Name: in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues
Uploaded: 2017-03-21T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

The project was supported by National Research Fund OTKA (K 104045).

1.细胞培养和试剂的制备<ol><li>保持在制造商推荐的培养基中的细胞培养物，补充有10％（体积/体积）胎牛血清和抗生素（称为完全培养基）。保持细胞培养烧瓶在湿润的5％CO 2气氛中培养器在37℃。按照倒置显微镜下细胞的增殖和汇合（使用10X相衬物镜）。 </li><li>当细胞达到足够汇合，取出培养基，并用2-3毫升，无菌磷酸盐缓冲盐水（PBS）洗涤培养物。移除PBS并加入0.5毫升，无菌0.25％胰蛋白酶-EDTA溶液向细胞培养，并在37℃下孵育直至细胞分离（约10分钟）。 </li><li>暂停细胞3-4毫升无菌完全培养基停止胰蛋白酶和他们转移到无菌离心管中。离心细胞，在150×g离心，在室温（RT）下4分钟。小心弃上清，并暂停在pellet在2-3毫升无菌完全培养基。 </li><li>取出100μL的细胞悬浮液，并用100微升0.4％（M / V）台盼蓝溶液混合，以染色死细胞。负载10微升该混合物成血球和确定存活细胞倒置显微镜的数量。 </li><li>稀释细胞悬浮液，在步骤1.3制得10毫升的无菌的完全培养基的所需量，含有4×10 4个细胞/毫升。 </li><li>加入每孔细胞悬液（步骤1.5准备）的250微升（10 4个细胞/孔）到七口井第一玻璃底部的8孔显微镜载玻片的。在使用促性腺激素释放激素摄入法这张幻灯片。 </li><li>添加每孔的细胞悬浮液（在步骤1.5中制备）的250微升（10 4个细胞/孔）到第二8孔显微镜载玻片的五井。这种滑动将在的GnRH-IR表达的方法中使用。 </li><li>加1，每孔的细胞悬浮液（在步骤1.5中制备）中的溶液（4×10 4个细胞/孔）成七个逢一个12孔板的LS。该板将在GnRH的量化方法中。 </li><li>让细胞附着在两个显微镜载玻片和板。孵育他们在湿润，5％的CO 2气氛中培养器在37℃下48小时。 </li><li>孵化后检查倒置显微镜下细胞（使用10X相衬物镜）。如果细胞是健康和连接，请继续下面的步骤。 </li><li>准备从每三个偶联物18的在二甲亚砜（DMSO）的10mM的GnRH-FITC储液。 （使用这些稀释浓度:16.43微克/μL[D-赖氨酸6（FITC） -的GnRH-I，16.97微克/μL[D-赖氨酸6（FITC） -的GnRH-II和16.47微克/μL[赖氨酸8（ FITC）〕 - 的GnRH-III）中保持这些储备溶液在暗处在室温下并在几周内使用它们。 </li><li>稀释每三个10毫米的GnRH-FITC股票解决方案1.7毫升完全培养基1.7微升。撼动这些解决方案。 （帖e为10μM的GnRH-FITC处理介质。）稀释每三个10μM的GnRH-FITC处理介质的150微升1.35中完全溶液中。摇这些解决方案，以及（这些是1μM的GnRH-FITC处理介质）。 注:含有处理介质的所有的GnRH-FITC应避光和几个小时内用完。 </li><li>预热的六个的GnRH-FITC处理介质（在步骤1.12的方法制备）和4毫升完全培养基中至37℃。 </li><li>稀的5mM的荧光探针溶液3微升在3毫升PBS中（在材料清单中提到核染剂）至5微米。该溶液应避光，其保持在RT，并在几个小时内使用完。 </li></ol> 2.激光共聚焦显微镜（CLSM） <ol><li> 促性腺激素释放激素摄入法 <ol><li>采取步骤准备1.6的第一个显微镜载玻片，并吸取了来自各的七的完全培养基。加入预热完整的中介的250微升微米到第一井。使用此作为阴性对照。添加预热的GnRH-FITC的250微升处理介质（从1.13）到每个未来六井（3个孔用1μM和3与10μM）。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃5小时。 </li><li>吸取出从每个孔的培养基，并用250微升PBS洗涤该细胞。加定影溶液（10％中性缓冲福尔马林）的250微升每孔，并培育在RT滑动，10分钟。 </li><li>吸取出从每个孔定影液，并用250微升PBS洗涤该细胞。添加250微升含在步骤1.14制备成​​每孔5微米的荧光探针溶液的PBS并孵育在RT滑动，10分钟。 </li><li>吸取了从每个荧光探针解决好，并用250微升PBS仔细清洗细胞两次。添加安装介质3-4滴到每口井，终于。