This work was supported by the Intramural Research Program at the National Institute on Drug Abuse. We thank Doug Howard, Chris Richie, Lowella Fortuno, and Josh Hinkle for their contributions to developing this method.

1. 体外试验 ：从一个稳定的SH-SY5Y细胞检测SERCaMP发布<ol><li>板的SH-SY5Y-GLUC-ASARTDL（SERCaMP）在组织培养物以每表面面积的平方厘米150,000个细胞处理的平板。为96孔板，例如，种子50,000个细胞每孔（图1A）。生长SH-SY5Y细胞在DMEM（高葡萄糖，含有GlutaMAX，丙酮酸）+ 10％牛生长血清+ 1×青霉素/链霉素。 <ol><li>传代细胞的15倍（图1B）。更高的通道数尚未经过测试。 </li><li>返回细胞湿度培养箱在37℃下用5.5％的CO 2孵育过夜。 </li></ol></li><li>实验性治疗（S）前，以及通过将5微升培养上清到一个不透明的收集每一个基准样品寨96孔板。在收集，轻轻敲击板各方和漩涡，避免渐变效果。用粘合剂印章不透明板伊灵片，并储存于4℃直至酶法测定的时间。 注意：分泌GLUC-SERCaMP是在细胞培养基中非常稳定。我们以前报道GLUC活性约5-10％被损失了72小时培养后，在37℃（培养上的SH-SY5Y细胞）12。 </li><li>根据需要检查ER钙枯竭治疗的细胞（ 如环境 ，药物，遗传操作）。避免长时间暴露在外界环境（外控孵化器）作为pH值的变化会迅速发生在大气中的二氧化碳。添加HEPES到培养基中，在20毫米，来稳定pH值14，如果长时间操作是必需的。在培养液中15使用HEPES时，应避免暴露于光。 <ol><li>阳性对照：把细胞用100nM毒胡萝卜素（TG）或50μM环匹阿尼酸（CPA）4-6小时，在SH-SY5Y细胞的实验。在其它细胞系最大应答可能需要更长的孵育或不同concen化合物trations。 </li><li>阴性对照：来自父母的SH-SY5Y细胞收集培养基。在我们的经验中，背景发光该测定是从稳定的SH-SY5Y-GLUC-SERCaMP细胞基础分泌小于0.05％。 </li></ol></li><li>收集和条件培养基的储存器5微升样品在期望的时间点，如在步骤1.2中概述。 </li><li>在中期评估酶活性，如第5概述。 </li><li>检查细胞内的胰高血糖免疫印迹或发光测定法。 <ol><li>对于免疫印迹：裂解细胞在含有50mM Tris（pH值7.4），150 mM氯化钠，0.25％脱氧胆酸钠，1mM的EDTA，1％NP40改性RIPA缓冲液，和蛋白酶抑制剂。单独的上SDS-聚丙烯酰胺凝胶，并转移到0.20微米PVDF膜上。孵育膜GLUC抗体（1：2000稀释）过夜，在4℃。执行二抗孵化和膜洗涤根据用户定义的协议选择的检测试剂。 </li><li>供发光测定法（ 图2）：执行在冰上以下步骤： <ol><li>从组织培养板的孔中取出所有平台和冲洗用200μl冷PBS中。 </li><li>添加75微升含有50mM Tris（pH值7.5），150毫摩尔NaCl，1％NP40裂解缓冲液和蛋白酶抑制剂每个孔中。 </li><li>旋转板在定轨振荡器（120转），在4℃进行20分钟。 </li><li>轻轻吸取溶解物上下使用多通道移液器，避免产生气泡。将5微升裂解液为不透明板和评估，如第5条所列发光。 </li></ol></li></ol></li></ol> 2.