Name: parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures
Uploaded: 2016-11-11T15:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

我们非常感谢我们来自日内瓦大学的同事:雅克·菲利普对这项工作的建设性意见，Ueli SCHIBLER用于与灌流系统的发展和科学的激励宝贵的帮助，安德烈利亚尼对于已经构思设计，制造和调试灌注系统，莱萨科技有限公司公司在灌流系统和滴灌biolumicorder软件开发的协助下，乔治·塞韦用于与灌流实验援助，乌苏拉Loizides - 曼戈尔德批判地阅读手稿，和安妮 - 玛丽·马赫卢夫的慢病毒制剂;到艾蒂安Lefai，萧蔷Chanon和休伯特·维达尔（INSERM，里昂）制备人主要成肌细胞;和多梅尼科黄宗泽和蒂埃里·伯尼（人胰岛移植中心，日内瓦大学医院）提供人胰岛。这项工作是由瑞士国家科学基金会批准号资助31003A_146475 / 1，硅nergia瑞士国家科学基金会批准号CRSII3-154405，基金会罗曼德倒拉河畔RECHERCHE糖尿病反，博Hjelt基金会，基金会Ernst等露西斯密德亨尼，和兴业Académique日内瓦（CD）。

伦理学声明:包含在该协议手法是由日内瓦大学医院伦理委员会和伦理委员会的SUD EST IV（协议一百一十一分之一十二）19的批准。正如我们参考16,18从商业来源描述，或者获得人胰岛从日内瓦大学医院（瑞士）在胰岛移植中心脑死亡的多器官捐献者的胰脏中分离。 1.原代细胞培养制备<ol><li>人类胰岛分离，解离和文化 注:涂料每管，塑料尖端或吸管诺医学研究实验室（CMRL），以防止胰岛或胰岛细胞粘到塑料表面上，这可导致细胞材料的显著损失介质。 <ol><li>胰岛细胞解离加入1ml富含层粘连蛋白-5 - 细胞外基质的前一天（从804G细胞作为descri衍生床每3.5厘米菜参考29）。电镀细胞之前，吸出基质并用无菌双蒸馏水洗涤培养皿3次。允许盘到层流柜下干燥5分钟。 </li><li>层流柜内，分发带有CMRL中所获得的胰岛分为15毫升管（S）。离心在272×g离心5分钟。 </li><li>吸出上清液，然后重悬沉淀在1无菌的Dulbecco磷酸盐缓冲盐水中的溶液（DPBS）预热至37℃的无钙和镁。离心在272×g离心5分钟。 </li><li>吸出上清液，重悬细胞沉淀在1ml的DPBS。 </li><li>计数的胰岛，吸管10微升从1ml的胰岛悬浮液的总数量成新3.5厘米菜。计数的胰岛的数目在显微镜下10微升下降，并从该计算中1ml的胰岛悬浮的胰岛的总数量。加入14毫升DPBS和离心细胞悬液多一个时间在272×g离心5分钟。 </li><li>胰岛细胞分离，吸出上清液，并加入1毫升细胞分离液最多为1000胰岛当量（IEQ）。将管水浴在37°C和移液器向上和向下几次每分钟，6-10分钟内轻轻拌匀小岛。 注意:要检查消化质量，吸管载玻片上悬架的2微升下降，并且所有细胞被很好地分离，在显微镜下检查，并且没有双峰或细胞团块仍然。 </li><li>通过加入14毫升冷CMRL与补充剂（10％胎牛血清（FBS），1％的反应停止L-丙氨酰-L-谷氨酰胺二肽，1％青霉素 - 链霉素（P / S），1％庆大霉素，1 ％丙酮酸钠）。离心在425×g离心5分钟。吸出上清液，并加入15mL CMRL培养基到细胞沉淀。 </li><li>重悬沉淀在小体积CMRL与补充剂。计数细胞，在显微镜下用血细胞数计，调整CMRL音量以获得〜650，000个细胞/ ml的细胞浓度。 </li><li>吸管3分隔每个100微升的液滴从在3.5 cm培养皿预涂与层粘连蛋白在步骤1.1.8中获得的分散细胞悬浮液。 注:细胞附着在培养皿中约24小时。 </li><li>孵育在组织培养箱细胞（ 图1A），在37℃在潮湿的腔室中。改变细胞的培养基中，每2-3天滴通过从每滴抽吸加入100μl并用相同体积的新鲜培养基替换。 </li></ol></li><li>人类初级成肌细胞培养及诱导分化成肌管<ol><li>组织活检，卫星细胞分离和培养成肌细胞 注意:从组艾蒂安Lefai（INSERM，法国里昂）19获得肌肉活检。 <ol><li>根据先前描述的过程30净化主骨骼肌成肌细胞。 </li></ol></li><li>区分primary人的成肌细胞为肌管<ol><li>取保存在液氮中的人类成肌细胞中的一个管形瓶（1×10 6个细胞），并通过将30秒至1分钟的小瓶在水浴中在37℃下搅拌快速解冻细胞。 </li><li>吸管细胞（1毫升）到24毫升生长培养基组成的HAM F-10补充有20％FBS，1％P / S，0.5％庆大霉素和0.2％两性霉素B </li><li>离心5分钟，在150×g的。 </li><li>用15毫升每2.5×10 5个细胞的新鲜生长培养基除去上清液和重悬细胞。 </li><li>每F75粘附烧瓶板至少2.5×10 5个细胞。保持在细胞培养箱成肌细胞在37℃和5％的CO 2。 </li><li>一旦细胞达到60-80％汇合，解离用胰蛋白酶-EDTA 0.05％的细胞为1-2分钟，并在上粘附3.5厘米培养皿2毫升生长培养基的板他们。 </li><li>达到汇合后，取出生长培养基。 </li><li>开始分化原通过在培养它们人成肌成肌管的栏2毫升含1克/ L葡萄糖，2％FBS，1％P / S，0.5％庆大霉素和0.2％两性霉素B（分化培养基）的Dulbecco改良的Eagle培养基（DMEM）的在37℃和5％CO 2的细胞培养箱中。改变介质每2至3天。 注:肌管通常在7-10天内形成。 </li><li>通过观察成肌细胞的融合成肌管polynucleated 19检查显微镜（ 图2A）下肌细胞的分化。 </li></ol></li></ol></li></ol> 2.小​​干扰RNA（siRNA）转染<ol><li>人体胰岛细胞的转染 注意:转染协议在细胞解离之后，第二天加入100μl（步骤1.1.1-1.1.10）滴进行。 <ol><li>抽吸将100μl来自每滴CMRL培养基中，用相同体积的无血清的最低必需培养基（MEM）2小时的替换吹打转染前。 </li><li>制备转染试剂的基于MEM混合和靶的siRNA（SICLOCK）或50nM的50纳米非靶向siControl根据制造商的说明。 <ol><li>一碟3滴准备2个1.5毫升管用200微升MEM每个。 </li><li>添加到这些管4微升转染试剂中的一个。 </li><li>加入的合适的siRNA原液（20μM）的第二管1微升。 </li><li>慢慢搅动这两个管上的轨道摇床5分钟，然后混合管的内容一起，搅拌20多分钟。 </li></ol></li><li>从每滴吸100μl的MEM中，用相同体积的在上一步骤，通过移液获得的转染混合物的更换。 </li><li>后在37℃温育4小时替换CMRL培养基中的转染溶液。重复步骤2.1.1-2.1.3翌日为小区重新转染。 </li></ol></li><li>人类肌管siRNA转染<ol><li>转染前，更换介质（见步骤1.2.2.8），用2毫升，每3.5厘米培养皿的新鲜分化培养基中。 </li><li>在一个无菌1.5毫升管，准备为20nM的siRNA（siControl或SICLOCK），其对应于2.4微升20μM的siRNA的溶液中，并转染试剂12μl的在100μl分化培养基稀释的混合。在室温下孵育该溶液温和搅拌15分钟。 </li><li>转染细胞在37℃114.4微升每3.5厘米培养皿和位置细胞与siRNA混合到组织培养箱中和5％的CO 2 24小时。 </li></ol></li></ol> 3.连续长期昼夜生物发光录制演出在并行与激素的分泌在活的人类原代细胞的评估<ol><li>引入生物发光昼夜记者进入人力原代细胞慢病毒转导 注:使用的慢病毒颗粒的所有步骤必须在生物安全2级机构承担的工作额外的预防措施与构成温和的潜在危险对人员和环境的代理商来执行。 <ol><li>通过记者准备的慢病毒颗粒共转染兴趣pLenti6.4的矢量/ R4R2 / V5-DEST / BMAL1吕克或pLV156-Per2基因-DLUC（分别称为BMAL1吕克和Per2基因-吕克 ，）与慢病毒载体质粒pMD2G 31和psPAX到使用聚乙烯亚胺法293T细胞（详细过程见参考文献16）。 </li><li>滴定所获得的慢病毒颗粒（可在http://lentilab.unige.ch/得到的滴定详情）。进一步的实验中，使用慢病毒滴度测距四月 10日至5月10日传感单元[TU /微升。 </li><li>地方菜的层流舱内人胰岛细胞或人成肌细胞（在30-50％汇合）等和替换用2ml新鲜补充的CMRL培养基的培养基（参见步骤1.1.7）或生长培养基（见步骤1.2.2.2），分别。 </li><li>计算感染复数（MOI）（ 即感染性颗粒（转导单位）细胞/数）。 </li><li>通过以获得一个MOI = 3吹打慢病毒溶液的菜转导原代细胞培养物（例如，65,000附着的细胞加3微升病毒溶液与6.5×10 4至100μl介质下降的效价）。 </li><li>在组织培养箱孵育过夜。介质改变第二天。 注意:转导的生物发光记录前至少4天，以人的胰岛细胞，以实现所述报告构建的足够的表达。成肌细胞是在扩张阶段转，然后发展到汇合，随后分化成肌管。 </li></ol></li><li> 人原代细胞的体外同步<ol> &lt;利&gt;添加腺苷酸环化酶激活剂的10μM的每含有先前在步骤3.1.5转导的原代细胞3.5厘米培养皿2ml培养基。 </li><li>在细胞培养培养箱孵育60分钟，在37℃。 </li><li>用2.5毫升含有100μM的荧光素的记录介质的更改包含腺苷酸环化酶活化剂的介质。 注:对于人胰岛细胞利用CMRL补充有10％FBS，1％L-丙氨酰-L-谷氨酰胺二肽，1％P / S，1％庆大霉素;人类肌管使用酚红 - 的DMEM用1克/升葡萄糖补充有2％FBS，2％L-丙氨酰-L-谷氨酰胺二肽，1％P / S，0.5％庆大霉素和0.2％两性霉素B </li></ol></li></ol>花样人分泌原代细胞生物发光昼夜录音和激素分泌型材4.平行评估<ol><li>设置长期恒定灌流和生物发光录音人类原代细胞。 注意:工作时出局IDE中的层流柜，清洁所有的接触表面，并限制培养物或培养基的暴露在空气中，以避免污染。 <ol><li>为了制备表面灌流介质，添加100μM的荧光素的培养基中。 注:对于人胰岛细胞利用CMRL补充有10％FBS，1％L-丙氨酰-L-谷氨酰胺二肽，1％P / S，1％庆大霉素;人类肌管使用酚红 - 的DMEM用1克/升葡萄糖补充有2％FBS，2％L-丙氨酰-L-谷氨酰胺二肽，1％P / S，0.5％庆大霉素和0.2％两性霉素B </li><li>层流柜内，打开包含转导，转染和同步原代细胞培养物（胰岛细胞或肌管），为上述3.5 cm的培养皿。将无菌的金属帽（内部开发）（ 图1B2）成均配备了硅胶流入/流出连接管（ 图1B1 / B5）3.5 cm的培养皿。 </li><li>将测量平台上的菜在37°C L飞行紧孵化器。通过使用一个螺接适配器（ 图1B3）固定碗碟到该平台。插入表面灌流系统的流入/流出管到盖（ 图 1B1 / B5）的适当硅胶管，并设置了泵的速度的流速约0.5毫升，每1小时培养基。 </li><li>打开记录来自光电倍增管（PMT）探测器的信号对内部开发的滴灌biolumicorder软件。选择了其中数据将被存储，并开始连续生物发光通过点击"开始"图标从各皿的记录的目录。 注意:可替换的滴灌biolumicorder软件，其它程序（ 如 LumiCycle），可用于记录从PMT检测信号。 </li><li>放置无菌6孔组织培养板中在冰上收集箱。 </li><li>打开控制所述收集井之间的自动开关的定时的控制的软件。成立时间瓦特中集（SEC）的indow。开始收集流出介质的每4小时（14,400秒;每个时间点〜2毫升）单击"运行"图标。 </li><li>吹打从每个采集传输，测量流出中早已进入无菌2ml管。保持在-20℃冷冻试管开始下一步骤之前。重复步骤4.1.5-4.1.6每24小时。 </li><li>停止生物发光录音，并通过点击相应的软件"停止"图标介质流向。取下金属盖和吸从菜残留网上平台。 </li><li>为了正常化在不同的实验中获得的分泌蛋白的值，无论是DNA提取（正常化由基因组DNA含量为肌管19），或（按总激素含量为胰岛细胞18正常化）将加入1 ml裂解酸-乙醇缓冲液菜肴。 </li></ol></li></ol> 5.测量胰岛激素和Myokine水平的外流由人原发性内分泌细胞连续灌流媒体获得<ol><li>胰岛素<ol><li>通过使用按照制造商的说明书的人胰岛素酶联免疫吸附测定（ELISA）试剂盒量化从所收集的时间点流出介质基础胰岛素水平。 </li><li>正常化的数据采集平台的每个孔中，并与总胰岛素含量的绝对量，在实验（步骤4.1.9）18的端部从酸-乙醇处理的细胞中提取。 </li></ol></li><li>白细胞介素-6（IL-6） <ol><li>量化 基础的IL-6在从所收集的时间点流出中等水平通过使用人IL-6 ELISA试剂盒按照制造商的说明进行操作。 </li><li>正常化数据到每个孔中，并在实验（步骤4.1.9）的末端基因组DNA含量收集介质的绝对音量。 </li></ol></li></ol> 6.昼夜数据集分析了Biolumines萤光及荷尔蒙分泌简介<ol><li>生物发光分析<ol><li>用随附的软件19分析生物发光轮廓。 </li></ol></li><li> JTK周期分析<ol><li>通过使用JTK_CYCLE算法分析32荷尔蒙分泌配置文件和生物发光录音效果。 </li><li>在20-24小时将昼夜周期宽度。 注意:如果该实验条件被记录在平行，能够进行配对的统计分析的实验进行比较。 </li></ol></li><li> CosinorJ分析<ol><li>可替代地，分析激素分泌，并使用CosinorJ软件28昼夜生物发光轮廓。 </li></ol></li></ol>

