这项工作是支持批卫生研究院（R01 HL086699，HL086699 01A2S1，1S10RR027327 - 01）的MM。我们的文章部分是由卡尔蔡司显微成像公司的支持。

活性氧可测氧化敏感染料（1＆2）使用或使用质粒传感器（3＆4），共聚焦显微镜的活细胞。 1。 J774细胞胞质ROS的可视化<ol><li>生长在玻璃底部35 mm培养皿（哈佛大学仪器），48小时J774.1小鼠单核细胞衍生的巨噬细胞（10 6细胞/ ml） </li><li>挑战细胞TLR激动剂（2微克/毫升，磷壁酸锂TLR2激动剂;10μg/ml，保利（I：C）- TLR3激动剂; 1微克/ ml的LPS - TLR4的激动剂）为6小时37 ° C 注：类似条件下处理的巨噬细胞共培养模型的Ca 2 +动员。 </li><li>添加氧化敏感的染料H 2 DCF - DA（Invitrogen公司）给ECM终浓度为10μm（外介质：121毫米氯化钠，碳酸氢钠3，5毫米10毫米钠HEPES，氯化钾4.7毫米，1.2毫米的KH 2 PO 4，硫酸镁 1.2毫米，2毫米氯化钙2，10葡萄糖和2.0％右旋糖酐，pH值7.4;取得所有自Sigma）含2％BSA的菜肴和共聚焦显微镜下可视化的前20分钟的孵育细胞。 </li><li>染料加载后，可视化放在一个适当的成像系统上的一个温度控制阶段的细胞。我们使用卡尔蔡司LSM 510 META激光共聚焦成像系统，氩离子激光源加上488 DCF的荧光纳米40X油目标 13（图1A和D）的激发。 </li><li>分析图像使用ImageJ软件（1.44），从国立卫生研究院提供免费软件（Rasband，WS，ImageJ，美国，马里兰州贝塞斯达，美国国家卫生研究院<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij /</a> 1997-2009）。 </li></ol> 2。 J774细胞在可视化mROS <ol><li>生长在玻璃底部35 mm培养皿（哈佛大学仪器），48小时J774.1小鼠单核细胞衍生的巨噬细胞（10 6细胞/ ml） </li><li>挑战的TLR配体细胞诱导mROS生产（2微克/毫升，锂，磷壁酸- TLR2的10μg/ml，保利（I：C）- TLR3的; 1微克/ ml的LPS - TLR4）的为6 h，在37 °; C。 </li><li>线粒体超氧指标MitoSOX红（Invitrogen公司）给ECM终浓度的5微米，含2％BSA的菜肴和培育细胞30分钟，在37 ° C至允许装载MitoSOX红。 </li><li>染料加载后，可视化细胞上的一个温度控制的卡尔蔡司LSM 510元共聚焦成像系统与氩离子激光器MitoSOX红色荧光，使用40倍的石油目标（图1B和E）的561纳米的激发源夫妇阶段。 </li><li>对于控制实验后，染料加载添加PBS或DMSO（阴性对照）或2μm的抗霉素（线粒体超氧发生器阳性对照）之前向细胞图像样本在2.4（图1C）所述。 </li><li>使用ImageJ软件（1.44），分析图像。 </li></ol> 3。 Visualizati胞浆内活性氧在稳定的Hyper - CYTO MPMVECs <ol><li>我们使用pHyPer CYTO（Evrogen）哺乳动物表达载体编码一个荧光传感器超定位在细胞质和专门感官submicromolar浓度的H 2 O 2月 14日 。 </li><li>转染小鼠肺微血管内皮细胞（MPMVECs）pHyPer - CYTO使用或者电击或转染试剂的载体。通过使用BioRad公司基因脉冲electroporator系统（250V; 500μF），电击，我们使用10 × 10 6细胞的pHyPer CYTO10μg染。辅以10％胎牛血清，非必需氨基酸，内皮细胞生长补充和抗生素的DMEM培养基培养MPMVECs。 注意：以下转染细胞，低密度板，以方便菌落生长。 </li><li>用500微克/ ml的G418，转染后的48小时内完成文化培养基介质。屏幕克隆H的比例最高<html> yPer阳性细胞，并通过超阳性克隆，以增加超阳性细胞（图2）。 注：我们采用连续稀释的细胞，单细胞克隆选择和稳定细胞高表达的Hyp​​er屏幕。 </li><li>生长在0.2％明胶涂层盖玻片（80％汇合）稳定的Hyper - CYTO MPMVECs。第二天挑战细胞的TLR配体（;10μg/ml，保利（2微克/毫升，脂磷壁酸TLR2的：C）- TLR3的; 1μg/ ml的LPS - TLR4的）为6小时，在37 ° C。