这项工作是由R01 DC013339，R03 DC011373，和露丝属Kirschstein国家研究服务奖T32 DC000014支持。使用莫耐尔化学感受受体信号的核心，这是支持，部分由美国国立卫生研究院，NIDCD核心格兰特P30 DC011735资金进行的工作的一部分。作者感谢C. Sezille的帮助，收集数据。

Hana3A细胞1。文化<ol><li>通过补充的最小​​必需培养基（MEM），用10％（V / V）FBS的制备M10介质。 </li><li>文化维护<ol><li>维持细胞M10的媒体。注意：对于RTP1L，RTP2，REEP1和G表达载体αolf赋予Hana3A细胞嘌呤霉素抗性，但保持了细胞的这种抗生素没有显著影响测定结果。 </li><li>亚文化在1:8的10厘米菜每2-3天的比率。 </li><li>在37℃，5％CO 2。 </li></ol></li></ol> 2，用于转染细胞的电镀<ol><li>吸媒体从Hana3A细胞100％汇合10cm皿。 </li><li>通过加入10ml PBS，涡旋盘中，并吸取PBS洗涤细胞。 </li><li>加入3毫升0.05％胰蛋白酶/ EDTA，等待细胞解离（约1分钟）。 </li><li>通过加入5ml M10灭活胰蛋白酶和拆散细胞团块通过捣碎大约10倍和＃160;用10毫升吸管。吸管小心，以避免引入气泡进入媒体。 </li><li>对于每个96孔板，转让1毫升细胞入15ml锥形管中，离心分离机，在200×g离心5分钟，吸出上清液，而不会干扰细胞沉淀。 </li><li>每1ml在步骤2.5转移细胞重新悬浮于6ml M10细胞。 </li><li>移液管加入50μl细胞到96孔板的每个孔中并孵育过夜，在37℃，5％的CO 2。 </li></ol>嗅觉受体3。转<ol><li>质粒DNA的制备<ol><li>通过无内毒素的协议制备质粒DNA。注意：使用质粒DNA制备试剂盒指定的“无内毒素”，或添加一个酚 - 氯仿提取步骤，质粒DNA制备的协议。 </li><li>稀释的DNA至100毫微克/微升的TE缓冲液中的浓度。 </li></ol></li><li>观察接种细胞（步骤2.7），以确保近似的正确汇合tely 30-50％，每孔并返回到孵化器。注意：虽然此汇合不是最佳的脂质转染试剂，30-50％在此步骤中，汇合是最优的，用于测量荧光素酶活性24小时后转染。 </li><li>转染混合物的制备<ol><li>移液器RTP1S-PCI，M3-R-PCI，PCRE-LUC和PSV40-RL质粒导入MEM培养基每详述于表1中，使质粒混合物的体积（体积表示为每96 -孔板）。 <table border="0" cellpadding="0" cellspacing="0" style="width:356px;"><tbody><tr height="22"><td colspan="3" height="22" style="height:22px;width:356px;"> 质粒组合 </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td> 每孔 </td><td> 每个96孔板 </td></tr><tr height="21"><td height="21" style="height:21px;"> MEM </td><td> - </td><td> 500微升</td></tr><tr高度=“21”&gt; <td height="21" style="height:21px;"> RTP1S-PCI </td><td> 5纳克</td><td> 480纳克</td></tr><tr height="21"><td height="21" style="height:21px;"> M3-R-PCI </td><td> 2.5纳克</td><td> 240纳克</td></tr><tr height="21"><td height="21" style="height:21px;"> PCRE-LUC </td><td> 10毫微克</td><td> 960纳克</td></tr><tr height="22"><td height="22" style="height:22px;"> PSV40-RL </td><td> 5纳克</td><td> 480纳克</td></tr></tbody></table> 表1质粒混合元件。