保持室温在黑暗的幻灯片，直到成像。 </LI&gt; </ol></li><li> 促性腺激素释放激素-IR表达法 <ol><li>取在步骤1.7制得的第二8孔显微镜载玻片和吸取出从各五个井的完全培养基。 </li><li>添加预热的完全培养基的250微升到第一井，用此作为阴性对照。加入预热的完全培养基250μL进入第二也很好，并使用此GnRH的治疗前检查的GnRH-IR表达。 </li><li>加入这三个预热10μM的GnRH-FITC处理介质的250微升到下一个三口井。使用这些井检查的GnRH-IR表达之前，用预处理的GnRH-FITC结合物的细胞。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃1小时。 </li><li>吸取出从每个将被用于检查的GnRH处理后的GnRH-IR表达并用预热完全培养基250μL的洗涤这些孔中的三个孔的处理介质。加入250μL的预热完全培养基到每个三口井的。孵育在湿润，5％的CO 2气氛中培养器滑动，在37℃1小时。 </li><li>吸取出从每五个孔的培养基，并用250微升PBS洗涤该细胞。添加250微升定影溶液导入各孔中，并孵育在RT滑动，10分钟。 </li><li>吸取出从每个孔定影液并用PBS 250微升洗细胞。添加的PBS 250微升含5％牛血清白蛋白（BSA）封闭溶液导入各孔中，并孵育在RT滑动，1小时。 </li><li>稀的GnRH-IR初级抗体的10μL的1毫升PBS中（1:100的比例）。将其保持在RT，并在几个小时内使用完。 </li><li>吸取出从每个孔除外阴性对照（第一阱）的BSA封闭溶液。洗净用250微升PBS细胞（除阴性对照好）。加入250包含步骤2.2.7制备成EAC的GnRH-IR一抗的PBS微升ħ井（除了阴性对照）。孵育所述滑动在RT，1小时，在黑暗的地方。 </li><li>稀释的Alexa 546标记的第二抗体的2.5微升在1.25毫升的PBS（1:500的比例）。 注意:此溶液应该避光，其保持在RT，并在几个小时内使用完。 </li><li>吸取出从每五个井的解决方案，并用250微升PBS洗涤该细胞。添加的PBS 250微升含在步骤2.2.9制备成每个孔的AF 546标记的第二抗体。孵育所述滑动在RT，在黑暗1小时。 </li><li>吸取出从每个孔中的溶液，并用250微升PBS洗涤该细胞。添加包含在步骤1.14制备成​​每孔5微米的荧光探针溶液的PBS 250微升，孵育滑动10分钟，在RT。 </li><li>吸取出从每个孔的荧光探针溶液，并用250微升的PBS小心洗细胞两次。添加安装介质3-4滴进每口井，后来。避光幻灯片在室温下，直到显微成像。 </li></ol></li><li> 成像 <ol><li>图像的单元倒置共聚焦激光扫描显微镜（使用63X油浸物镜） <ol><li>使用下面的激发/发射波长。 GnRH的结合物:五百十四分之四百八十八纳米，Alexa的546标记的抗体:五百四十六分之四百八十八纳米（仅使用促性腺激素释放激素-IR表达法），DRAQ5荧光探针:680分之633纳米。 </li><li>设置使用阴性对照细胞中的摄像参数。使用这些参数来图像处理的细胞（ 图3）。重新调整上在分析的开始每个显微镜载玻片成像参数。微调和出口的图像与在必要时由制造商提供的软件。 </li></ol></li></ol></li></ol> 3.荧光激活细胞分选（FACS） <ol><li> GnRH的定量方法 <ol><li>采取步骤准备1.8板，并吸取了来自EA的完全培养基七口井的通道。添加1毫升预热的完全培养基到第一井。使用此作为阴性对照。加入1毫升每个预热六个处理介质（3口井1μM和3口井，10μM）进入下一个六口井的。孵育在湿润，5％的CO 2气氛中孵化该板在37℃5小时。 </li><li>吸取出从每个孔的介质。用2毫升的PBS仔细清洗细胞两次。加的胰蛋白酶-EDTA溶液500微升到每个孔中。在37℃孵育板，直至细胞分离（约10分钟）。 </li><li>加入1毫升完全培养基到每口井停止胰蛋白酶。摇板。轻轻悬浮细胞用吸管，并从各孔转移悬浮液到FACS管。离心管，在150×g离心4分钟，在4℃。 </li><li>小心倒出上清液，用一招。添加冰冷的PBS 500微升到每个FACS管中，轻轻地重新悬浮细胞。保持冰管，直到结束的实验。 </li></ol></li><li> 分析计算 <ol><li>分析通过流式细胞术细胞。激发，使用488nm的氩激光器波长和用于检测使用530nm的波长。通过运行阴性对照细胞建立细胞仪参数的流动。使用这些参数来分析处理的细胞（ 图4A）。用制造商的软件对数据进行评估，确定平均荧光强度（MFI）值，并且计算相对的MFI值。 </li></ol></li></ol>

