这项研究是由美国国立卫生赠款AI - 52381，CA138623和肯塔基州的肺癌研究委员会和机构支持从格雷厄姆詹姆斯布朗癌症中心的支持。

方法显微镜的说明活细胞成像实验用的TE - FM系统连接到尼康的Eclipse TE300倒置显微镜落射荧光。显微镜配备加热阶段。一个凉爽的总部管理单元数字B / W CCD（罗伯科学）相机和LAMDA 10-2光学过滤器更换（萨特仪器公司）是连接到显微镜。激发和发射波长控制滤光轮和兰巴10-2滤光轮控制器控制仪器有限公司，萨特曝光时间为500 ms应该足够，以查看RFP或GFP在活细胞。 MetaMorph软件控制，硬件控制和图像采集。本研究过滤器的选择，过滤器设置S480/20x，S525/40m和S565/25x，GFP和RFP S620/60m，分别EGFP / DsRed的双分色，色度技术。该过滤器轮最多可容纳6个过滤器设置。显微镜连接之前Proscan阶段控制器操纵杆。 MetaMorph软件可以控制所有这些硬件附件。显微镜还附有Micromanipulators举行的微量。 A.测量白三烯B 4导演的树突状细胞迁移使用上面介绍的显微镜设置，我们开发了实时配体定向的树突状细胞迁移的方法。 micromanipulators（Narishige）连接到显微镜。小鼠骨髓骨髓衍生的树突状细胞对的LTB 4梯度（BMDCs）的趋化作用被记录下来。在这里，我们描述的方法准备微移液器使用微量移液器拉马，装入微量移液器的配体和设立显微镜监测细胞的定向迁移的加热阶段的趋化实验。这种方法可应用于诱导迁移以及在活细胞趋化抑制剂的影响，以测试在不同的趋化因子的功效。已在若干出版物，包括朱庇特22 21 BMDCs从鼠标的隔离。 1。准备的BMDCs <ol><li>板BMDCs盖玻片底部氟菜，让他们增长提前16小时的实验（培养后10天）。 </li><li> 3毫升的1X PBS至少2倍（加入3毫升的1X PBS板和吸缓冲区中）洗细胞。 </li><li>加入2毫升的1XPBS板块。现在这些细胞为实验做好准备。细胞保持在37 ° C培养箱前实验。 </li></ol>微量加样器的配装的筹备工作： <ol start="4"><li>修复的玻璃毛细管（玻璃标准，0.75毫米× 0.4毫米; 6“，猫＃625500，调幅系统，INC）在垂直的微量拉马（Narishige国际USA，INC）。 </li><li>温度保持在52℃，使玻璃熔化和拉开，形成锋利的刀刃提示。 </li><li>修正到微量移液器支架移液器。 </li><li>微量加样器的使用伪造微粗糙的边缘平滑的M - 900（Narishige国际USA，INC）在显微镜下观看。 </li><li>准备每个实验至少10-20移液器。注：微量，也可以从世界精密仪器（μ提示，T1801TW1F）购买。 </li><li>考虑到1毫升effendorf管所需的配体浓度（500μL100纳米，在目前的实验LTB 4），并保持溶液中的微枪头，并允许进入微量加样器（回填土）的解决方案。 </li><li>取1毫升配体和进入注射器（1毫升结核菌素注射器），慢慢地填写使用微量加样器，从顶部的微填写针（MF 28G - 5，世界精密仪器）的配体。 </li><li>将油管，紧固移液器吸液管和管针（21 G，11 / 2）1 mL注射器，这是与配体填充。 </li><li>现在，仔细地将微量移液器显微镜阶段和修复填充到微型机器人的配体。 </li><li>申请从注射器小的压力，以确保配体是慢慢从针尖释放。 </li><li>带镀不成熟BMDCs显微镜，并继续加热板（37℃）阶段。 </li><li>删除氟盘的盖子。 </li><li>使用油浸60X物镜聚焦细胞。 </li><li>小心地装入吸管使用“粗手动机器人MN - 188NE细胞附近的配体。 </li><li>聚焦使用“液压精细的微操作机器人MN - 188东北”的吸管。 </li><li>申请从注射器小的压力，以确保配体释放入菜。 </li><li>设置图像采集参数，以获得明亮的申请在每15秒（10毫秒曝光），2小时使用MetaMorph软件。 </li><li>随时监控每30分钟的公关ogression对配体的迁移。 </li><li>如果在拥挤枪头的细胞，改变针尖的位置，在该板块继续实验的迁移。 </li></ol> B.测量G蛋白偶联受体（BLT1）和胞浆蛋白（β- arrestin的）相互作用，在活细胞易位 1。质粒DNA结构的制备 DNA质粒结构的详细描述在加拉等人 （2005年）18。 2。人力BLT1 RFP和β- arrestin的- GFP的转染RBL - 2H3细胞<ol><li>保持大鼠嗜碱性Leukomia（RBL - 2H3）细胞（60％至70％汇合）在37 ° C的95％空气加湿气氛中，5％的CO 2作为生长介质的单层培养（15％胎牛血清，2补充的DMEM MM L -谷氨酰胺，100 U / mL青霉素和100μg/ mL链霉素在T75烧瓶）。 </li><li>从菜/烧瓶细胞分离使用6毫升的胰蛋白酶- EDTA（0.05％胰蛋白酶，0.53毫米EDTA）和孵化5分钟，在37 °在95％空气饱和湿度，5％的CO 2 。轻轻摇动烧瓶监察支队细胞的程度。 </li><li>添加6毫升培养基含有胰蛋白酶- EDTA 6毫升的容量瓶中，并收集细胞混合移液器上下多次。转移到细胞15毫升猎鹰管。 </li><li>使用血球计数细胞，在15 mL离心管和离心3分钟和200μL转染介质中的悬浮480克RPM（DMEM培养液，20％FBS，50毫米的HEPES）4 × 10 6细胞。如果您计划执行3转染，准备相应的细胞和转染媒体，增加细胞数量和转染介质的体积。 </li><li>准备至少为1.5μg/μL的浓度转染质粒。我们准备通过使用QIAGEN公司马克西DNA质粒试剂盒的DNA质粒。新增的个人或两个人BLT1 - RFP（25微克）和β- arrestin1 - GFP（15微克）的质粒DNA编码的无菌电泳试管，（BIO - RAD＃16552088），其中有0.4厘米的电极间隙。 </li><li>取200μL上述悬浮细胞，并含有要么hBLT1 RFP或β- arrestin1 - GFP或两者一起试管，轻轻混合，用1 mL无菌吸管。 </li><li>留在室温为10分钟以上的试管。 </li><li> Electroporate细胞基因脉冲电压250 V和500μF的能力。 </li><li>后电击让细胞停留在室温为10分钟。 </li><li>取1 mL的正常生长培养基，在试管电穿孔细胞混合。 </li><li>分配到35毫米组织玻璃底培养皿中含有2毫升的正常生长介质（氟菜，世界精密仪器），这种混合物300μL孵育1小时在95％的空气加湿的气氛在37 ° C，5％CO 2。允许细胞坚持以盘底部。 </li><li>更换后孵育1 h，用正常生长介质的媒体，让他们成长在37 ° C在95％空气饱和湿度，5％CO2培养箱中培养18-24小时。 </li></ol> 3。活细胞成像收购的图像<ol><li> 18-24小时后，用2毫升含10毫米HEPES，pH值7.55（直接加入2毫升暖媒体板和吸媒体重复2〜3次）温暖的无酚红的RPMI细胞2或3倍。添加1.8毫升，含10毫米的HEPES温暖的无酚红的RPMI。 </li><li> 30毫米的玻璃盖玻片底培养皿中含有RBL - 2H3细胞，转染hBLT1 RFP和加热显微镜阶段的β- arrestin1 - GFP（37℃）。 </li><li> 600X放大倍率，使用60 X客观尼康计划阿婆60X/1.4数值孔径油浸镜头（可在我们的显微镜系统）收集的细胞图像。 </li><li> 60 ×客观透镜放在一滴油，重点与常规明场细胞传送光，触摸盘底。 </li><li>监察转蛋白的荧光，荧光过滤器（RFP的过滤器，如果你想看到受体或GFP，如果你想看到β- arrestin1）从明亮的领域（透射光）切换到。 </li><li>选择明亮而健康的细胞，这hBLT1 - RFP的表达对细胞表面使用RFP的过滤器和捕获图像。 </li><li>然后转移到GFP的过滤器，确保在细胞质中的β- arrestin的- GFP的表达和捕捉图像。然后都是伪彩色图像，捕获时间失效前活细胞图像，以确保通过荧光流血未服用RFP和GFP之间发生。 </li><li>一旦你的手机选择，设置参数，根据你的目的使用软件（Metamorph软件在这种情况下）。 </li><li>在目前的示范，16个位图像的来收购RA分级设置为1 × 1 60 X客观尼康计划阿婆60X/1.4数值孔径油浸镜头相结合。 </li><li>收集30秒的时间间隔（如果易位/相互作用太快，一方面可以减少的时间间隔）MetaMorph软件控制这些使用滤光轮同时RFP和GFP荧光的荧光图像。 </li><li> RFP和GFP，相机的曝光时间设置为1000毫秒。 （根据RFP和GFP的荧光强度，设置特定的曝光时间）。 </li><li>没有加入配体，它为0的时间点，首先收集1分钟的图像。 </li><li> 1分钟后，而不会干扰盘/或单元格的位置，添加配体的200μL（股票从10微米至终浓度为1μm）。 </li><li> 60分钟后，加入配体荧光图像采集，存储为TIFF图像文件名的顺序增加。 </li><li>所有这些文件都可以作为与使用该软件的时间邮票的个人/组合荧光图像的堆栈文件。 </li></ol>图像和定量荧光蛋白质和本地化的过程<ol><li> RFP和GFP荧光过滤器与普通介质（无细胞）的图像采集，并保存为背景图像。使用这些图像中减去从上面获得的实际数据图像。 </li><li> RFP和GFP图像单独准备的图像的堆栈。 </li><li>选择/确定各地区（表面VS胞质）来衡量RFP和GFP的荧光强度（BLT1和β- arrestin的易位） </li><li>剧情作为时间的函数量的荧光强度。该图将提供有关的特定分子的易位动力学的信息。 </li></ol>代表性的成果 A.测量白三烯 B 4导演的树突状细胞迁移在本议定书中所描述的方法使用，以确定对树突状细胞白三烯 B 4（图1和附视频 1）21的迁移。我们可以运用类似的方法，以确定细胞趋化的能力，特别是在活细胞配体。 <img src="/files/ftp_upload/2315/2315fig1.jpg" alt="图1" /> 图1。小鼠骨髓衍生树突状细胞的迁移，对100纳米的 LTB 4。 视频1。活细胞迁移对LTB 4 BMDCs。 <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Boddulrui_Video1.AVI">点击这里看录像</a> 。 B.测量G蛋白偶联受体（BLT1）和胞浆蛋白（β- arrestin的）相互作用，在活细胞易位此过程完成后，可以得到以下信息在G蛋白偶联受体信号事件。 <ol class="lroman"><li> GPCR和胞浆蛋白和细胞核的定位（图2）。 </li><li>配体诱导的相互作用的表面G蛋白偶联受体（在这种情况下BLT1）与胞浆蛋白（β- arrestin的在这种情况下）（图3）。 </li><li>其次是膜受体的内化和β- arrestin的易位动力学的内化受体（图4）的（ 贝特贾拉等 。2005）18 。 </li><li>人能确定在活细胞配体依赖的受体激活状态（附视频2），并确定关键的图案，或在受体激活的（ 贝特贾拉等。2005 ）18的进程。 </li></ol><img src="/files/ftp_upload/2315/2315fig2.jpg" alt="图2" /> 图2。BLT1 - RFP（一），β- arrestin的GFP（二），pNuc - CFP（三）到RBL - 2H3细胞中的表达。颜色相结合的图像显示面板D. <img src="/files/ftp_upload/2315/2315fig3.jpg" alt="图3" /> 图3配体受体和β- arrestin的诱导易位。在细胞内的荧光强度的线扫描显示。 <img src="/files/ftp_upload/2315/2315fig4.jpg" alt="图4" /> 图4。动力学后，除1微米的LTB 4受体的内化和β- arrestin的易位。的荧光强度进行测量的时间在膜和细胞的胞质地点的功能。 视频2 BLT1 - RFP和β- arrestin的- GFP的表达后，除了1微米的LTB 4的细胞的实时<a href="https://www.jove.com/files/ftp_upload/2315/Jala and Bodduluri_Video2.AVI">成像。点击这里</a>观看视频