每井和每RTP1S-PCI，M3-R-PCI，PCRE-LUC和PSV40-RL，和MEM的96 -孔板的卷。 </li><li>对于每个96孔板，稀释18微升脂转染试剂在450微升的MEM培养基。 </li><li>移液器质粒组合（从步骤3.3.1）的PCI质粒（RHO-OR-PCI）视紫红质标记的嗅觉受体和脂质转染混合物（从步骤3.3.2），使在复杂细节<sTRONG&gt;表2。混合溶液通过研磨和在室温下孵育15分钟。停止反应，根据表2中加入M10请注意：该反应中是对时间敏感的，不应该被允许继续进行30分钟以上。井+ 10％的计算是非常重要的，以确保足够的容积用于随后的步骤。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="21"><td colspan="3" height="21" style="height:21px;width:472px;"> 复杂 </td></tr><tr height="21"><td height="21" style="height:21px;"></td><td> 每孔 </td><td> 每孔+10％ </td></tr><tr height="21"><td height="21" style="height:21px;">质粒组合</td><td> 4.2微升</td><td> 4.58微升</td></tr><tr height="21"><td height="21" style="height:21px;"> ρ-OR-PCI </td><td> 0.05纳克</td><td> 0.06纳克</td></tr><tr高度=“21”&gt; <td height="21" style="height:21px;">脂质转染混合物</td><td> 4.2微升</td><td> 4.58微升</td></tr><tr height="22"><td height="22" style="height:22px;"> M10 </td><td> 41.7微升</td><td> 45.83微升</td></tr></tbody></table> 表2。复杂的组件。每口井，每个质粒组合（ 表1）以及±10％体积，嗅觉受体质粒（RHO-OR-PCI）和脂质转染混合物。 M10被加入以淬灭下列一个15分钟温育在室温下进行反应。 </li></ol></li></ol><ol start="4"><li>挖掘出的电池板媒体。 </li><li>移液管加入50μl的复合物至每孔中，并孵育过夜，在37℃，5％的CO 2。 </li></ol> 4，气味刺激<ol><li>观察转染细胞，以确保每孔50-80％正确的汇合，并返回到培养箱中。注意：如果电池是低于50％汇合，萤火虫荧光素酶和海肾荧光素酶的读数可能会太低，受体激活的测量。考虑放弃平板。 </li><li>准备1个月股票的解决方案在DMSO每个气味。 </li><li>准备在CD293中型刺激气味的解决方案。 <ol><li>用于筛选实验中，稀释气味的储备液至100μM的。还（仅CD293），以控制或激活后台准备一个没有气味控制。注：对于筛选实验，每个OR /气味对只每一次实验测试。因为整个井某些气味扩散是可能的，所以建议以刺激与一种加臭剂对每个板。 </li><li>对于剂量响应实验，制备的气味原液，一式三份开始以1mM每种受体7 10倍连续稀释。还准备一式三份空载体转染细胞相同的气味稀释液，以控制气味的背景激活。注：对于剂量反应实验中，每个Ø多尔浓度治疗应一式三份进行。 </li></ol></li><li>挖掘出的电池板媒体。 </li><li>移液管将25μl的刺激气味液到每个孔中并孵育4小时。 </li></ol> 5，测量或活动通过萤光素酶检测<ol><li>根据制造商的在-80℃下的指令和存储重悬萤火虫荧光素酶底物</li><li>解冻1毫升萤火虫荧光素酶底物的每个96孔板中。 </li><li>准备新鲜的萤火虫荧光素酶反应淬灭剂和海肾萤光素酶底物试剂（5微升淬灭剂荧光素酶/ 海肾萤光素酶每1 ml缓冲液底物）。注：约1毫升试剂是需要每96孔板。 </li><li>准备发光酶标仪。打开酶标仪软件。系统内的图标： <ol><li>在“预热”选项卡，勾选为“ON”的框中，然后设定机器的温度为25°; C。 </li><li>下的“分配器”标签中，首要每个分配器有1,000微升70％乙醇，随后由1,000微升蒸馏水。注意：使用醇与水的分离等份的每个分配器。乙醇是用来消毒的分配器，和水去除残留的乙醇。 </li><li>首相每个饮水机与1500微升空气（从液体中移除掌柜）。注：与空气吸确保荧光素酶底物不与残留水稀释。 </li><li>总理饮水机1与1,080微升萤火虫荧光素酶底物（从步骤5.2）中。素分配器2与海肾荧光素酶底物（来自步骤5.3）。注意：注意不要交叉污染的荧光素酶的底物。用荧光素酶底物吸填补了试剂掌柜的死角。 </li></ol></li><li>设置以下协议来读取这两种萤火虫和海肾萤光素酶发光。内用酶标仪，取消相关的软件DER“文件”菜单中，单击“新任务”。突出显示“协议”，然后单击“新建”。在接下来的窗口，旁边的“标准协议”圆应选择。单击“确定”。在屏幕的左侧双击“程序”。 <ol><li>将10μL萤火虫荧光素酶底物的利用分配器1的所有油井。在“操作”菜单上，单击“配给”。在“分装步”窗口中，设置：“饮水机”1，“灌注”到无“，分配容量”，以10微升和“速度”，以225微升/秒。点击“确定”。 </li><li>摇板30秒。在“操作”菜单上，单击“摇”。在“步摇”窗口中，将“强度”，以中和“持续时间”到0时30 MM：SS。点击“确定”。 </li><li>阅读所有孔的每孔0.5秒的发光。在“操作”菜单上，单击“读取”;。在“阅读步骤”窗口中，设置：“检测方法”来发光，“读类型”设置为端点，“积分时间”，以零时00分50秒MM：SS：SS，“过滤设置”设为1，“排放”以孔，“光学位置”上面，“增益”到135，和“读取高度”为1.00毫米。点击“确定”。 </li><li>将10微升海肾萤光素酶底物的利用分配器2的所有油井。设置条 ​​件，如步骤5.6.1，集“饮水机”，以2的除外。 </li><li>摇板30秒。设定的条件，如步骤5.6.2。 </li><li>阅读所有孔0.5秒的发光每口井。设定的条件，如步骤5.6.3。 </li></ol></li><li>从96孔板中除去盖子，然后将板在微量板读数器。启动程序设置步骤5.5读板发光。 </li><li>清洁剂分配器泵。从“饮水机”选项卡下的系统图标： <ol><li>清除1,000微升的f从萤火虫荧光素酶分配器到回收管irefly荧光素酶底物。注：萤火虫荧光素酶可以储存在-80℃并重复使用。 </li><li>素每个分配器有100​​0微升的蒸馏水，接着1,000微升70％的乙醇，最后1500微升的空气（从液体移除分配器）。注：去除水中的荧光素酶底物的试剂泵，乙醇消毒杀菌，空气干燥残留的乙醇。 </li></ol></li></ol> 6，数据分析<ol><li>数据导出<ol><li>在酶标仪软件，双击屏幕左侧的“报告/导出建设者”。 </li><li>点击按钮“新建导出到Excel”，然后单击“确定”。 </li><li>突出EXPORT1并单击“编辑”。 <ol><li>在“内容”检查“系统说明”，“程序”，“盘说明”和“反应板布局矩阵”。包括“原始数据”一ND“计算数据”。 </li><li>在“工作流”，勾选“自动执行的程序的完成”。在导出模式，勾选“在同一工作簿中的所有板”和“作为一个新的工作表”。 </li><li>在“文件”选择文件名格式和文件位置，然后单击“确定”。 </li><li>关闭“报告/导出建设者”窗口。 </li></ol></li></ol></li><li>为了得到归一化的荧光素酶值，由海肾荧光读数为每口井（步骤5.6.6）将萤火虫荧光素酶发光读数每口井（步骤5.6.3）。 </li></ol>

