我们感谢阅读这篇稿子的关键松波实验室的成员。

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，?...

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<td>Cover slip 22 X 22 mm (#1)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>72200-11</td>
<td>&nbsp;</td>
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<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>16000</td>
<td>&nbsp;</td>
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<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
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<td>Minimal essential medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M4655</td>
<td>With L glutamate, Earle&#x2019;s salt and bicarbonate</td>
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<td>Mowiol</td>
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<td>Hanks Balanced Salt Solution (HBSS) 1X </td>
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<td>15630</td>
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<td>Poly D Lysine Hydrobromide</td>
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<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
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<td>Sodium Azide</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0299-1</td>
<td>&nbsp;</td>
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<td>Tissue culture dish 35 X 10 mm</td>
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<td>Falcon</td>
<td>353801</td>
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<td>Trypsin-EDTA </td>
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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

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

含量自发活动，研究昼夜节律和睡眠参数果蝇</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

科研

神经科学

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二：外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

本体感觉和张力感受器在蟹四肢：学生实验室练习

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

使用生物素化测定原代星形细胞培养物中蛋白质的细胞表面表达和吞噬率

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...