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The authors have nothing to disclose.

本文所述实验中使用的选择性标记GnRH类似物进行筛选粘附 细胞培养体外 。共聚焦显微镜和流式细胞仪方法适合于跟踪和量化以时间和浓度依赖的方式，这些的GnRH-FITC缀合物的细胞摄取。这些实验有以下关键步骤:1）保持无菌的，健康的细胞培养物; 2）的GnRH肽必须是高质量的; 3）合理的浓度和孵育时间必须遵循; 4）一种治疗和洗涤步骤; 5）以及调整仪器设置。 ?...

<html>基于肽靶向给药系统已成为癌症治疗中一个快速显影和希望的领域，在过去几年中1,2。人促性腺激素释放激素受体I型（GnRH的-IR）是主要位于脑垂体，但也存在于其他几个它们负责自我繁殖3的组织。的GnRH-IR也表示在许多癌组织中，相关或无关的生殖系统4,5。促性腺激素释放激素-IR的几种恶性肿瘤细胞高表达与正常组织相比，提供了有针对性的治疗5,6的机会。 许多促性腺激素释放激素（GnRH）类似物已在过去的几十年中，它可以用来作为靶向部分开发F"&gt; 7，8，9，这些肽是能够提供以高选择性抗癌剂为恶性肿瘤细胞，其过量表达的GnRH-IR 6。几种的GnRH偶联的抗肿瘤药物具有更高的选择性和更高的效率比相应的未缀合游离药物已经报道了7，8，9。 有关的GnRH肽及其受体以前的出版物报道，GnRH的红外可以假设具有不同的选择性GnRH类似物10的各种构象。高度可变的GnRH-IR已复杂和各种信号通路被赋予了对他们的自然和人工配体11个不同的活动。这些事实使得基于促性腺激素释放激素系统的调查具有挑战性。另一方面，在<html> Ÿ具有前途的治疗潜力。用放射性标记的GnRH肽几个实验以前报道12， 第13， 第 14，第 15，但实验，其中使用荧光标记的GnRH类似物仍然有限。而放射性标记具有高灵敏度，荧光标记有几个其他的优点，例如容易处理，并用不同的荧光团给染液的能力。已成功地被用于药物递送三种常见GnRH类似物是[D-赖氨酸6] -GnRH-I，D-赖氨酸6] -GnRH-II和GnRH的-Ⅲ，但这些肽的作为靶向部分的有效性是很少相比16，17。另一方面，在其中不同的癌细胞和GnRH类似物被用于从不同的实验结果是多样的。 ntent"&gt;基于这些考虑，我们集中在肿瘤靶向和药物递送的潜在这些的GnRH肽的，并且由此合成和表征的[D-赖氨酸6（FITC）〕 -的GnRH-I，D-赖氨酸6（FITC ）〕 -的GnRH-II和[赖氨酸8（FITC）〕 -的GnRH-III肽缀合物18这些类似物有选择地与FITC标记上的他们的赖氨酸或D-赖氨酸（肽-FITC比率侧链1:1每个。缀合物），其思想是，选择性荧光标记可以提供有关这些肽新颖的信息，并允许他们的良好的跟踪和可靠的定量。这些结合物具有安全处理和可靠探测，这使得它更容易比较他们的肿瘤靶向效率，并且许多类型的恶性肿瘤细胞的筛选，我们希望达至最新的实验与这些肽偶联物可能有助于新型癌症的靶向发展促性腺激素释放激素药物结合物，并有助于发现新的治疗焦油得到为好。 本手稿演示用的GnRH-FITC结合物以及一些重复性好，快速实验。的GnRH-R的细胞表面表达是关于GnRH的摄取决定性条件，因此我们同时研究了所测试的细胞系中的GnRH-1R的细胞表面水平。我们可视通过共聚焦激光扫描显微镜（CLSM）GnRH的IR和GnRH的-FITC缀合物和使用荧光激活细胞分选（FACS）定量的GnRH-FITC缀合物的细胞摄取。

