Name: live-cell measurement of odorant receptor activation using a real-time camp assay
Uploaded: 2017-10-02T14:00:00.000+00:00
Duration: 9 min 11 s
Description: Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

这项工作得到了中国国家科学基金会 (31070972)、上海市科学技术委员会 (16ZR1418300)、上海市教育委员会创新研究小组项目、上海东部学者计划 (J50201) 和中国国家基础研究计划 (2012CB910401)。

1. Hana3A 细胞的培养和维护 <ol> <li> 将10毫升的最小必需培养基 (FBS)、100和 #181 的细胞维持在10% 胎牛血清中; g/毫升青霉素-链霉素, 以及1.25 和 #181; g/毫升两性霉素 B 在100毫米细胞培养皿在37和 #176; C 细胞培养孵化器与 5% CO 2 。每隔一段, 添加1和 #181; 嘌呤以保持质粒的稳定转染 (参见 简介 ). 
	注意: 在第二类生物安全柜中执行涉及细胞培养的所有步骤, 以确保无菌环境. </li>
	<li> 亚文化的比例为10-20% 在100毫米的菜肴每2-3 天. </li>
</ol> 2。用于转染的电镀细胞 <ol> <li> 在相衬显微镜下观察 Hana3A 细胞, 以确保细胞活力并估计汇流. </li>
	<li> 从细胞培养皿中吸取所有培养基. </li>
	<li> 通过在盘子上加入10毫升磷酸盐缓冲盐水 (pbs) 来洗涤细胞, 旋转盘子, 并吸出 pbs. </li>
	<li> 在板上加入3毫升的0.05% 胰蛋白酶-乙烯二胺乙酸酸 (EDTA)。混合1分钟或直到所有细胞漂浮. 
	注意: 在必要的显微镜下观察细胞从底板底部分离的进展情况. </li>
	<li> 在 10% FBS 和吸管上加5毫升的记忆液来破坏胰蛋白酶, 从而分解细胞块. 
	注: 对于在转染前电镀, 所使用的培养基不应含有抗生素。添加抗生素可降低转染效率. </li>
	<li> 将适当数量的电池转移到 15 mLl 管, 具体取决于要转印的板材数量。对于每个96孔板, 板 2 x 10 6 单元格, 或约1/5 的100% 汇合 100 mm 盘, 每井提供 2 x 10 4 单元格数, 或每井大约15-30% 汇合点的密度。离心管在 200 x g 为5分钟, 并吸取上清, 而不干扰细胞颗粒. 
	注: 计算正确数量的细胞被镀上 96-井板, 以避免 overgrowing 或 undergrowing 细胞之前的刺激. </li>
	<li> 在15毫升管和吸管中加入适量的 10% FBS, 以分解细胞块。对于每个96井板, 重6毫升的记忆与 10% FBS 的细胞. 
	注意: 小心不要在管子里产生气泡. </li>
	<li> 将悬浮的单元格添加到库中。使用多通道吸管, 吸管50和 #181; L 的细胞到每一个96井板的井。在37和 #176 的夜间孵育; C 与 5% CO 2 . </li>
</ol> 3。转染质粒 <ol> <li> 在转染前, 观察镀膜细胞以确保在相衬显微镜下每井大约30-50% 的适当汇合, 并返回孵化器. </li>
	<li> 预先准备好ρ标记或构造 11 , 21 , 22 , 28 , 辅助因子构造 (RTP1S 22 , 36 和 M3R 23 ) 和生物传感器变体构造 29 30 由 miniprep。通过分光光度计对 dna 浓度进行量化, 并根据需要调整质粒 dna 浓度 ( 例如 , 以 100 ng/和 #181; L). </li>
	<li> 转染混合物的制备 <ol> <li> 在500和 #181 中准备质粒转染混合物; 根据 表 1 , 每个 96-井板的记忆体 L。