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R., 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=Activation+state+of+the+M3+muscarinic+acetylcholine+receptor+modulates+mammalian+odorant+receptor+signaling.">Activation state of the M3 muscarinic acetylcholine receptor modulates mammalian odorant receptor signaling.</a> Science signaling. 4 (155), (2011).</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> Nature. 3 (9), 1402-1413 (2008).</li><li>Haddad, R., Khan, R., Takahashi, Y. 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M., Hashimoto, M., Mikoshiba, K., Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+of+a+mammalian+odorant+receptor.">Functional expression of a mammalian odorant receptor.</a> Science. 279 (5348), 237-242 (1998).</li><li>Touhara, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+identification+and+reconstitution+of+an+odorant+receptor+in+single+olfactory+neurons.">Functional identification and reconstitution of an odorant receptor in single olfactory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 4040-4045 (1999).</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=Synergism+of+accessory+factors+in+functional+expression+of+mammalian+odorant+receptors.">Synergism of accessory factors in functional expression of mammalian odorant receptors.</a> The Journal of biological chemistry. 282 (20), 15284-15293 (2007).</li><li>McRae, J. F., Mainland, J. D., Jaeger, S. R., Adipietro, K. A., Matsunami, H., Newcomb, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+the+odorant+receptor+OR2J3+is+associated+with+the+ability+to+detect+the+&amp;#34;grassy&amp;#34;+smelling+odor,+cis-3-hexen-1-ol.">Genetic variation in the odorant receptor OR2J3 is associated with the ability to detect the &amp;#34;grassy&amp;#34; smelling odor, cis-3-hexen-1-ol.</a> Chemical senses. 37 (7), 585-593 (2012).</li><li>Adipietro, K. A., Mainland, J. D., 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=Functional+evolution+of+mammalian+odorant+receptors.">Functional evolution of mammalian odorant receptors.</a> PLoS genetics. 8 (7), (2012).</li><li>Zhuang, H., Chien, M. -. S., 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=Dynamic+functional+evolution+of+an+odorant+receptor+for+sex-steroid-derived+odors+in+primates.">Dynamic functional evolution of an odorant receptor for sex-steroid-derived odors in primates.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21247-21251 (2009).</li><li>Oka, Y., Nakamura, A., Watanabe, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+odorant+derivative+as+an+antagonist+for+an+olfactory+receptor.">An odorant derivative as an antagonist for an olfactory receptor.</a> Chemical senses. 29 (9), 815-822 (2004).</li><li>Oka, Y., Omura, M., Kataoka, H., Touhara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+receptor+antagonism+between+odorants.">Olfactory receptor antagonism between odorants.</a> The EMBO journal. 23 (1), 120-126 (2004).</li><li>Fawcett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+ROC+analysis.">An introduction to ROC analysis.</a> Pattern Recognition Letters. 27 (8), 861-874 (2006).</li><li>Baghaei, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+Receptors.">Olfactory Receptors.</a> Olfactory Recept. Methods Protoc. 1003, 229-238 (2013).</li><li>Dey, S., Zhan, S., 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=Assaying+surface+expression+of+chemosensory+receptors+in+heterologous+cells.">Assaying surface expression of chemosensory receptors in heterologous cells.</a> Journal of visualized experiments JoVE. (48), (2011).</li></ol>