<table><tbody><tr><td>EBC-1</td><td>JCRB Cell Bank</td><td>JCRB0820</td><td>Human lung squamous cell carcinoma</td></tr><tr><td>BxPC-3</td><td>ATCC</td><td>CRL-1687</td><td>Human pancreatic adenocarcinoma</td></tr><tr><td>Detroit-562</td><td>ATCC</td><td>CCL-138</td><td>Human pharyngeal carcinoma</td></tr><tr><td>RPMI-1640 Medium with L-glutamine</td><td>Lonza</td><td>BE12-702F</td><td>Culture medium for BxPC-3 cells</td></tr><tr><td>Eagle&#039;s Minimum Essential Medium</td><td>Lonza</td><td>BE12-611F</td><td>Culture medium for EBC-1 cells, and complemented with 0.1% sodium pyruvate for Detroit-562 cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Euro Clone</td><td>ECS0180L</td><td>Complements the culture medium</td></tr><tr><td>MycoZap Plus-CL&amp;nbsp;</td><td>Lonza</td><td>VZA-2012</td><td>Complements the culture medium (antibiotics)</td></tr><tr><td>Standard Line Cell Culture Flasks</td><td>VWR</td><td>10062-872</td><td>Provide consistent, sterile growth environment for cells</td></tr><tr><td>Trypsin-EDTA Mixture&amp;nbsp;</td><td>Lonza</td><td>BE17-161E</td><td>Remove attached cells</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza</td><td>BE17-516F</td><td>Solvent</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418-100ML</td><td>Solvent</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td>Used to counting cells</td></tr><tr><td>[D-Lys&lt;sup&gt;6&lt;/sup&gt;(FITC)]-GnRH-I</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>[D-Lys&lt;sup&gt;6&lt;/sup&gt;(FITC)]-GnRH-II</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>[Lys&lt;sup&gt;8&lt;/sup&gt;(FITC)]-GnRH-III</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>glass bottom 8-well microscopic slide</td><td>Ibidi</td><td>80826</td><td>Use in CLSM experiment</td></tr><tr><td>Corning Costar cell culture plate, 12 well</td><td>Sigma-Aldrich</td><td>CLS3513-50EA</td><td>Use in FACS experiment</td></tr><tr><td>Fixing solution (10% neutral buffered formalin)</td><td>Bio-Optica</td><td>01V60P</td><td>Fix adherent cells</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-10G</td><td>Prevent the nonspecific binding of the antibodies</td></tr><tr><td>GnRHR Antibody</td><td>Proteintech</td><td>19950-1-AP</td><td>Immunocytochemistry reagent, bind to the human type-I GnRH receptors</td></tr><tr><td>Secondary Antibody, Alexa Fluor 546 conjugate</td><td>Thermo Fisher Scientific</td><td>A11035</td><td>Immunocytochemistry reagent, bind to the primary antibody</td></tr><tr><td>Fluorescent probe solution (Draq5)</td><td>Thermo Fisher Scientific</td><td>62254</td><td>Counterstain nuclei</td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Sigma-Aldrich</td><td>F4680-25ML</td><td>Use in CLSM experiment, preserving fluorescence</td></tr><tr><td>Inverted microscope</td><td>Elektro-Optika Ltd.&amp;nbsp;</td><td>Alpha XDS-2T</td><td>Check the phenotype and confluency of cell cultures</td></tr><tr><td>Inverted confocal laser scanning microscope</td><td>Zeiss</td><td>LSM-710</td><td>Use in CLSM experiment</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>BD FACSCalibur</td><td>Use in FACS experiment</td></tr></tbody></table>

