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1。程序: 该过程包括三个部分，超过3天的总时间完成:转移细胞，转染和免疫细胞化学，每天进行一个步骤。转移和细胞的转染前两天，必须在层流室的无菌条件下进行。 第1天:转让HEK293T细胞表面表达检测。 <ol><li>传输介质（M10）:混合辅以10％（体积比）牛血清胎儿在无菌条件下的最低限度的基本介质。 </li><li>种植细胞所需的汇合在一个100mm的培养板，根据需要设置的板块数量。重要的是要确定避免杂草丛生或稀疏的细胞被转移到细胞的一小部分:例如，从100％的融合100毫米菜，一可转移约3％至35毫米的菜中获得约30％的汇合后者。 </li><li>在此之前转移细胞，在无菌层流室，大衣22 x 22毫米玻璃与无菌ð聚赖氨酸（1毫克/毫升）和每35毫米的细胞培养皿中的地方，有涂层的一面电镀细胞盖玻片（图1 ）。允许干5 - 10分钟的解决方案。在此期间，可打开紫外光源在层流室，消毒盖玻片，如果需要的话。 ð聚赖氨酸有助于异种细胞粘到盖玻片。 </li><li>转移细胞，吸出为100mm碟现有媒体，通过仔细加入8 mL无菌PBS，吸出PBS洗，用3毫升0.05％胰蛋白酶EDTA trypsinize的细胞，细胞分离从盘时，阻止进一步酶促反应加入5毫升的M10。磨碎的细胞和所需量转移到无菌的锥形管;在200克离心5分钟沉淀细胞。吸出上清液含有胰蛋白酶EDTA，使颗粒细胞完好无损。细胞转移至35 mm培养皿中，每道菜添加1毫升M10和磨碎游离的细胞。加入1毫升的悬浮细胞每一道菜。孵育细胞过夜，于37℃，5％的CO 2。 <img src="/files/ftp_upload/2405/2405fig1.jpg" alt="图1" /> 图1。大衣22 x 22毫米，与聚ð赖氨酸和地方每盘35毫米的有涂层的一面盖玻片。 </li></ol>第2天:转染HEK293T细胞。 <ol><li> 18-24小时后，转移，细胞是准备要测定受体的转。使用受体在哺乳动物表达载体PCI编写使用Qiagen公司小量Kit中的下列协议前4描述的细菌培养，克隆RTP1S 。使用，是N末期与20个氨基酸长的视紫红质的抗原表位标记，以方便免疫受体。 </li><li>使用受体的构建，250纳克RTP1S构建绿色荧光蛋白转和50毫微克1000ng。执行转用脂质体2000，为制造商手册。 </li></ol>第3天:免疫组化，可视化细胞表面染色受体。 <ol><li>在开始之前，准备染色和洗涤解决方案，并冷却至4 ° C染色介质:最低限度的基本中等，45毫升;胎牛血清，5毫升; HEPES（1M），500μL（终浓度为10毫米）;叠氮化钠（1.5M），500μL（终浓度为15毫米）。洗涤液:HBSS中，500毫升; HEPES（1M），5毫升（终浓度为10毫米）;叠氮化钠（1.5M），5毫升（终浓度为15MM）。这两种解决方案都可以存储在4 ° C重用。准备所需的抗体染色介质的100倍稀释的抗体解决方案。 </li><li>开始表面约24小时后转染的气味受体免疫组织化学染色。要执行的活细胞染色，实验的设立，必须冷却至4 ° C时:填补了大型载冰和拍拍冰面，使其尽量均匀，放置一个托盘上的冰，并喷洒70％乙醇。剪下一块封口膜（小于托盘的长度），并加盖乙醇喷托盘上，以获得一个光滑的表面。传输的玻璃盖玻片含有转染细胞上的封口膜，一次一个，细胞一侧，用小镊子。层每个盖玻片用100μL冷主要染色液，挤压滑（图2）从一个角落仔细。分层抗体溶液与盖玻片后，包括托盘，以防止干燥空气中的解决方案。开展初级孵化45-60分钟。 </li><li>初级孵化后，迅速转移到原来的35毫米培养皿盖玻片，包含媒体。吸媒体和添加2毫升冷冻洗涤液从盘边缘，轻轻地从细胞的盖玻片，以防止丢失。吸出洗涤液，重复两次完成三洗。第三洗结束，不吸洗涤液。 </li><li>准备二次抗体soluti。在染色媒体和盖玻片从洗涤液转移到一个新贴的封口膜层在同一托盘。再次轻轻挤出100μL抗体溶液层像初级孵化的角落，每个盖玻片。二次孵化开展30 - 45分钟，并传回在原有的35毫米菜的洗涤液。再次，洗盖玻片三倍。 </li><li>吸洗最后一次洗涤的解决方案，并添加2 mL 1％的冷冻多聚甲醛。修复细胞冷冻1％多聚甲醛溶液中，冰浴15分钟。 </li><li>装入清洁显微镜的玻璃盖玻片幻灯片使用安装试剂Mowiol，与细胞的一侧，谨慎地排除所有气泡（图3）。一旦Mowiol晾干，轻轻洗盖玻片表面上加入蒸馏水，立即吸。 <img src="/files/ftp_upload/2405/2405fig2.jpg" alt="图2" /> 图2层每个盖玻片，用100μL冷主染色液。 <img src="/files/ftp_upload/2405/2405fig3.jpg" alt="图3" /> 图3安装与细胞方面的盖玻片上干净的玻璃显微镜下来幻灯片使用Mowiol安装试剂。 </li></ol> 2。代表性的成果: 活细胞染色使一个可视化的细胞表面的蛋白质，与伴侣（在此情况下气味受体Olfr62与受体的运输蛋白RTP1S）（图4）受体的转染。受体细胞表面染色的特点是点状染色格局（图4B，插图）。染色进行不适当，可能会导致更高的噪声，使观察者难以区分真正的表面染色从噪音。 <img src="/files/ftp_upload/2405/2405fig4.jpg" alt="图4" /> 图4转染受体与伴侣允许使用活细胞染色的细胞表面蛋白质的可视化。转染Olfr62单独的嗅觉受体（A）和与RTP1S伴侣（二）转染的Olfr62。 GFP表达水平作为一个转控制（A&#39;和B&#39;）。