作者什么都没有透露。

气味识别是通过嗅觉受体激活模式编码，但受体的激活模式，包括受体被激活到什么程度，被称为人类的嗅觉受体1-11少于6％。努力来表征嗅觉受体已经由劳动密集型方法或适用性不限于嗅觉受体家族17,23,24,33,34的一个子集。该Hana3A异源表达系统支持大多数测试嗅觉受体的鲁棒表达，并且可以在同一个的cAMP响应性荧光素酶报告系统，以监测嗅觉受体活化19,26,27一起使用。该...

哺乳动物嗅觉系统具有响应恶臭刺激广大，允许用于检测和数以千计的加臭剂的鉴别能力。嗅觉受体（OR值）是通过在嗅觉上皮12的嗅感觉神经元表达的分子传感器。哺乳动物的气味识别是通过激活不同的OR ​​通过的气味，而OR基因家族是广泛的，有大约1000小鼠和400人的受体12-16。在嗅觉神经元，并在异源细胞手术室以前的功能分析表明，不同的气味是由独特的认可，但重叠的OR ​​值10,17-20合奏。选配配体OR值是理解的嗅觉代码和建立嗅觉可行的模型必不可少的关键。由于表达OR值在异源系统，以及大量两者的气味和OR的困难，这一数据已经从F基本上不存在IELD;的确，人的口服补液盐少于6％已公布配体1-11。这个协议描述了使用荧光素酶测定法的表征气味/或相互作用。此法使手术室的高通量表征，任务是必不可少的理解气味/或相互作用以及发展气味编码的典范。 手术室的高通量研究面临的三大挑战。首先，表达的异源细胞的ORs被保留在ER中，并随后降解的蛋白酶体21,22，防止从OR值与在测定系统23-25 ​​气味物质相互作用。这个问题是由辅助蛋白，以促进广泛的ORs 19,26,27的稳定的细胞表面表达的发现处理。受体转运蛋白1和2（RTP1和2）促进或响应于刺激的气味19的细胞表面表达和活化。在此基础上，将HEK293T细胞修饰以稳定表达RTP1长（RTP1L）和RTP2，受体表达增强蛋白1和Gαolf，导致Hana3A细胞系19,27。此外，3型毒蕈碱型乙酰胆碱受体（M3-R）进行交互的ORs在细胞表面，并增强活化响应于加臭剂26。共转染的或用RTP1S和M3-R的进Hana3A细胞的结果在宽范围的ORs的在细胞表面27的健壮的，一致的，和功能性表达。第二，哺乳动物或剧目都相当大。在人类中，例如，或剧目是一个数量级比味觉受体库越来越多，2个数量级比视觉受体剧目更多了的。虽然克隆一个OR是一个相对简单的协议，需要显著的前期工作，以产生一个综合库。第三，虽然我们知道，在视觉，波长转换成颜色和在试音的频率转换成沥青，气味的组织是知之甚少，使得研究人员很难从气味物质的代表性样品进行插值。虽然有些已经取得了进展在这方面10,28，嗅觉景观的地图仍然是不完整的。筛选分子数万数百对口服补液盐是一项艰巨的任务;在这一领域的高通量筛选需要仔细界定的活动。其余的主要挑战是那些后勤和成本的，而不是固有的技术问题。虽然异源筛查还没有被广泛使用的学术团体，以确定配体，一间私人公司使用同样的技术来识别配体的100人的OR ​​值29。不幸的是，这些数据仍然是专有的。 这里所概述的高通量荧光素酶测定具有超过用于评估或激活的替代方法的几个优点。虽然respon本地嗅感觉神经元的SES使用电和钙成像进行了测量，这些技术有困难戏弄除了这或将导致由于对嗅觉神经元反应特性的重叠神经元的反应。虽然敲在GFP-标记的受体型30,31，通过腺病毒递送的特异性受体鼠嗅觉神经元32,33，或记录后进行RT-PCR 17,24,33可以连接到记录单受体类型，这些方法低通量和不适合于大型画面。异源筛查系统更具扩展性，以及两种主要形式被发现在文学：cAMP途径记者和三磷酸肌醇（IP3）途径记者。一旦刺激气味，OR值激活A Gαolf转导信号级联导致生产环磷酸腺苷（cAMP）的12。通过共转染交流电的控制下，萤火虫荧光素酶报告基因AMP应答元件（CRE），荧光素酶的生产可测气味响应的函数，允许进行定量或激活。或激活也可通过共表达G蛋白如Gα15/16或A Gα15-OLF嵌合体24,25,34相连的IP3通路。我们选择了这里提出的测定法基于以下三个因素：（1）共表达RTP1用的Rho-标记的嗅觉受体改善嗅觉受体在细胞表面19,27的表达; （2）使用的cAMP应答报告基因可用于测量或通过规范的第二信使通路的激活;和（3）的测定是非常适合于高通量筛选。 这样的高通量荧光素酶测定法是适用于各种研究有价值的嗅觉的字段。第一，大量的ORs可以确定一个SP受体激活图案进行筛选针对单一的加臭剂ecific气味。这种类型的研究发现OR7D4作为或负责响应类固醇气味雄8。相反，一种或可以以确定的受体响应曲线图10来筛选对加臭剂的一个面板。当候选嗅气味/或通过对这些屏幕识别，相互作用可以通过进行剂量反应实验检查或对气味的浓度增加的反应得到证实。剂量响应曲线也可以评估一个或遗传变异如何影响体外加臭剂的反应8,9,11,35，和这些研究可以扩展到种间OR变化，从而对受体的进化的跨物种和因果突变的进化的检查36,37，最后，此测定法可用于筛选气味拮抗剂能够拮抗或响应于对一个已知的加臭剂/受体对38,39的特定气味。总之，这种高通量的荧光素酶测定法是适用于一系列的研究，这将有助于表征或激活模式，并提供一个更好的理解气味编码的嗅觉系统。