in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues

GnRH类似物是有效的靶向部分和能够提供抗癌剂选择性为恶性肿瘤细胞，其高表达的GnRH受体。然而，GnRH类似物&#39;细胞摄取的定量分析以及基于GnRH的药物递送系统的研究的细胞类型，目前的限制。以前介绍过，选择性荧光标记的GnRH I，-II和-III衍生产品提供了很大的可探测性，和他们有重复性的和健壮的实验合适的化学性质。我们还发现，适当的向上的最新方法与这些标记GnRH类似物能够提供有关基于促性腺激素释放激素的药物递送系统的新信息。该原稿介绍关于[D-赖氨酸6（FITC）]的细胞摄取了一些简单的和快速的实验-促性腺激素释放激素I，D-赖氨酸6（FITC）〕 -的GnRH-II和[赖氨酸8（FITC）〕 -的GnRH -III在EBC-1（肺），在中BxPC-3（胰腺）和底特律-562-（咽）恶性Ťumor细胞。在与这些的GnRH-FITC缀合物平行，促性腺激素释放激素-1受体的细胞表面水平也之前和促性腺激素释放激素治疗通过共聚焦激光扫描显微镜后检查了这些细胞系。的GnRH-FITC缀合物的细胞摄取是通过荧光激活细胞分选进行定量。在这些实验中观察到GnRH类似物和细胞类型之间的主要区别之间的微小差别。其中细胞系的显著差异有其独特的细胞表面的GnRH-I受体水平相关。引入的实验包含实用的方法来可视化，量化和在各种粘附细胞培养以时间和浓度依赖的方式进行比较的GnRH-FITC缀合物的吸收效率。这些结果可以预测在给定的细胞培养物的GnRH偶联物的药物靶向效率，并提供用于在基于促性腺激素释放激素的药物递送系统的检查的进一步实验的良好基础。

激光共聚焦扫描显微镜（CLSM）获得的图像提供有关的GnRH-FITC结合物在给定的细胞培养的时间和浓度依赖性摄取壮观信息。在与这些的GnRH-FITC缀合物的同时，促性腺激素释放激素-1R的细胞表面上的存在也由CLSM实验验证。另外，通过使用一远红DNA染色荧光探针，能够为了对细胞的细胞核以外的GnRH和GnRH的-1R。然而，虽然共焦图像不是量化的，简单的"GnRH的摄入法"可以容易地估...

Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

选择性标记的荧光的GnRH-I，-II和-III衍生物对于跟踪可靠的工具和量化它们的细胞摄取。这个手稿介绍实验来可视化，量化和比较在各种细胞系这些GnRH的缀合物的吸收效率。

体外成像和荧光标记的GnRH类似物的药物靶向性的定量分析

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

in-vitro-imaging-quantification-drug-targeting-efficiency

Research

JoVE Journal

Cancer Research

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

7.6K 次观看.  Semmelweis University. 选择性标记的荧光的GnRH-I，-II和-III衍生物对于跟踪可靠的工具和量化它们的细胞摄取。这个手稿介绍实验来可视化，量化和比较在各种细胞系这些GnRH的缀合物的吸收效率。 

视频: In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

科研

化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

生物化学

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

使用体外活体细胞成像技术研究脂纳米粒子化疗

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

医学

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

一个荧光识别GIRK通道调制器的筛选试验

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

生物学

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

自体影像学作为一种简单而有力的药理靶标可视化和表征方法

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

神经科学

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

追踪药物诱导Colocalizational变化分析受体后内部贩运

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

活细胞成像和血管紧张素的三维分析受体1a型贩运转染人胚肾细胞使用共聚焦显微镜

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...