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A., Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+peripheral+clocks:+applications+for+studying+circadian+phenotypes+in+physiology+and+pathophysiology.">Human peripheral clocks: applications for studying circadian phenotypes in physiology and pathophysiology.</a> Front Neurol. 6, 95 (2015).</li><li>Brown, S. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock+starts+ticking+at+a+developmentally+early+stage.">The circadian clock starts ticking at a developmentally early stage.</a> J Biol Rhythms. 25 (6), 442-449 (2010).</li><li>Mannic, T., 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=Circadian+clock+characteristics+are+altered+in+human+thyroid+malignant+nodules.">Circadian clock characteristics are altered in human thyroid malignant nodules.</a> J Clin Endocrinol Metab. 98 (11), 4446-4456 (2013).</li><li>Parnaud, G., 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=Proliferation+of+sorted+human+and+rat+beta+cells.">Proliferation of sorted human and rat beta cells.</a> Diabetologia. 51 (1), 91-100 (2008).</li><li>Agley, C. 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The authors have nothing to disclose.

此处所述的实验设置是由慢病毒递送的昼夜生物发光记者到培养的人原代细胞中，接着后续体外同步和几天生物发光的连续记录，并通过在同一细胞激素分泌的平行分析。他们代表了探索的分子机制和人原代细胞的生物钟功能方面的有效方法。 供体材料的质量是可行的主组织培养的制剂的一个重要问题。人胰岛的质量应在开始实验前每次进行评估。不推荐这些实验与?...