<table border="0" cellpadding="0" cellspacing="0" style="width:685px;" width="685">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">Hana3A cells</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">RTP1S-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:265px;">M3-R-pCI</td>
			<td style="width:135px;">Avaiable from the Matsunami Laboratory upon request</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pCRE-luc</td>
			<td style="width:135px;">Agilent</td>
			<td style="width:119px;">219076</td>
			<td><a href="http://www.chem.agilent.com/store/ProductDetail.aspx?productID=219076%28Agilent%29">LUC</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">pSV40-RL</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2231</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-vectors-and-cell-lines/prl-renilla-luciferase-control-reporter-vectors/">RL</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Media, Eagle</td>
			<td>Sigma Aldrich</td>
			<td>M4655</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m4655?lang=en&amp;region=US">MEM</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">FBS</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/16000044?ICID=search-16000044">FBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (without Ca2+ and Mg2+)</td>
			<td>Cellgro</td>
			<td>21-040-CV</td>
			<td><a href="http://cellgro.com/products/balanced-salt-solutions/phosphate-buffered-saline/phosphate-buffered-saline-1x-2.html">PBS</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin (0.05% Trypsin EDTA)</td>
			<td>Life Technologies</td>
			<td>25300</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/25300054?ICID=search-product">Trypsin</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CD293</td>
			<td>Life Technologies</td>
			<td>11913-019</td>
			<td><a href="http://products.invitrogen.com/ivgn/product/11913019?ICID=search-11913019">CD293</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">96 well PDL white/clear plate</td>
			<td style="width:135px;">BD BioCoat</td>
			<td style="width:119px;">356693</td>
			<td><a href="http://www.thomassci.com/Supplies/Tissue-Culture-Cellware/_/BD-Biocoat-Poly-D-Lysine-Culture-Dishes?q=356693">plates</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Lipid transfection reagent: Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td><a href="https://products.invitrogen.com/ivgn/product/11668019?ICID=search-11668019">Lipofectamine</a></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:265px;">Firefly luciferase substrate, firefly luciferase quencher/Renilla luciferase substrate: Dual-Glo Assay</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">E2980</td>
			<td><a href="http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_glo-luciferase-assay-system/?__utma=1.1932750276.1376486173.1376486173.1376486173.1&amp;__utmb=1.1.10.1376486173&amp;__utmc=1&amp;__utmx=-&amp;__utmz=1.1376486173.1.1.utmcsr=google|utmc">dual glo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synergy S2&#160;</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">SLAD</td>
			<td><a href="http://www.biotek.com/products/microplate_detection/synergy2_multimode_microplate_reader.html">BioTek S2</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">Microplate reader software: Gen5 Data Analysis Software</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">Gen5</td>
			<td><a href="http://www.biotek.com/products/microplate_software/gen5_data_analysis_software.html">Gen5</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">BIOSTACK</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">BIOSTACK2WR</td>
			<td><a href="http://www.biotek.com/products/microplate_software/biostack_microplate_stacker.html">BioStack</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Multiflo</td>
			<td style="width:135px;">BioTek</td>
			<td style="width:119px;">MFP</td>
			<td><a href="http://www.biotek.com/products/liquid_handling/multiflo_microplate_dispenser.html">MultiFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">4414</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_4_e.html">GripTips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">300ul GripTips ViaFlo96</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6433</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">12.5ul GripTips 384 XYZ</td>
			<td style="width:135px;">Integra</td>
			<td style="width:119px;">6403</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/pipette_tips_griptips_XYZ_4_e.html">XYZ tips</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">384ViaFlo</td>
			<td style="width:135px;">Integra</td>
			<td>6030</td>
			<td><a href="http://www.integra-biosciences.com/sites/us/96_384_channel_pipette.html">384ViaFlo</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer</td>
			<td>Macherey Nagel</td>
			<td>740797.1</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/d2650?lang=en&amp;region=US">DMSO</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">forskolin</td>
			<td>Enzo Life Sciences</td>
			<td>BML-CN100-0010</td>
			<td><a href="http://www.thomassci.com/007635F0-AA41-4B24-B46A-05723D685997/_/FORSKOLIN-10MG?q=c002e24">FOR</a></td>
		</tr>
	</tbody>
</table>

high-throughput analysis of mammalian olfactory receptors: measurement of receptor activation via luciferase activity