		注: 当不同的口服补液盐在相同的96孔板上进行测试时, 转染混合物的体积和添加的质粒 DNA 的数量必须适应于在给定或. </li>
		<li> 在500和 #181 中用18和 #181 的试管制备转染混合试剂; 我的记忆. </li>
	</ol> </li> <li> 移将质粒混合物与转染混合物混合。室温孵育15分钟. </li>
	<li> 停止反应, 加入5毫升的记忆与 10% FBS. </li>
	<li> 在细胞培养罩中摊开一层厚的无菌纸巾。从孵化箱中取一个96井板。轻轻地、反复地在纸巾上把盘子倒过来敲击, 使介质完全被纸巾吸收. 
	注意: 不要用力或突然敲打盘子, 因为你可能会失去细胞. </li>
	<li> 传输50和 #181; 将混合转染混合物的 L 转到96井板的每个井中, 并在夜间孵育18-24 小时, 在37和 #176; C 与 5% CO 2 . 
	注: time-managed 转染和刺激计划应在测量之前, 超过一个 96-井板在一个实验中测试, 以使所有的板块测量后, 相同的转染时间和刺激曝光时间. </li>
</ol> 4。使用实时野营试验的刺激和测量或活动 <ol> <li> 在相衬显微镜下观察转染细胞, 以确保每井50-80% 的正确汇合并返回孵化器. </li>
	<li> 将10毫米羟乙基 piperazineethanesulfonic 酸 (HEPES) 和5毫米葡萄糖添加到汉克和 #39 的平衡盐溶液 (HBSS), 以制备刺激培养基. </li>
	<li> 解冻 real-time 阵营化验基质试剂等分在冰上。在-80 和 #176 上贮存底物试剂; C 在55和 #181; 在 PCR 管中每管 L。每盘使用1管. </li>
	<li> 2% 平衡解决方案通过混合55和 #181; 基体试剂和2750和 #181 的 l; HBSS/HEPES/葡萄糖溶液. </li>
	<li> 在长凳上摊开一层厚的纸巾。轻轻地反复敲击纸巾上的盘子, 使转染介质完全被纸巾吸收. </li>
	<li> 移50和 #181 清洗细胞; HBSS/HEPES/葡萄糖溶液对每一个井. </li>
	<li> 轻轻地反复从96井板上 HBSS/HEPES/葡萄糖溶液. </li>
	<li> 吸管25和 #181; 每井2% 平衡溶液, 在室温下孵育 2 h. </li>
	<li> 提前准备1米气味库存解决方案, 并在-20 和 #176 中存储, 直到使用. </li>
	<li> 在孵化时间结束之前, 将气味的库存溶液稀释到 HBSS/HEPES/葡萄糖刺激培养基中的工作浓度. 
	注: 在这一步骤中制备的气味稀释的浓度应加倍, 以便在每个井中产生正确的最终浓度. </li>
	<li> 使用化学发光板读写器和在气味之前, 测量板材的基发光水平连续2次, 以1000年毫秒为单位的速率 34 . </li>
	<li> 快速从板读取器中取出板, 并添加25和 #181; 气味稀释每口井, 并立即启动所有井的连续发光测量20周期在30分钟内 
	注意: 在同一板块中使用不同的气味和/或不同浓度的同一气味时, 小心移液器避免污染相邻的水井. </li>
</ol> 5。数据分析 <ol> <li> 从化学发光板读取器软件导出数据. </li>
	<li> 使用公式计算每个时间点的规范化或响应 
	(发光的 N 和 #8211; 发光 基 )/(发光 最高的 和 #8211; 发光 基底 ) 在其中 N = 某一井的发光值; 基底 = 两个基底的平均发光值发光值;最高 = 一盘或一组板材的最高发光值. 
	注: 根据实验的目的, 可采用可选的图形表示。例如, 对于筛选分析 (参见 图 1 ) 和生成浓度-响应曲线, 在动态测量期间某一特定时间点的发光值 (如最大值或最终值, 可以使用. </li>
</ol>