<ol>
	<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65, 175-187 (1991).</li><li>Malnic, B., Hirono, J., Sato, T., Buck, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+receptor+codes+for+odors.">Combinatorial receptor codes for odors.</a> Cell. 96, 713-723 (1999).</li><li>Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RTP+family+members+induce+functional+expression+of+mammalian+odorant+receptors.">RTP family members induce functional expression of mammalian odorant receptors.</a> Cell. 119, 679-691 (2004).</li><li>Zhuang, H., Matsunami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell-surface+expression+and+measuring+activation+of+mammalian+odorant+receptors+in+heterologous+cells.">Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells.</a> Nat Protoc. 3, 1402-1413 (2008).</li></ol>

每一步都必须小心进行，以确保突出和独特的表面染色。整个染色过程中，必须在寒冷的开展（冰），和盖玻片奠定托盘必须被冷却之前使用，以确保细胞活路。此外，固定做表面染色过程结束。这是内部染色，其中固定先染色通透细胞膜的小学和中学抗体形成鲜明对比的。盖玻片暴露在空气中的时间必须最小化，以防止从干涸的细胞，因此盖玻片与抗体的解决方案的分层，转让盖玻片洗涤液，?...

<table><tbody><tr><td>Cover slip 22 X 22 mm (#1)</td><td>Thermo Fisher Scientific, Inc.</td><td>72200-11</td><td></td></tr><tr><td>Fetal bovine serum</td><td>GIBCO, by Life Technologies</td><td>16000</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Minimal essential medium</td><td>Sigma-Aldrich</td><td>M4655</td><td>With L glutamate, Earle&amp;rsquo;s salt and bicarbonate</td></tr><tr><td>Mowiol</td><td>Calbiochem</td><td>475904</td><td></td></tr><tr><td>Paraformaldehyde</td><td>EMS</td><td>19208</td><td></td></tr><tr><td>Hanks Balanced Salt Solution (HBSS) 1X </td><td>GIBCO, by Life Technologies</td><td>14025</td><td>With calcium chloride and magnesium chloride</td></tr><tr><td>HEPES Buffer Solution</td><td>GIBCO, by Life Technologies</td><td>15630</td><td></td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Cellgro</td><td>21-040-CV</td><td>Without calcium and magnesium</td></tr><tr><td>Poly D Lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P7280</td><td></td></tr><tr><td>Sodium Azide</td><td>Electron Microscopy Sciences</td><td>SX0299-1</td><td></td></tr><tr><td>Tissue culture dish 35 X 10 mm</td><td>Falcon BD</td><td>353801</td><td></td></tr><tr><td>Trypsin-EDTA </td><td>GIBCO, by Life Technologies</td><td>25300</td><td>0.05%</td></tr></tbody></table>