创建的气味嗅觉受体激活的独特和重叠的图案，让一个家庭的约1,000名鼠和400人的受体识别数以千计的气味的。加臭剂的配体已经公布人类受体1-11少于6％。这种缺乏数据的部分原因是由于功能上的困难表达这些受体在异源系统。在这里，我们描述了用于表达大多数嗅觉受体家族中Hana3A细胞，然后用荧光素酶报告基因分析嗅觉受体活化的高通量评估的方法。此法可用于（1）屏幕对嗅觉受体面板的气味的面板; （2）通过剂量反应曲线确定气味/受体相互作用;和（3）中的受体的变体比较受体的激活水平。在我们的样本数据，328的嗅觉受体进行了筛选对26的气味。气味/受体对具有不同的反应得分均选择位泰德和剂量反应测试。这些数据表明，一个屏幕是丰富对加臭剂/受体对，将通过剂量反应实验， 即受体具有一种加臭剂善意响应的有效方法。因此，这种高通量荧光素酶测定法是表征嗅觉受体 - 一个基本朝向气味编码在哺乳动物嗅觉系统的一个模型的步骤的一种有效方法。

一个主屏幕测试328对手术室26气味在100μm的浓度。这种气味浓度已被证明有效激活OR值与已知配体10的相当大的比例。首先，标准化的荧光素酶活性计算方式为将萤火虫荧光素酶的阅读由海肾萤光素酶的阅读。接着，基线值的计算中减去归一化的荧光素酶的读数用于从归一化的荧光素酶的读数没有气味控制每个孔（ 图1）。剂量反应曲线上48加臭剂进行与/或对随机分布在?...

Watch this Scientific Journal Video about High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity at JoVE.com

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

嗅觉受体的激活模式进行编码气味的身份，但缺乏公布的数据识别气味的配体哺乳动物的嗅觉受体的阻碍气味编码进行了全面的发展模式。这个协议描述了一种方法，以促进嗅觉受体的配体和受体活化的定量高通量鉴定。

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 ...

high-throughput-analysis-mammalian-olfactory-receptors-measurement

Research

JoVE Journal

Neuroscience

12.3K 次观看.  Monell Chemical Senses Center. 嗅觉受体的激活模式进行编码气味的身份，但缺乏公布的数据识别气味的配体哺乳动物的嗅觉受体的阻碍气味编码进行了全面的发展模式。这个协议描述了一种方法，以促进嗅觉受体的配体和受体活化的定量高通量鉴定。 

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

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

FIM Imaging and FIMtrack: Two New Tools Allowing High-throughput and Cost Effective Locomotion Analysis

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to a certain degree, many animals have to be analyzed, to obtain statistically significant data. This in turn requires a computer assisted automated quantification of locomotion patterns. To obtain high contrast images of almost translucent and small moving objects, a novel imaging technique based on frustrated total internal reflection called FIM was developed. In this setup, animals are only illuminated with infrared light at the very specific position of contact with the underlying crawling surface. This methodology results in very high contrast images. Subsequently, these high contrast images are processed using established contour tracking algorithms. Based on this, we developed the FIMTrack software, which serves to extract a number of features needed to quantitatively describe a large variety of locomotion characteristics. During the development of this software package, we focused our efforts on an open source architecture allowing the easy addition of further modules. The program operates platform independent and is accompanied by an intuitive GUI guiding the user through data analysis. All locomotion parameter values are given in form of csv files allowing further data analyses. In addition, a Results Viewer integrated into the tracking software provides the opportunity to interactively review and adjust the output, as might be needed during stimulus integration. The power of FIM and FIMTrack is demonstrated by studying the locomotion of Drosophila larvae.

FIM is a novel, cost effective imaging system designed to track small moving objects such as C. elegans, planaria or Drosophila larvae. The accompanying FIMTrack program is designed to deliver fast and efficient data analysis. Together, these tools allow high-throughput analysis of behavioral traits.

FIM成像和FIMtrack：两个新的工具允许高吞吐量和成本效益分析步态

The analysis of neuronal network function requires a reliable measurement of behavioral traits. Since the behavior of freely moving animals is variable to ...

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 ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

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. ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.