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作者没有什么可透露的。

在接触到某气味时, 准确地测量或激活是破译嗅觉信息编码的第一步。本研究中所示的实验表明了一个例子, 说明如何使用一个体外或表达系统来识别人类或有气味的化学物质的反应性口服补液盐, 并随后表征该受体药理学使用各种浓度的化学物质。我们的结果证实 OR5AN1 作为一个善意受体的麝香。这与以前的报告39一致, 并提供了进一步的证据, 推测只有少数的受体参与了嗅麝香气味2740。与 Akuhara et al.的筛查数据相比, 在类似或异源表达系统上的荧光检测结果显示, 我们的筛选效果略差的信噪比。除了所涉及的不同技术所固有的差异, 输出的差异可能是由于我们使用了较低的麝香浓度 (30 µM vs. 100 µM)。事实上, OR5AN1 对麝香的浓度-反应曲线的 EC50值与我们通过 real-time 阵营法获得的结果是相同的数量级, 与佐藤-Akuhara et al.在其荧光化验系统中获得的数值相同,即使在使用不同的检测方法时, 也会显示相同的异源或表达系统的可比结果。在另一项研究中, 我们小组发现, real-time 阵营系统可能比荧光报告基因系统更敏感, 在评估 OR2T11 受体选择性, 鉴于少数气味只活跃在前者, 但不在后者系统35。与 Geithe et al.报告的一系列数据进行比较时31对 OR1A1 (+)-芹, 我们的 OR5AN1 对麝香的数据显示了到达高原所需时间的延迟, 这可能是由不同种类的口服补液盐和气味所使用的直接结果。在其他 real-time 阵营分析数据从我们的小组报告, 我们也显示了不同的时间峰值取决于口服补液盐和气味34,35。另外, 不同的细胞系用于表达口服补液盐, 不同的基质使用, 不同的 real-time 阵营生物传感器变体质粒, 不同的敏感性的化学发光板读取器, 和/或不同的实验条件, 如变化室温, 可能会导致发光输出的变化。
real-time 阵营化验显示了一些比其他测量或活化方法的优势。首先, 类似荧光记者基因检测, real-time 阵营化验提供了额外的体外手段的高通量识别或曲目响应气味。大规模的受体和/或气味屏幕的 real-time 阵营的分析可以提供大量的信息的受体-气味配对与使用少量的板块。当先进的 luminometers 和移机器人可用时, 这里描述的96井协议可以很容易地升级到384井格式, 其中更少的质粒 DNA 和试剂是每单位的数据输出所必需的。第二, real-time 营法能够实时测量营地内细胞浓度的变化。虽然大多数用于测量或活动到目前为止的检测都是荧光-或 chemiluminescence-based 的终点发现需要细胞裂解, real-time 营化验 isimplemented 下 non-lytic, 活细胞条件更类似的内生情况, 并能够监测难民营水平的变化。第三, 执行 real-time 方法需要较短的时间。另一种高通量分析方法或功能, 依赖于一个荧光的记者基因表达, 需要额外的4小时后, 气味刺激完成一个实验。在某些情况下, 长时间的气味潜伏期可能会对细胞产生潜在的气味毒性, 在瞬态照射下有效缓解。此外, 在较长时间的试验中, 在同一板块中使用不同的气味和/或不同浓度的同一气味时, 相邻油井的挥发污染几率也增加。气味添加后立即检测, 使 real-time 阵营的试验快速和可靠的范例。
real-time 阵营测试的限制为测量或活化是如下。首先, 我们的体外检测是基于一个 HEK293T-derived 细胞系, 它缺乏本机嗅觉神经元的许多内生分子。此外, 可溶性蛋白和酶的转化发生在鼻腔粘液可能与气味和/或影响或对气味的亲和力。因此, 在异源系统中的激活可能不同于在体内的灵敏度和气味调节的情况。其次, 对某一特定气味的口服补液盐的鉴定需要至少一个特定物种的完整或汇辑。口服补液盐是一个大家庭的受体, 人和老鼠的数量或蛋白质是非常大的4,5,41。有相当多的时间和精力可以参与克隆每一个或所有感兴趣的剧目。第三, 虽然或外源表达系统包括或序列修改 (如ρ标签) 和一些关键或辅助蛋白质 (如 RTP1S 和 M3R), 我们怀疑并非所有的口服补液盐在功能上表达的细胞膜HEK293T-derived 细胞。因此, 由于某些口服补液盐可能只是在细胞表面表达得不好, 无法识别其配体, 因此没有对某些气味的反应, 就没有必要排除或响应体内。因此, 只要可能的 real-time 阵营应该与其他在体外和体内的方法结合使用, 以获得更有说服力的结果。
在 real-time 营区试验中, 应特别注意几个关键步骤。首先, 在一般的发光实验中, 温度是影响实验结果的一个关键因素, 因为温度的升高会降低基础和诱导的光输出水平, 温度增加光输出。因此, pre-equilibrating 板的稳态工作温度为仪为了避免温度变化对结果的影响, 必须在气味之前加入 nt。其次, 应根据实际情况调整 real-time cAMP 测定基质试剂的浓度。当基底发光不能达到化学发光板读取器的最低检测阈值时, 应考虑增加基片浓度以增加信号。最后, 尽管是黏附细胞线, Hana3A 细胞可能分离在实验期间。当吸气或添加培养基时, 将吸管尖端放在井侧, 可以减少细胞单层的破坏。同样重要的是, 在适当和均匀的密度下, 以避免 overgrowing 细胞的细胞密集斑点的细胞往往剥离在介质的变化。
除了识别或 (s) 气味的利益, real-time 阵营的分析也可以用来筛选同源配体的给定或当图书馆的化学品是可用的。最后, 当使用适当的细胞类型和转染条件时, 该方法还可以检测表达或内源性化学激活, 并给予激动剂和拮抗药的浓度依赖性反应曲线。