未经处理的细胞作为控制。 </li><li>治疗后，安装到Attofluor细胞室（Invitrogen公司）的盖玻片。新增预1毫升回暖汉克斯平衡盐溶液（HBSS）总商会和地方商会卡尔蔡司LSM 510 META激光共聚焦成像系统上的一个温度控制阶段。可视化和图像在使用40X的石油目标（图3A和D）的488 nm的激发荧光的变化。 </li><li>分析图像使用ImageJ软件（1.44）（见第6节）。 </li></ol>书名“&gt; 4。线粒体ROS的可视化稳定的Hyper - dMito MPMVECs <ol><li>我们使用pHyPer dMito（Evrogen）一个哺乳动物表达载体编码线粒体定位超定位在线粒体和专门感官submicromolar浓度的H 2 O 2。 </li><li>转染与pHyPer dMito MPMVECs和产生稳定的Hyper - dMito MPMVECs 3.2-3.3节所述的步骤。 </li><li>生长在0.2％明胶涂层盖玻片（80％汇合）稳定的Hyper - dMito MPMVECs。 </li><li>第二天挑战细胞的TLR配体（;10μg/ml，保利（2微克/毫升，脂磷壁酸TLR2的：C）- TLR3的; 1μg/ ml的LPS - TLR4的）为6小时，在37 ° C。未经处理的细胞作为阴性对照。添加2μm的抗霉素（线粒体超氧诱导阳性对照）之前，以可视化的一个样本。 </li><li>治疗后，安装到Attofluor细胞室（Invitrogen公司）的盖玻片。新增预热汉克斯平衡盐溶液（HBSS）1ml到​​室地方室温度卡尔蔡司LSM 510 META激光共聚焦成像系统的控制阶段。可视化和图像使用40倍的石油目标（图3B，C和E）的488 nm的激发荧光的变化。 </li><li>分析图像使用ImageJ软件（1.44）（见第6节）。 </li></ol> 5。共培养模型内皮细胞旁分泌衍生活性氧的可视化触发的Ca 2 +信号对于可视化和测量的[Ca 2 +] i的变化，我们使用的Ca 2 +敏感的荧光染料荧光凌晨4点（Invitrogen公司） <ol><li>生长在0.2％明胶涂层25毫米盖玻片（80％汇合）MPMVECs。 </li><li>盖玻片为30分钟在ECM含有5微米荧光凌晨4点（Invitrogen公司）终浓度为2％BSA和0.003％嵌段酸在室温孵育成长的细胞，让加载荧光凌晨4点。加载后，用细胞实验成像解决方案（ECM的含0。25％BSA），其中包含sulfinpyrazone以最大限度地减少染料损失。 </li><li> Attofluor细胞室（Invitrogen公司）搬上荧光4，摩盖玻片后。加入800μl预热预实验成像解决方案，会议厅和卡尔蔡司LSM 510 META激光共聚焦成像系统上的一个温度控制阶段。 </li><li>在一个单独的管标签nonactivated或激活巨噬细胞中分离从野生类型或gp91 PHOX - / -小鼠的细胞跟踪红色CMTPX（500ng/ml）（Invitrogen公司）在ECM中含2％BSA（LPS 6小时处理）和0.003％的嵌段共聚物酸。与实验成像解决方案200μL洗涤和重悬细胞。 </li><li>添加标记巨噬细胞上的荧光凌晨4点加载在商会大会MPMVECs。 </li><li>记录的绿色和红色荧光，每3S为10-20分钟，用氩离子激光源与激励使用卡尔蔡司LSM510 META共焦成像系统在488和561 nm的荧光凌晨4点和细胞跟踪红色荧光respectivelY（图4交流电）。 注：另外，J774.1细胞系可用于引出MPMVECs的Ca 2 +动员。 </li><li>分析荧光- 4荧光强度的变化，使用ImageJ软件（1.44）（见第6节）。 </li></ol> 6。使用ImageJ软件进行数据分析转让共聚焦系统的数据分析。LSM格式的文​​件获得的图像。 2009年和禅ZEN的2010软件与LSM 510元共聚焦显微镜或LSM提供710多光子共聚焦显微镜分别保存。LSM格式的文​​件。我们建议使用这种原生图像分析LSM文件，为保留原有的品质。这种文件格式可以直接打开图像分析程序中的图像J.以下步骤提供了一个例子单幅图像定量分析。无需额外的插件需要此​​数据分析。 <ol><li>打开“。LSM或TIFF文件中使用”文件“&gt;”打开“程序图像J </li><li>点击“选项卡图像&gt;二氧化碳LOR&gt;分割渠道和分裂的绿色和DIC渠道。从绿色通道，以供后续分析使用的图像。 </li><li>双击，选择“椭圆刷选择”标签的像素值设置为20。 </li><li>对于超的细胞图像，点击一个单元格定义测量的圆形区域。然后按“M”键来衡量所选区域的强度。值保留在一个窗口中的新成果。 </li><li>通过举办不同的细胞“Shift”键单击以选中的另一个领域，按“M”键下一步。强度记录在结果窗口。重复步骤4-5 50种不同的细胞。 </li><li>对于Hyper D -美图图像，像素值设置至10日在椭圆刷选择。感兴趣区域（ROI）以手动方式追查，包括特定的细胞线粒体。 </li><li>使用FreeHand，跟踪区域周围的线粒体，然后按“M”。重复步骤6-7 50种不同的细胞。 </li><li>复制的我<html>从结果窗口，并粘贴在一个Excel工作表或任何软件绘制的结果值。我们使用要么SigmaPlot或Graphpad棱镜情节作为图形数据。 </li></ol> 7。代表性的成果图1显示胞质和线粒体ROS产生的TLR配体所面临的挑战时。代表共聚焦图像显示DCF（胞浆）和MitoSOX红（线粒体）的刺激后6小时的荧光增加。荧光变化正常化，并表示作为倍的变化。 图2显示了稳定的Hyper -的细胞和内皮细胞超dMito代。小鼠内皮细胞转染与pHyPer CYTO或通过电穿孔pHyPer dMito质粒结构。 G418两个星期的稳定表达质粒的细胞被选中。选择后，稀释后的细胞接种于96孔板。单个菌落进行分离和同质化的表达成像。 e_content“&gt;图3显示了内皮细胞的细胞质和线粒体H 2 O 2水平的测量。代表共聚焦图像显示增加的Hyper -的细胞（胞浆）和Hyper - dMito（线粒体）的刺激后6小时的荧光，荧光强度的变化正常化并表示作为倍改变。 图4显示了评估的巨噬细胞源性活性氧诱导的内皮细胞胞浆内的Ca 2 +动员。 LPS刺激的巨噬细胞，分别加入到肺微血管内皮细胞（PMVEC）以前已经加载与细胞内 Ca 2 +（CA 2 +] i的）指示剂荧光4。应用野生型LPS激活巨噬细胞诱发的[Ca 2 +] i的上升在PMVECs功能NADPH氧化酶基因敲除减毒（gp91 PHOX - / - ）。 <img src="/files/ftp_upload/3511/3511fig1.jpg" alt="图1" /> 图1。 Visualizati巨噬细胞产生的细胞活性氧和线粒体超氧。 J774.1细胞TLR2，3和4配体与挑战（2微克/毫升，脂磷壁酸- TLR2的;10μg/ml，保利（I：C）- TLR3的; 1μg/ ml的LPS - TLR4的6 h后）。 37 ° C。 （一）治疗后细胞装入的细胞活性氧指标（H 2 DCF - DA的绿色荧光）或（B）线粒体超氧指标（MitoSOX红色，红色荧光） 。 （三）抗霉素A作为阳性对照线粒体ROS产生。使用共聚焦显微镜，荧光强度的变化进行了评估。 （四）及（E）的平均荧光变化的定量分析。 <img src="/files/ftp_upload/3511/3511fig2.jpg" alt="图2" /> 图2。产生的内皮细胞的胞质和线粒体ROS的指标稳定表达的示意图 。 <img src="/files/ftp_upload/3511/3511fig3.jpg" alt="图3" /> 图3。 V内皮细胞的细胞质和线粒体H 2 O 2水平isualization MPMVECs稳定表达（一）超细胞质或（B）的Hyper - dMito，与TLR2的，3和4配体（2微克/毫升，脂磷壁酸- TLR2的挑战;10μg/毫升，保利（I：C）- TLR3的; 1微克/ ml的LPS - TLR4的）为6小时，在37 ° C。 （三）抗霉素A作为阳性对照线粒体ROS产生。治疗后使用共聚焦显微镜，荧光强度的变化进行了评估。 （四）及（E）的平均荧光变化的定量分析。 <img src="/files/ftp_upload/3511/3511fig4.jpg" alt="图4" /> 图4。巨噬细胞源性活性氧引起内皮细胞受损的Ca 2 +动员。 （一）6小时1μg/ ml的LPS示意图（二）从WT和gp91phox null小鼠（杰克逊实验室）新鲜分离的小鼠巨噬细胞被激活内皮细胞/巨噬细胞共培养模型的研究Macrophages标记细胞跟踪Red（红色）和添加到荧光4加载PMVECs（绿色），以评估旁分泌Ø 2• -的信号（三 ）从WT小鼠中分离出的内毒素激活巨噬细胞，引发大和迅速的Ca 2 +动员从gp91phox null小鼠的巨噬细胞相比PMVECs。巨噬细胞活化的验证先前已被证明。