在体外试验 ：永生化细胞与SERCaMP的瞬时转染注意：我们已观察到，瞬时转染程序可诱导的细胞应激和钝随后GLUC-SERCaMP响应。下面的方法被开发，以尽量减少转EFF<html>学分在SH-SY5Y细胞。可能需要此过程（包括选择转染试剂），用于替代细胞系的优化。如果可能，建议使用分别在3和第1中概述的稳定细胞株或病毒转导的方法。 <ol><li>板SH-SY5Y细胞在100mm培养皿在4×10 6个细胞。这个菜将足以重新播种约300孔中（96孔板形式）以下转染过程。 </li><li>转染细胞用20微克质粒DNA（编码GLUC-SERCaMP）Xfect试剂和6微升Xfect试剂。规模的DNA和Xfect试剂相应的较大或较小的培养容器。 </li><li>返回细胞培养箱中培养48小时。 </li><li> Trypsinize细胞和补种到96孔板中，每孔60,000个细胞。按照第1列出后续步骤。 </li></ol> 3.在体外试验 ：对病毒载体介导的表达GLUC-SERCaMP 帐篷“&gt;注意：腺相关病毒（AAV）载体包装16和纯化12和慢病毒的生产13先前已经报道。 <ol><li>使用下列引物和探针滴度GLUC-SERCaMP AAV：正向引物，5&#39;-CACGCCCAAGATGAAGAAGT-3&#39;;反向引物，5&#39;-GAACCCAGGAATCTCAGGAATG-3&#39;;探针（5&#39;-6-FAM / 3&#39;-BHQ-1标记的），5&#39;-TACGAAGGCGACAAAGAGTCCGC-3&#39;用于定量PCR。 <ol><li>通过线性化含有GLUC的质粒，并使用旋转柱（例如Machery-纳格尔NucleoSpin凝胶和PCR净化）纯化该DNA制备的标准曲线。通过在沿着一个质量梯状琼脂糖凝胶分离定量的DNA。从100微克/毫升，以PBS中的10 FG /毫升制备1点10稀释的DNA。计算的GLUC质粒DNA的拷贝/ ml，在每一个基于所述质粒的分子量的浓度。 </li><li>稀释所述AAV股在PBS（适当稀释将依赖于病毒制备的参数和根据经验确定）。 </li><li>使用以下条件运行在实时PCR系统进行PCR反应：95℃×5分钟，94℃×20秒，和60℃×1分钟的41个循环。利用标准曲线计算毫升拷贝/。 </li></ol></li><li>慢病毒滴度与P24伦蒂-X快速滴度试剂盒。解冻慢病毒等分冰和稀的SH-SY5Y介质中的p24调控蛋白。 <ol><li>稀释慢病毒，使得其将落入标准曲线（如1：20000或1：100）上。所需的稀释因子将基于所述慢病毒制剂参数而变化，并且必须凭经验确定。遵循制造商的指示对所有后续步骤。 </li></ol></li><li>板的细胞的96孔板。对SH-SY5Y细胞，在第1节中概述电镀程序可以遵循。对大鼠原代皮质神经元，细胞应隔离并接种于适当涂层板一小号先前描述的16。维持在补充有1×B27和500μM的L-谷氨酰胺Neurobasal培养基大鼠原代皮质神经元。执行中的一半交流隔日1次。 </li><li>转导细胞病毒。病毒转导的目标是实现低水平表达，如荧光素酶Gaussia提供健壮信号。 <ol><li> AAV转导SH-SY5Y细胞：转导以下电镀的一天。稀释的AAV至6.0×10 7的vg /微升在AAV稀释缓冲液（PBS + 0.5mM的MgCl 2的）。加入5微升稀释的AAV（3.0×10 8的vg;约6000 MOI）每96孔平板（图3A）良好。用10％漂白粉灭活病毒载体的浪费。 </li><li>大鼠原代皮层神经元的AAV转导：电镀转导后6-8天。 AAV稀释至4.0×10 6 VG /微升AAV稀释液。