嗅觉在动物的生存中起着重要作用, 因为它们依赖嗅觉才能获得食物, 避免捕食者和危险, 区分物种, 并选择交配1,2。这些功能的实现取决于气味受体 (口服补液盐), 这是单独表达在纤毛表面的嗅觉感觉神经元 (OSNs) 位于嗅觉上皮 (OE)。口服补液盐组成的 G 蛋白偶联受体 (化学) 超家族的最大的家庭与大约400和1200多样或基因在人和小鼠, 分别3,4,5。气味激活的口服补液盐, 通过嗅 g 蛋白 (g阵线) 和 III 型腺苷 (ACIII) 的顺序激活, 导致细胞内 cAMP 水平增加。细胞内 cAMP 的增加可以作为第二信使, 在细胞表面打开核苷酸门控通道, 触发阳离子的流入, 包括 Ca2 +和动作电位, 并最终启动神经-潜在的传播和嗅觉知觉。检测和鉴别大量气味的过程被认为是嗅觉知觉的第一步6,7。
由于降压和一周半跳8首次成功地克隆了气味受体, 阐明了由口服补液盐引发的嗅觉知觉机制, deorphanization 或家族成为该领域的热点之一。各种体内, ex 体内和体外测量或激活方法已被报告9,10,11,12。传统的方法, 使用 Ca2 +成像后, 单细胞 RT PCR 在 OSNs 启用了识别不同的口服脂肪族气味13,14,15。最近, large-scale 转录分析的问世促进了更高通量的在体内方法的发展。肯塔基试验鉴定了丁香酚和麝香反应的老鼠, 使用了 S100a5-tauGFP 报告鼠株和微阵列分析9。根据气味暴露后的下降或 mRNA 水平, 梦想技术采用了一种组的方法来确定或激活脊椎动物和无脊椎动物物种的剖面图16。同样, 考虑到 S6 在神经元激活中的磷酸化, Matsunami 组从磷酸酯核糖体 immunoprecipitations 测序基因, 以确定反应灵敏的口服补液盐12。最后, 该组报告了超级嗅探小鼠, 可以作为一个平台, 研究气味编码在体内, 称为 MouSensor 技术17。
在体外领域, 培养 OSNs 的挑战使一个异源表达系统模仿或功能表达在体内一个理想的解决方案, 进行 large-scale 筛选气味化学品的口服补液盐。然而, 因为培养的细胞系 non-olfactory 起源不同于当地的 OSNs, 或蛋白质被保留在内质网并且不能交通对血浆膜, 导致或退化和损失受体作用18,19. 为了解决这一问题, 在外源细胞系中对细胞膜的复制或功能表达做了大量的工作。Krautwurst et al.首先将前20氨基酸的视 (ρ标签) 附加到 N 终端或蛋白质, 这促进了一些口服补液盐在人类胚胎肾脏 (HEK) 细胞的细胞表面表达,20。通过对单 OSNs、斋藤et al的基因表达 (SAGE) 库分析进行序列分析。首先克隆受体转运蛋白 (RTP) 家族成员, RTP1 和 RTP2, 和受体表达增强蛋白 1 (REEP1), 促进或贩运到细胞膜和增强气味介导的口服补液盐在 HEK293T 细胞的反应21. 根据这些发现, Matsunami 集团成功地建立了 Hana3A 细胞系, 稳定地转染了 RTP1、RTP2、REEP1 和 Gα阵线HEK293T, 并瞬时转染了ρ标记的口服补液盐, 用于高效或功能表达.随后的研究揭示了 1) 一种较短的形式的 RTP1, RTP1S, 可以更有力地促进或功能比原 RTP1 蛋白和 2) 类型3毒蕈乙酰胆碱受体 (M3R), 可以加强或活动通过抑制β-arrestin-2招聘, 这两个被引入到外源表达系统优化实验输出22,23。
几种检测方法已被用来量化受体激活在异源系统。分泌的胎盘碱性磷酸酶 (SEAP) 检测工作与一个记者酶转录调节的阵营反应元素 (CREs), 使它成为一个有吸引力的选择评估或激活。荧光在培养基样品中容易地被发现在培养媒介以后与 SEAP 检测试剂24。使用这种方法, 口服补液盐的功能以及第二类化学受体表达的 OE-微量胺相关受体 (TAARs) 已被描绘成25,26,27。另一种常见的方法, 荧光检测, 使用了萤火虫荧光报告基因的控制下的阵营反应元件 (综合考试)。测量荧光生成的发光提供了一种有效且健壮的方法来量化或激活10、11、28。
实时营区检测也被广泛应用于动态监测外源或内受体的功能。这种先进的分析的一个例子是利用基因编码的生物传感器变体, 它拥有一个阵营结合的领域融合到一个突变形式的荧光。当阵营束缚, 构象变动导致激活荧光, 发光从然后可以被测量用化学发光的读者29,30。据报道, real-time 营技术适用于 HEK293 和 NxG 108CC15 细胞中人类气味受体的 de-orphaning驴子 = "xref" >> 31,32,33, 以及在 HEK293T-derived Hana3A 单元格34,35。Krautwurst 组还详细描述了 real-time 阵营技术适合双向 large-scale 或筛选方法32,33。
在这里, 我们描述了一个用于测量或激活使用 real-time cAMP 测定 Hana3A 细胞的协议。在本协议中, pre-equilibrated 活细胞的发光是动力学测量的30分钟后, 特定的挥发性化合物的治疗, 代表一个更有效和准确的分析或活化, 不太容易在细胞环境中发生的工件, 具有长时间和气味诱导的细胞毒性。这种 real-time 的测量允许 large-scale 筛选的口服补液盐和配体, 以及表征特定或配基对的兴趣。使用这种方法, 我们成功地确定了 OR5AN1 作为麝香化合物麝香受体, 通过对379人体口服补液盐进行筛选, 并随后确认阳性筛查结果。