昼夜定时系统（来自拉丁美洲"近似行乐"）已经出现在所有的光敏感的生物，如自适应机制，地球的旋转。在哺乳动物中，它在一个分层的方式组织，包括中央时钟，这是坐落在腹侧丘脑的视交叉上核和外设（或从站）是在不同的器官手术振荡器。此外，这些细胞中自主自我维持的振荡器是在主体1的几乎每一个细胞中有功能。光的信号代表的SCN神经元支配同步提示（ 授时 ），而来自SCN发出神经，体液的信号复位外设时钟。除了休息，活动节律，这反过来喂养空腹开车的周期，对于外设时钟2进一步同步。据我们目前的了解，核心时钟的分子组成是基于转录和translaTIONAL反馈回路，这是生物体之间是保守的。这包括转录激活因子BMAL1和CLOCK，它们共同激活负内核时钟PER和CRY基因的转录。 PER和CRY蛋白质含量高将通过抑制BMAL1 / CLOCK复杂的抑制自己的转录。辅助回路包括核受体REV-ERBS和RORS，这也规范BMAL1和CLOCK的转录。此外，翻译后的事件，包括磷酸化，SUMO化，乙酰化，O-GlcNAc糖基，降解和核心时钟蛋白质的入核建立了24小时的振荡周期3代表一个附加的重要的调节层。 越来越多的证据来自于啮齿动物模型研究茎和突出了昼夜系统中的代谢和内分泌功能4-5的协调的关键作用。许多的LARG电子秤转录组分析表明，馈送-空腹循环发挥周振荡器6-8的同步的中心作用。在这些研究中的协议，在啮齿动物和人类代谢组学和脂质组分析已经揭示，大量的代谢产物在组织，血浆和唾液振荡在一个昼夜方式9-11。重要的是，大多数表现出的激素在血液中5,12-13昼夜节律。此外，生产外周组织相应的激素生物钟可能局部调节激素的分泌。细胞自主昼夜振荡器在啮齿动物和人类的胰岛细胞14-16被描述。这些振荡器在调节胰岛转录发挥重要的作用和功能15,17-18。此外，由人骨骼肌管myokine分泌最近已证实表现出昼夜节律模式，它是由细胞自主oscillato调节RS在这些细胞中19可操作。 为在体内的人类研究的生理节律的几种方法已被广泛使用。例如，血浆褪黑激素或皮质醇水平以及胸部皮肤表面温度（在参考文献3,20中综述）进行了研究，以评估内源性生物钟。虽然这些方法可以研究在体内系统性的昼夜波动，他们远离提供在不同器官和组织自由运行的自主昼夜节律的可靠评估。然而，来自全身调节这种夹层会理解对这些细胞的功能的细胞内分子的时钟的具体效果的不可或缺的工具。因此，大量的努力已经进行制定体外同步永生或原代培养细胞研究人类钟表可靠的方法。重要的是，它已被证明在原代培养的皮肤成纤维细胞测量时钟特性密切地反映整个机体21的单个时钟性能。荧光和生物发光昼夜记者的发展极大地推进这种做法22-27。此外，个从不同的外围器官衍生的研究主小区时钟允许人类组织特异性时钟3,5,16,19-20,28的分子性质的研究。因此，在体外同步初级外植体或细胞生物钟的评估，通过使用生物发光的记者，表示一个非常有用的方法来研究人末梢时钟的分子组成和它们对器官功能的影响。 在本文中，我们将介绍详细的协议， 用于体外同步以及自主胞脉的影响，对人体主要的胰岛细胞和骨骼肌细胞评估节律基因表达中断对这些细胞的分泌功能。