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没有利益冲突的声明。

这里介绍的方法，允许快速和定量测量活性氧，在活细胞使用的氧化敏感的染料或质粒传感器。的Toll样受体激动剂（Toll样受体）的化合物，通过TLRs的细胞表面上的刺激细胞和触发下游信号通路15。在我们的协议中，我们使用三种不同的TLR激动剂，即，锂，磷壁酸TLR2激动剂;聚（I：C）。TLR3激动剂; LPS - TLR4的激动剂据报道，作为一个模型系统诱导ROS证明活性氧测量。使用氧化剂敏感染料的?...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> Attofluor细胞室</td><td> Invitrogen公司</td><td> A7816 </td></tr><tr><td>抗霉素A </td><td>西格玛爱秩序</td><td> A8674 </td></tr><tr><td> DMEM低血糖液</td><td> Invitrogen公司</td><td> 10567-014 </td></tr><tr><td>内皮生长因子的补充（ECGS） </td><td>上州</td><td> 02-102 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 12662011 </td></tr><tr><td> G418 </td><td> Invitrogen公司</td><td> 10131-027 </td></tr><tr><td>明胶</td><td>西格玛爱秩序</td><td> G1393 </td></tr><tr><td> H 2 DCFDA </td><td> Invitrogen公司</td><td> D - 399 </td></tr><tr><td>脂多糖</td><td>西格玛爱秩序</td><td> L4516 </td></tr><tr><td>陆路交通管理局</td><td>西格玛爱秩序</td><td> L2515 </td></tr><tr><td> MitoSOX红</td><td> Invitrogen公司</td><td> M36008 </td></tr><tr><td> OPTI - MEM我降低血清培养基</td><td> Invitrogen公司</td><td> 51985091 </td></tr><tr><td> PEN /链球菌（10X） </td><td> Invitrogen公司</td><td> 15140163 </td></tr><tr><td> pHyPer - CYTO </td><td> Evrogen </td><td> FP941 </td></tr><tr><td> pHyPer - dMito </td><td> Evrogen </td><td> FP942 </td></tr><tr><td>保利（I：C） </td><td>西格玛爱秩序</td><td> P0913 </td></tr><tr><td> Prism软件5.0 </td><td> GraphPad软件公司</td><td></td></tr><tr><td> SigmaPlot 11.0 </td><td> SYSTAT软件公司</td><td></td></tr><tr><td>胰蛋白酶EDTA（10X） </td><td> Invitrogen公司</td><td> 15400054 </td></tr><tr><td> T - 25培养瓶</td><td>康宁</td><td> 430639 </td></tr><tr><td> T - 75培养瓶</td><td>屋宇署猎鹰</td><td> 353136 </td></tr><tr><td> 96孔TC微型孔板</td><td>屋宇署猎鹰</td><td> 353072 </td></tr><tr><td>禅2009软件</td><td>卡尔蔡司510元共聚焦显微镜</td><td></td></tr></tbody></table>