<table><tbody><tr><td>Amphotericin B</td><td>Sigma</td><td>A2942</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10099-141</td><td></td></tr><tr><td>GloSensor cAMP reagent</td><td>Promega</td><td>E1290</td><td></td></tr><tr><td>pGloSensor-20F cAMP plasmid</td><td>Promega</td><td>E1171</td><td></td></tr><tr><td>Hana3A cells</td><td></td><td></td><td>available from authors upon request</td></tr><tr><td>HBSS, without calcium or magnesium</td><td>GIBCO</td><td>14175095</td><td></td></tr><tr><td>HEPES</td><td>Hyclone</td><td>SH30237</td><td></td></tr><tr><td>Lipofectamine2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>M3R plasmid</td><td></td><td></td><td>cloned into a mammalian expression vector such as pCI</td></tr><tr><td>MEM, with EBSS and L-glutamine</td><td>Hyclone</td><td>SH30024</td><td></td></tr><tr><td>Muscone</td><td>Santa Cruz</td><td>sc-200528</td><td></td></tr><tr><td>Musk tibetene</td><td>Sigma-Aldrich</td><td>S359165</td><td></td></tr><tr><td>OR plasmids</td><td></td><td></td><td>cloned with a Rho-tag into a mammalian expression vector such as pCI</td></tr><tr><td>PBS, without calcium or magnesium</td><td>Cellgro</td><td>21-040-CV</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Plasmid miniprep kit</td><td>Tiangen</td><td>DP103-03</td><td></td></tr><tr><td>Puromycin</td><td>Sigma</td><td>P8833</td><td></td></tr><tr><td>RTP1S plasmid</td><td></td><td></td><td>cloned into a mammalian expression vector such as pCI</td></tr><tr><td>Trypsin-EDTA</td><td>Hyclone</td><td>SH30236</td><td></td></tr><tr><td>0.2-mL PCR tube</td><td>Axygen</td><td>PCR-02-C</td><td></td></tr><tr><td>1.5-mL Eppendorf tube</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>15-mL 17 mm x 120 mm conical tube</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>8-well and/or 12-well multichannel pipetman</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>96-well flat-bottomed white cell culture plate</td><td>Greiner</td><td>655098</td><td></td></tr><tr><td>100 mm x 20 mm cell culture dish</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Class II biological safety cabinet with laminar flow</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator, with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge, with swinging bucket rotor for 15-ml conical tubes</td><td></td><td></td><td></td></tr><tr><td>Infinite F200 plate reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Phase-contrast microscope with x10 and x20 objectives</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Sterile reagent reservoirs for multichannel distribution</td><td></td><td></td><td></td></tr><tr><td>Sterile paper towel</td><td></td><td></td><td></td></tr></tbody></table>