加入5微升稀释AAV（2.0×10 7 VG;约350 MOI）每井96孔板（图3B）。最优MOI应凭经验其它细胞类型来确定。用10％漂白粉灭活病毒载体的浪费。 </li><li> SH-SY5Y细胞的慢病毒转导：转导以下电镀的一天。稀慢病毒至20微微克/微升的p24中Hank氏平衡盐溶液（用滴定试剂盒浓度测定）。各5μl每孔的稀释的病毒添加（100皮克的p24，相当于125万​​的慢病毒颗粒，或〜1250 IFUs）96孔平板含有100微升体积。因此规模较大的格式。用10％漂白粉灭活病毒的浪费。 </li></ol></li><li>收集介质的预处理样品，并开始实验处理48小时（SH-SY5Y）或5-7天（大鼠原代皮质神经元）转导之后。 </li></ol> 4. 体内 SERCaMP分析注意：在开展任何动物的程序一定要通过您的机构，以获得适当的批准。都生存手术是有足够的麻醉无菌条件下进行。下面描述的所有程序已获得批准，并符合美国国立卫生研究院ACUC指引。 <ol><li>高压釜手术器械启动手术前（121℃，30分钟灭菌，20分钟干燥时间）。清洁的超声发生器紧随其后的所有程序，并在高压灭菌前所有仪器。保持在生存手术无菌条件。对于需要多个动物手术，擦拭用70％的酒精，ultrasonicate和胎圈之间的大鼠消毒手术器械。请参阅材料清单进行必要的手段。 </li><li>准备大鼠进行手术。这里我们使用雄性SD（180-200克）。 <ol><li>麻醉用异氟烷气体大鼠3分钟（4-5％异氟烷在1000毫升/分钟输送），然后加入甲苯噻嗪（8毫克/千克）和氯胺酮（80毫克/千克）的腹膜内注射。应用眼药膏，以防止角膜干燥。乙egin手术一旦老鼠是深度麻醉具体表现为以下尾巴或脚捏缺乏撤退反射的。 </li><li>剃略低于肋（下胸区域）到中间腹部区域腹部的区域。擦洗手术区三次，交70％的酒精和优碘擦洗。将大鼠在无菌手术单无菌手术区仰卧位。 </li></ol></li><li>稀的AAV-GLUC-SERCaMP至7.6×10 9的vg /毫升（最终浓度）。颠倒离心管充分混匀。 注意：病毒浓度的范围可以变化（ 图4）。 <ol><li>吸取105微升稀释AAV到无菌培养皿。使用30号针头收集病毒到注射器。 </li></ol></li><li>进行手术暴露肝脏和注入AAV-GLUC-SERCaMP。 <ol><li>在此之前做腹部切口，应用0.25％布比卡因的切口区域。使用手术刀，使下面的肋骨水平切口（AP近因2-3厘米）。生硬的解剖，从皮下组织分离结缔组织。 </li><li>切腹部肌肉，露出右内侧叶肝。 注：另外叶可根据最终应用注入。 </li><li>放置动物手术显微镜下，并调整以获得正确的内侧叶肝的视场。病毒注入到内侧叶实质内; 3个独立的站点，每个站点约33微升。 注：保留针组织5-10秒以下注射，以确保交付的整个注射量。 </li><li>分别缝合腹部肌肉和皮肤，用棉签添加抗生素软膏。将大鼠在恢复室，直到意识恢复了和大鼠能够保持直立的姿势。不要让老鼠（S）无人看管，直到它重新获得足够的意识，以保持胸骨斜卧。众议院单独直至切口愈合和缝线已被删除（7-14天）。 <br/&gt;注：根据美国国立卫生研究院手术后的指导方针，保持手术记录。用程序，日期，实验标识符，体重，和手术天注释笼卡。痛/窘迫，屎生产，活动和食物和水的消耗将被监测和记录3天后手术。术后镇痛通过加入对乙酰氨基酚到饮用水（450毫克/ 100毫升）提供，尽管我们不通常观察疼痛或痛苦的手术后的迹象。 </li></ol></li><li>注射后4-7天开始尾采血。用标签和前称量制备血液采集管。加入50微升肝素（1000单位/毫升），每管。 </li><li>地方大鼠在异氟烷麻醉室3分钟（4-5％异氟烷在1000毫升/分送到）。