visualization of vascular ca2+ signaling triggered by paracrine derived ros

氧化应激有牵连的一些包括缺血/再灌注损伤和败血症的病理条件。氧化应激的概念是指异常形成的活性氧（活性氧），其中包括 Ø 2• - ，H 2 O 2，和羟基自由基。活性氧的影响多种细胞过程，包括信号转导，细胞增殖和细胞死亡 1-6 。活性氧有可能直接损伤血管和器官的细胞，并能启动中学的化学反应和遗传变异，最终导致初始ROS介导的组织损伤的放大。放大级联，加剧了不可逆的组织损伤的一个关键组成部分是招募和循环炎性细胞的激活。在炎症，炎性细胞产生细胞因子如肿瘤坏死因子-α（TNFα）和IL - 1，激活内皮细胞（EC）和上皮细胞，并进一步增强炎症反应 7 。血管内皮功能障碍是一种急性炎症的建立功能。巨噬细胞促进血管内皮功能障碍，在炎症的机制仍不清楚。 • -和各种促炎性细胞因子，从而触发在相邻细胞的病理信号 8激活巨噬细胞外释放的O 2的结果。 NADPH氧化酶是ROS的主要的主要来源，在大多数的细胞类型。最近，它是我们和其他9,10所示，由NADPH氧化酶产生的活性氧诱导线粒体ROS在许多病理生理条件的生产。因此，测量线粒体ROS产生同样重要的是，除了测量胞质ROS的。巨噬细胞产生ROS的黄素酶NADPH氧化酶在炎症反应中起着主要作用。一旦被激活，吞噬细胞NADPH氧化酶产生丰富的O 2• - 11,12在宿主防御机制的重要。虽然旁分泌衍生Ø 2• -扮演重要的作用，在血管疾病的发病机制，包括CA 2旁分泌ROS诱导的细胞内信号可视化 +动员仍然是假说。我们已经开发出了激活巨噬细胞作为氧源•使用模型-信号转导的一个相邻的内皮细胞。使用这个模型，我们表明，巨噬细胞源性Ø 2• -在邻近的内皮细胞钙信号。

Watch this Scientific Journal Video about Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS at JoVE.com

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

一种有效的方法获得可视化的旁分泌衍生活性氧诱导内皮细胞Ca2 +信号的见解的描述。这种方法测量旁分泌衍生活性氧引发共培养模型在血管内皮细胞内Ca2 +动员的优势。

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

visualization-vascular-ca2-signaling-triggered-paracrine-derived

Research

JoVE Journal

Biology

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

15.4K 次观看.  Temple University. 一种有效的方法获得可视化的旁分泌衍生活性氧诱导内皮细胞Ca2 +信号的见解的描述。这种方法测量旁分泌衍生活性氧引发共培养模型在血管内皮细胞内Ca2 +动员的优势。

视频: Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

技术成像钙 2 +信号在人类精子

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

科研

生物学

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI灌注作为血管可视化的方法<em&gt;墨西哥恶性肿瘤</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

高压低温电子层析成像对蓝藻 DNA 压实的可视化研究

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

视网膜低温切片、全支架和低渗隔离血管毛细血管微血管组织免疫组化的制备

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

一种研究细胞间分泌信号的近距离培养方法

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

palt1 副酶活性对髓系先天信号通路的调控

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...