live-cell measurement of odorant receptor activation using a real-time camp assay

哺乳动物气味受体 (或) 家庭的巨大尺寸在许多挥发性化学物质中发现其同源配体存在困难。为了有效和准确地 deorphanize 口服补液盐, 我们结合使用异种细胞线表达哺乳动物口服补液盐和基因修饰的生物传感器质粒来测量营地生产的下游或实时激活。这种方法可以用于筛选气味对口服补液盐和反之亦然. 通过对各种气味浓度的测试, 产生浓度-反应曲线, 可以证实从屏幕上的正气味受体相互作用。在这里, 我们使用这种方法进行高通量筛选的气味化合物对人类或图书馆表达的 Hana3A 细胞, 并证实, 积极反应受体是同源受体的化合物的兴趣。我们发现这种高通量的检测方法在评估或激活时是有效和可靠的, 我们的数据提供了它的潜在用途或功能研究的一个例子。

麝香是天然麝香的主要芳香成分。最近的研究发现 OR5AN1 作为人类受体的麝香和其他环麝香化合物的同源性的鼠标或, MOR215-1, 克隆从麝香反应性肾小球的行为小鼠37,38。通过筛选人类或曲目, 我们的小组和 Touhara 组也确定 OR5AN1 作为主要受体的两个环麝香化合物, 酮和麝香, 分别使用荧光化验系统38,39.
使用这里描述的 real-time 阵营分析, 我们筛选了人或剧目反对30µM 麝香 (图 1)。在379人筛查中, OR5AN1 出现了对麝香的一个突出的反应, 而其他口服补液盐和负控制 (no-odor 控制和空的矢量控制) 没有显示出重大的反应。阳性对照试验 OR5AN1 对30µM 麝香麝香, 一个配体 OR5AN1 (未公布的数据), 并被用来正常化口服补液盐的反应麝香。对于这组数据的图形表示, 我们选取了 OR5AN1 30 µM 麝香麝香的最大发光读数的时间点。
我们接下来研究了气味-或对3不同浓度的麝香的浓度-反应关系。对于每一个浓度的麝香测试, 在前20分钟的麝香加法, 响应由 OR5AN1 逐渐增加, 然后平稳在最后10分钟 (图 2A), 而跟踪为 no-odor 加法控制 (0 µM 麝香) 保持相对平坦。高浓度的麝香诱发强或反应比较低的, 这是可区分的整个测量。利用最后的时间点从动力学测量, 我们产生了浓度-反应曲线和浓度为50% 的最大效果 (EC50) 的价值的曲线估计是20.82 µM (图 2B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55831/55831fig1.jpg"> 
图 1: 麝香的人类口服补液盐的筛选。 379独特的人口服补液盐对30µM 麝香使用 real-time 阵营化验。沿x轴的彩色块表示不同的人或族。x轴上的每一列都表示单个或类型, 但最后三列除外, 这是 OR5AN1 到30µM 麝香麝香 (阳性对照) 的响应, OR5AN1 对 HBSS/HEPES/葡萄糖溶液的响应 (no-odor 负控制),对30µM 麝香 (一个空矢量负控制), 从左到右的反应。y轴表示当响应达到正控制 (N = 3) 的最大值时, 气味加法后30分钟的正常化发光。所有响应都规范化为正控制。误差线表示标准误差平均值 (SEM)。为清楚起见, 只显示正误差线。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55831/55831fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55831/55831fig2.jpg"> 
图 2: OR5AN1 对麝香反应的动力学测量。 (a) 对不同浓度麝香和 no-odor 负控制的 OR5AN1 活化进行实时测量, 在30分钟内进行气味加法。箭头表示气味加法的时间点。(B) 在气味加后30分钟, OR5AN1 对麝香的浓度反应曲线。y-轴表示正常化的发光 (N = 3)。所有响应都被规范化为对100µM 麝香的最高响应。误差线代表 SEM.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55831/55831fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>粒</td>
			<td>每 96-井板数量 (µg)</td>
		</tr>
		<tr>
			<td>ρ-或</td>
			<td>5</td>
		</tr>
		<tr>
			<td>RTP1S</td>
			<td>1</td>
		</tr>
		<tr>
			<td>M3R</td>
			<td>0。5</td>
		</tr>
		<tr>
			<td>阵营生物传感器变异</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
表 1: 转染混合物的组分。 每 96-井板数量的ρ-或, RTP1S, M3R, 和 real-time 阵营生物传感器变体质粒。

Watch this Scientific Journal Video about Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay at JoVE.com

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

表征气味受体的功能在 deorphanization 过程中起着不可缺少的作用。我们描述了一种方法来测量气味受体的激活, 在实时使用阵营分析。

