros Organics公司</td><td> CAS：7761-88-8 </td><td></td></tr><tr><td>硼氢化钠（加入NaBH 4） </td><td> Acros Organics公司</td><td> CAS：16940-66-2 </td><td></td></tr><tr><td>硝酸（HNO 3，远舰） </td><td>默飞世尔科技公司</td><td> A467-1 </td><td> ICP分析痕量金属级</td></tr><tr><td> 10,000微克毫升-1银标准，EnviroConcentrate </td><td>超科学</td><td>美IAA-047 </td><td></td></tr><tr><td> KrosFlo研究（二） 我切向流过滤系统</td><td>频谱检测实验室公司</td><td> <a href="http://www.spectrumlabs.com/pshop/RequestPrice/index.html?LiFrom=%2Ffiltration%2FKR2SystemO.html;FrName=Research+IIi+System;Pn=SYR2-U20-01N;" target="_blank">SYR2-U20-01N</a> </td><td></td></tr><tr><td> 0.05微米PS（0.5毫米）460厘米</td><td>频谱检测实验室公司</td><td> <a href="http://www.spectrumlabs.com/filtration/MidiKros.html?Pn=X30S-900-02N;" target="_blank">X30S-900-02N</a> </td><td></td></tr><tr><td>南部100 kD的PS 200平方厘米 </td><td>频谱检测实验室公司</td><td> X3-100S-901-02N </td><td></td></tr><tr><td> micro100 kD的PS 20 cm 2的 </td><td>频谱检测实验室公司</td><td> X1AB-300-10N </td><td></td></tr><tr><td> MASTERFLEX的C-Flex管L / S尺寸17 </td><td>科尔 - 帕默仪器有限公司</td><td> 06424-17 </td><td></td></tr><tr><td> MASTERFLEX的C-Flex管L / S尺寸14 </td><td>科尔 - 帕默仪器有限公司</td><td> 06424-14 </td><td></td></tr><tr><td>卡里50 UV-VIS-NIR分光光度计</td><td>瓦里安公司</td><td></td><td></td></tr><tr><td>的LabRam HR 800系统</td><td> HORIBA Jobin Yvon公司公司</td><td></td><td></td></tr><tr><td>瓦里安710ES ICP-OES </td><td>瓦里安公司</td><td></td><td></td></tr></table> 表1中。具体的试剂和设备。

tangential flow ultrafiltration: a  &ldquo;green&rdquo; method for the size selection and concentration of colloidal silver nanoparticles

如今，AGNPS被广泛用于制造消费类产品，水消毒剂，2疗法，1，3和生物医学设备由于其强大的抗菌性能。3-6这些纳米粒子的应用，我们强烈的AgNP大小和聚集状态的影响。存在许多挑战在受控制造7和未官能化的，同质的银奈米粒子从化学侵蚀性的上限/稳定剂或有机溶剂是免费的基于大小的隔离4,8 7-13摆脱限制成本高的毒性试剂，或减少的AgNP合成或分离方法（ 例如 ，离心，尺寸相关的溶解度，尺寸排阻色谱法，等）的效率。10,14-18为了克服这一点，我们最近发现，TFU允许更大的控制权的大小，浓度和（300克赖顿农业非点源污染的聚集态毫升15.3微克毫升-1 198.7微克毫升-1）下降到10毫升的19比传统的隔离方法，如超速离心。 TFU是常用的重量为基础的分离的蛋白质，病毒和细胞的再循环方法。20,21简单地说，该液体样品通过中空纤维膜的孔径范围从1,000 kD的10 kD的一系列。较小的悬浮或溶解的样品中的成分将通过多孔隔板与溶剂（滤液）一起，而较大的成分被保留（滞留）。 TFU可能被认为是“绿色”的方法，因为它既不损害样品，也不需要额外的溶剂，以消除有毒过量的试剂和副产品。此外，TFU可能被应用到了大量的各种纳米粒子，作为疏水性和亲水性两种过滤器是可用的。 本研究的两个主要目标是：1）说明实验方面的的TFU方法通过一个被邀请的视频体验和2）表现出较大的胶体纳米粒子的体积和更小的体积，截留TFU方法的可行性。首先，unfuctionalized银奈米粒子（4升，15.2微克毫升-1）的合成通过用NaBH 4还原的AgNO 3使用既定克莱顿方法22,23。 AgNP多分散性，然后最小化通过使用50纳米的过滤器（460厘米2），以除去银奈米粒子和AgNP-聚集体由两个100 kD的滤波器（200厘米2和20厘米2）大于50nm，接着的3个步骤TFU集中银奈米粒子。有代表性的样品进行了表征，利用透射电子显微镜，紫外 - 可见吸收光谱法，拉曼光谱法，电感耦合等离子体发射光谱。最终阻滞高度集中（4毫升，8,539.9微克毫升-1），但卑微的汇总和均匀银奈米粒子为1-20纳米的直径。这对应于约62％的银浓度收率。

Watch this Scientific Journal Video about Tangential Flow Ultrafiltration: A  â€œGreenâ€ Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles at JoVE.com

Tangential Flow Ultrafiltration: A  &amp;ldquo;Green&amp;rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

切向流超滤（TFU），是一个循环的生物样品的重量为基础的分离方法。 TFU适于大小选择（1-20 nm直径）和高度集中大量的多分散性的银纳米颗粒（4 L的15.2微克毫升<sup&gt; -1</sup8,539.9微克毫升下降到4毫升<sup&gt; -1</sup&gt;），以最小的聚集。

切向流超：A胶体银纳米粒子的大小选择与集中“绿色”方法

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

tangential-flow-ultrafiltration-green-method-for-size-selection

Research

JoVE Journal

Biology

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

17.7K 次观看.  Wright State University. 切向流超滤（TFU），是一个循环的生物样品的重量为基础的分离方法。 TFU适于大小选择（1-20 nm直径）和高度集中大量的多分散性的银纳米颗粒（4 L的15.2微克毫升 -1

视频: Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

通过颅窗口在新皮层的血流量为双光子成像方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

科研

生物学

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

一个微流体装置量化细菌趋稳定的浓度梯度

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

一个协议，用于噬菌体展示和亲和选择以重组蛋白诱饵

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

对肌红蛋白鲸类检测基于金免疫层析胶体试纸条的研制

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...