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H., Del Prete, A., Bock, C. B., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+leukotriene+B4+receptors+BLT1+and+BLT2:+a+critical+role+for+BLT1+in+collagen-induced+arthritis+in+mice.">Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice.</a> J Immunol. 176, 6254-6261 (2006).</li><li>Kim, N. D., Chou, R. C., Seung, E., Tager, A. M., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+requirement+for+the+leukotriene+B4+receptor+BLT1+for+neutrophil+recruitment+in+inflammatory+arthritis.">A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis.</a> J Exp Med. 203, 829-835 (2006).</li><li>Jala, V. R., Shao, W. H., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-independent+beta-arrestin+translocation+and+internalization+of+leukotriene+B4+receptors.">Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors.</a> J Biol Chem. 280, 4880-4887 (2005).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+G+protein-coupled+receptor+signaling+in+live+cells.">Real-time analysis of G protein-coupled receptor signaling in live cells.</a> Methods Mol Biol. 332, 159-165 (2006).</li><li>Del Prete, A., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+and+adaptive+immune+response+by+leukotriene+B4+receptors:+a+role+for+LTB4+in+up-regulation+of+CCR7+expression+and+function.">Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function.</a> Blood. 109, 626-631 (2007).</li><li>Salogni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+A+induces+dendritic+cell+migration+through+the+polarized+release+of+CXC+chemokine+ligands+12+and+14.">Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.</a> Blood. 113, 5848-5856 (2009).</li><li>Boudreau, J., Koshy, S., Cummings, D., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+myeloid+dendritic+cells+from+bone+marrow+precursors.">Culture of myeloid dendritic cells from bone marrow precursors.</a> J Vis Exp. , (2008).</li></ol>