assaying surface expression of chemosensory receptors in heterologous cells

是公认的嗅觉感的生动的气味世界。在小鼠的嗅觉是介导的由约1200克蛋白偶联受体（GPCRs）1，假设绑定挥发的气味分子，并通过与G蛋白耦合Gαolf的胞内信号转换成细胞外信号的剧目。绑定的气味受体被认为是按照组合规则，也就是说，一个气味可能几个受体绑定和一个受体可以绑定几个气味不同程度2。生化，信令和配体结合研究已方便进行最GPCRs的使用异种细胞。但是使用的气味受体的研究，异种细胞，排除了很长一段时间，因为他们在转没有出口到表面。 Saito等人已经证明单膜通过受体运输蛋白（RTP）的家庭伴侣显示在嗅感觉神经元和交通气味受体在异种细胞的表面，当联合一起转了3伴侣的行为表达增强。要利用异种细胞的受体进行生化检测，必须先确定，如果受体的细胞株显示了强大的表面表达。这可以检测高表达的受体与由活细胞染色，荧光标签或专门的外域外域标记的伴侣RTP1S。在这里，我们展示了一个协议，进行活细胞染色，可用于检测HEK293T细胞表面上的气味受体方便。此外，它也可能被用来检测其他化学感受受体或GPCRs的表面表达。

Watch this Scientific Journal Video about Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells at JoVE.com

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

在这里，我们展示了一个协议，进行活细胞染色，可用于检测HEK293T细胞表面上的气味受体方便。此外，它也可能被用来检测其他化学感受受体或GPCRs的表面表达。

测定在异种细胞表面的化学感应受体的表达

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors ...

assaying-surface-expression-chemosensory-receptors-heterologous

Research

JoVE Journal

Neuroscience

10.8K 次观看.  Duke University. 在这里，我们展示了一个协议，进行活细胞染色，可用于检测HEK293T细胞表面上的气味受体方便。此外，它也可能被用来检测其他化学感受受体或GPCRs的表面表达。

视频: Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性:含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

科研

生物工程

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 ...

化学

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

受体激活的测量通过荧光素酶活性:哺乳动物的嗅觉受体的高通量分析

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

神经科学

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

气相臭受体活化的实时体外监测

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

蚊子嗅觉附属物中的化学感应基因表达作图

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

作者聚焦：可视化蚊子的嗅觉受体表达 

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

杂交链式反应 RNA全山荧光 蚊子嗅附物化学感觉基因 的原位 杂交

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

测定在异种细胞表面的化学感应受体的表达

摘要

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此视频中的章节

腺苷酸环化酶的药物引起的过敏性:含量简化和小型化的小分子和siRNA筛选应用

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

受体激活的测量通过荧光素酶活性:哺乳动物的嗅觉受体的高通量分析

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

实时 cAMP 检测气味受体激活的活细胞测量

气相臭受体活化的实时体外监测

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

杂交链式反应 RNA全山荧光蚊子嗅附物化学感觉基因的原位杂交

测定在异种细胞表面的化学感应受体的表达

摘要

探索更多视频

此视频中的章节

腺苷酸环化酶的药物引起的过敏性:含量简化和小型化的小分子和siRNA筛选应用

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

受体激活的测量通过荧光素酶活性:哺乳动物的嗅觉受体的高通量分析

鼠标嗅觉感官神经元的完整神经上皮穿孔膜片钳记录:神经元表达鉴定的气味受体功能分析

实时 cAMP 检测气味受体激活的活细胞测量

气相臭受体活化的实时体外监测

抗体喂养方法研究分离原发性希马培养物中的谷氨酸受体贩运

通过异位细胞系统中的蛋白质聚合测量跨细胞相互作用

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

杂交链式反应 RNA全山荧光 蚊子嗅附物化学感觉基因 的原位 杂交

杂交链式反应 RNA全山荧光蚊子嗅附物化学感觉基因的原位杂交