<table><tbody><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td>For muscle biopsy digestion</td></tr><tr><td>DPBS no calcium no magnesium</td><td>Invitrogen</td><td>14190-094</td><td></td></tr><tr><td>HAM F-10</td><td>Invitrogen</td><td>41550-021</td><td>For myoblasts culture</td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270</td><td>Supplement to culture medium</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P0781-100</td><td>Supplement to culture medium</td></tr><tr><td>Gentamycin</td><td>Axon&amp;nbsp;</td><td>A1492.0001</td><td>Supplement to culture medium</td></tr><tr><td>Fungizone</td><td>Invitrogen</td><td>15290-026</td><td>Amphotericin B, supplement to culture medium</td></tr><tr><td>DMEM 1g/L glucose + Na pyruvate + glutamax&amp;nbsp;</td><td>Invitrogen</td><td>21885-025</td><td>For myotubes culture</td></tr><tr><td>DMEM 1g/L glucose -Na Pyruvate - glutamax</td><td>Invitrogen</td><td>11880-028</td><td>Recording medium for LumiCycle</td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-028</td><td>L-alanyl-L-glutamine dipeptide, supplement to recording medium</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td>Cell detachment solution, for islet cell dissociation</td></tr><tr><td>CMRL</td><td>Gibco</td><td>21530-027</td><td>Culture medium for islet cells</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360-039</td><td>Supplement to culture medium</td></tr><tr><td>15 ml High-Clarity Polipropylene Conical Tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>F75 flask</td><td>BD Falcon</td><td>353136</td><td></td></tr><tr><td>3.5 cm Petri dish&amp;nbsp;</td><td>BD Falcon</td><td>353001</td><td></td></tr><tr><td>Foskolin</td><td>Sigma</td><td>F6886</td><td>Adenylyl cyclase activator, used for synchronization</td></tr><tr><td>Luciferin</td><td>Prolume LTD</td><td>260150</td><td>Supplement to recording medium</td></tr><tr><td>OptiMEM&amp;nbsp;</td><td>Invitrogen</td><td>51985-026</td><td>Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection</td></tr><tr><td>Lipofectamine RNAiMAX reagent</td><td>Invitrogen</td><td>13778-150</td><td>Transfection reagent</td></tr><tr><td>HiPerFect reagent</td><td>Qiagen</td><td>301705</td><td>Transfection reagent</td></tr><tr><td>ON-TARGET plus siCLOCK smartpool&amp;nbsp;</td><td>Dharmacon</td><td>L-008212-00</td><td></td></tr><tr><td>ON-TARGET plus non targeting siRNA #1 (siControl)</td><td>Dharmacon</td><td>D-001810-01</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>For myotubes DNA extraction</td></tr><tr><td>RNeasy Plus Mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td>For myotubes RNA extraction</td></tr><tr><td>QIAshredder&amp;nbsp;</td><td>Qiagen</td><td>79654</td><td>For myotubes RNA extraction</td></tr><tr><td>2 ml collecting tubes</td><td>Axygen</td><td>311-10-051</td><td>To collect the medium with the perifusion</td></tr><tr><td>Tissue culture Plate, 6 Well</td><td>BD Falcon&amp;nbsp;</td><td>353046</td><td>To collect the medium with the perifusion</td></tr><tr><td>RNeasy Plus Micro kit&amp;nbsp;</td><td>Qiagen</td><td>74034</td><td>For islet RNA extraction</td></tr><tr><td>Human IL-6 Instant ELISA kit&amp;nbsp;</td><td>eBioscience</td><td>88-7066-22</td><td></td></tr><tr><td>Human Insulin Kit Mercodia</td><td>Mercodia</td><td>10-1113-01</td><td></td></tr><tr><td>Hydrochloric acid, min,37%,p.a.</td><td>Acros organics</td><td>124630010</td><td>Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>Ethanol (&gt;99.8%)</td><td>Fluka Analytical</td><td>02860-1L</td><td>Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>Human Islets for Research</td><td>Prodo Laboratories</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Heraeus</td><td>Megafuge 1.0R</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>1112A</td><td>&amp;nbsp;at 37 &amp;deg;C</td></tr><tr><td>Tissu culture hood</td><td>Faster&amp;nbsp;</td><td>SafeFastElite</td><td></td></tr><tr><td>Tissu culture incubator</td><td>Heraeus</td><td>HeraCell 150</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C, no water due to the LumiCycle installation</td></tr><tr><td>Tissu culture incubator</td><td>Heraeus</td><td>HeraCell 150</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C, no water due to the LumiCycle installation</td></tr><tr><td>Tissu culture incubator</td><td>Thermo Scientific</td><td>Hera Cell 150i</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C</td></tr><tr><td>Shaker</td><td>Heidolph Instruments</td><td>Unimax 1010</td><td>For agitation of the siRNA mix</td></tr><tr><td>LumiCycle</td><td>Actimetrics</td><td></td><td></td></tr><tr><td>LumiCycle software</td><td>Actimetrics</td><td></td><td></td></tr><tr><td>CosinorJ software</td><td>EPFL</td><td></td><td>Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/</td></tr><tr><td>Rheodyne titan MX&amp;nbsp;</td><td>ERC GmbH</td><td></td><td>Control software that controls the timing of the automated switch</td></tr></tbody></table>

parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures

生物钟是所有光敏感的生物功能，允许通过预测日常环境变化的适应外部世界。在我们的生物钟与生理的许多方面之间的紧密联系的理解相当大的进展已在该领域已经取得了在过去十年。不过，这解开在underlies人体昼夜节律振荡器功能的分子基础保持最高的技术挑战。这里，我们提供了长期（2-5天）的生物发光的记录和流出介质收集培养的人原代细胞的实验方法的详细描述。为了这个目的，我们已转导原代细胞用慢病毒萤光素酶报道即下一个核心时钟基因启动子，其允许激素分泌和昼夜生物发光的并行评估的控制。此外，我们描述的条件扰乱公关生物钟imary人体细胞转染的siRNA靶向时钟 。我们对人类胰岛分泌胰岛素的节律性的结果，人类骨骼肌细胞分泌myokine，在这里提出来说明这一方法的应用。这些设置可用于研究人末梢时钟的分子组成和分析的生理或病理生理条件下的原代细胞及其功能的影响。

胰岛激素分泌评估与并行昼夜生物发光录音从Perifused人体胰岛细胞提供生物钟的第一分子表征之后，执行在人胰岛细胞16，我们旨在研究时钟中断对胰岛功能和转录18的影响。我们建立了在分散的人胰岛细胞中的高效SICLOCK转染方案（见协议详情），这导致在时钟 mRNA的80％以上的击倒，...

Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

parallel-measurement-circadian-clock-gene-expression-hormone

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.2K 次观看.  University of Geneva Medical School, Institute of Genetics and Genomics in Geneva (iGE3). Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

视频: Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

科研

遗传学

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 ...

神经科学

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

用小鼠脾评估病原体相关分子模式对时钟基因表达的影响

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

生物工程

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

优化人肠道类肠生物用于昼夜节律研究

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

作者聚焦：多细胞系统中昼夜节律的体外研究

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

使用类器官中的生物发光报告基因监测昼夜节律振荡

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

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体外生物荧光法对乳腺上皮细胞昼夜节律的表征

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

用小鼠脾评估病原体相关分子模式对时钟基因表达的影响

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

在拟南芥原生质体转染昼夜的表型有荧光时钟记者的快速分析

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

使用类器官中的生物发光报告基因监测昼夜节律振荡

使用类器官中的生物发光报告基因监测昼夜节律振荡