活细胞成像是一个强大的工具，表现出特定的蛋白质的功能和互动，因为他们在实时发生。在这个手稿中描述的方法，清楚地表明，LTB 4能诱导树突状细胞的快速迁移。这些方法不仅扩大LTB 4个功能多样的细胞类型方面，它们允许类似的方法被应用到其他各种趋化因子和测试他们在不同的白细胞分人口趋化剂的功效。在这个系统中的荧光成像，可以实时监测的信号事件。此外，这?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

属于G蛋白偶联受体（GPCRs）的7个跨膜蛋白家族介导的细胞外信号传导到细胞内的反应。 GPCRs的控制多种生物学功能，如趋化，细胞内钙释放，基因调控配体依赖性通过异三聚体G蛋白的 1-2 。配体结合诱导了一系列构象变化，导致激活异三聚体G蛋白调节的第二信使，如环磷酸腺苷（cAMP）的，三磷酸肌醇（IP3）和二酰基甘油（DG）的水平。激活受体配体结合伴随还发起了一系列活动，以衰减信号通过脱敏，封存和/或内部化的受体。 GPCRs的脱敏过程中发生通过受体磷酸化G蛋白受体激酶（GRKs）和β- arrestins 3随后结合。 β- arrestins是胞浆蛋白转运到细胞膜G蛋白偶联受体激活后，磷酸化的受体结合（大多数情况下）通过促 ​​进受体的内化4-6存在。 白三烯B 4（LTB 4）是一种促炎性脂质分子，从花生四烯酸途径介导其行动派生通过GPCRs的，LTB 4受体1（BLT1;高亲和力受体）和LTB 4受体2（BLT2;低亲和力受体）7-9。 LTB 4 BLT1通路已被证明是在一些炎症性疾病，包括哮喘，关节炎，动脉粥样硬化10-17的关键。当前的纸张描述发展到显示器的LTB 4诱导白细胞迁移和BLT1的相互作用与β- arrestin的，在使用活细胞显微成像技术18-19中的受体易位的方法。 骨髓C57BL / 6小鼠的树突状细胞分离和培养作为先前描述 20-21 。这些细胞在活细胞成像方法进行了测试，证明LTB 4诱导细胞迁移。人类BLT1用红色荧光蛋白（BLT1 - RFP）的标签，在C -末端与绿色荧光蛋白（β- ARR - GFP）标记的β- arrestin1了两个质粒转染大鼠嗜碱性Leukomia（RBL - 2H3）细胞株18日至19日 。这些蛋白质和本地化之间的相互作用的动力学进行了监测使用的活细胞视频显微镜。在当前纸张的方法描述了利用显微技术研究G蛋白偶联受体在活细胞的功能反应。还介绍了当前的纸张MetaMorph软件的使用，以量化的荧光强度，以确定受体的动力学和胞浆蛋白的相互作用。

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with Î²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

本文介绍的方法，以确定具体的配体的白细胞趋化反应和识别细胞表面的受体，并用活细胞成像技术的胞质蛋白质之间的相互作用。

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

real-time-imaging-leukotriene-b4-mediated-cell-migration-blt1

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

12.7K 次观看.  University of Louisville. 本文介绍的方法，以确定具体的配体的白细胞趋化反应和识别细胞表面的受体，并用活细胞成像技术的胞质蛋白质之间的相互作用。

视频: Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

实时研究全血-内皮相互作用和血栓动力学的体外微流体疾病模型

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

使用实时细胞分析监测宿主外的流感病毒存活率

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

中性粒细胞中活性氧物种的实时定量 感染脑膜性 大肠杆菌

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...