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. ...

live-cell-measurement-odorant-receptor-activation-using-real-time

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.9K 次观看.  Shanghai Jiao Tong University School of Medicine. 表征气味受体的功能在 deorphanization 过程中起着不可缺少的作用。我们描述了一种方法来测量气味受体的激活, 在实时使用阵营分析。

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

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

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 (GPCRs) 1 that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein G&alpha;olf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees 2. Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together 3. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

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 ...

科研

神经科学

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 ...

生物工程

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 ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

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 ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

实时电:采用闭环协议来探测神经动力学和超越

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

使用 cADDis 进行 cAMP 分析:单细胞到高通量筛选

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer&#160;compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 &#181;M forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

作者聚焦:使用 cADDis 生物传感器推进细胞实时 cAMP 检测

The protocol presented in this study illustrates the effectiveness of the cAMP Difference Detector In Situ in measuring cAMP through two methods. One method utilizes a 96-well plate reading spectrophotometer with HEK-293 cells. The other method demonstrates individual HASM cells under a fluorescent microscope.

使用荧光 cAMP 原位差检测器在活细胞中实时 cAMP 动力学

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is ...

生物化学

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 ...

实时 cAMP 检测气味受体激活的活细胞测量

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测定在异种细胞表面的化学感应受体的表达

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

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

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

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

实时电:采用闭环协议来探测神经动力学和超越

使用荧光 cAMP 原位差检测器在活细胞中实时 cAMP 动力学

使用荧光 cAMP 原位差检测器在活细胞中实时 cAMP 动力学

使用荧光 cAMP 原位差检测器在活细胞中实时 cAMP 动力学

气相臭受体活化的实时体外监测