从腔室，并装在鼻锥（2-3％异氟烷以500cc /分钟提供）除去大鼠。如下尾部圆周率证明了缺乏撤退反射采血就可以开始，一旦老鼠被深度麻醉NCH​​。 <ol><li>用无菌剪刀尾（1-2毫米），切尖和滴采集血液进入预充肝素管。收集到的血液量达到大于2：1的血液肝素比（&gt; 100微升血液/ 50微升肝素）。采血卷可以基于实验设计进行调整。用湿棉签来止血粉适用于尾止血。 </li><li>库存血管在4℃，如果采集后续样品。剪刀擦拭用乙醇垫和珠集之间消毒。 </li><li>称重收集管和与肝素调整，以获得2:1的比例（血液：肝素）。这一步将正常化每种样品（表1）在肝素的量。 </li><li>离心管在2000×g离心5分钟，在4℃。转移上清液（血浆）到新的管中并储存在-80℃直至荧光素酶检测（第5部分）的时间。 注：样品达72小时之前，酶测定储存在4℃具有m上发光（图5A）inimal效果。至多3个冻融血浆样品的周期对发光没有影响（ 图5B）。 </li></ol></li><li>毒胡萝卜素施用（阳性对照）： <ol><li>通过在乙醇中稀释至2.5毫克/毫升的最终浓度制备毒胡萝卜素。注入毒胡萝卜素以1mg / kg腹腔进入下腹。 注意：thapsigargin触发增加凝血酶诱导的血小板凝聚17，并且可以从尾更难使采血。 </li></ol></li><li> Gaussia荧光素酶测定： <ol><li>解冻血浆样品在冰上。 注意：对于纵向研究，解冻并在同一天的所有时间点的样品（使用基板的单一制剂）进行发光测定。 </li><li>将10微升的血浆，以不透明的围墙板在部分每个血浆样品中5运行3-4技术重复描述测量酶活性。 </li></ol></li><li> Euthanasi一个 注意：SERCaMP技术的优点是，以纵向显示器的ER钙的能力。取决于实验参数和端点分析，动物是用适合的端点分析，并根据机构ACUC准则措施安乐死。 </li></ol> 5.发光检测<ol><li>通过在酸化的甲醇（30微升10当量盐酸至3毫升甲醇中）稀释所述化合物至20mM制备腔肠素（CTZ）储备液。准备一次性使用等分并储存在-80℃。 </li><li>准备上试验当天工作衬底。 <ol><li>用于体外测定 ，稀释腔肠至8微米的PBS，例如添加10微升20mM的CTZ库存到25毫升的PBS（图6A，B）。 </li><li> 对于体内测定法，腔肠素稀释至100μM的PBS，500mM的抗坏血酸，5mM的NaCl洗涤。 </li></ol></li><li>孵育制备的CTZ为至少30分钟在室温下开始测定前。 注意：该步骤通常包括在Gaussia萤光素酶测定法有快速基板衰减在制备后第一30分钟的报告。我们没有观察到这种效果在我们的系统（图6C）;然而，我们通常包括温育步骤，因为我们已发现对测定没有任何负面影响。 </li><li>使用读板器，其能够监测生物发光的，并配有一个基片喷射器。总理的线条与底物。 注意：通过行中的初始衬底可以容易降解。为了避免人为的低读数早期样品，测量实验样品之前注入20-30井空（装上读卡器空盘）。 </li><li>注入100微升底物为好，摇中速，持续5秒，并测量发光。为百特协同II板读取器，集成的光发射超过0.5秒（体外样品）和5秒（ 在体内的样品），用于读出步骤。优化平板阅读器参数理想的检测性能。 注：快速的信号衰减（相比，焕发出其他的荧光素酶观察动力学）Gaussia荧光素酶的展品闪烁动力学。 GLUC的闪光灯动力学是由血清细胞培养基中（图7A）的影响，突出在控制了横跨样品介质组合物的重要性。由于发光的迅速衰减加入底物（闪光动力学）后，注射和读取步骤必须均匀对于所有样品之间的时间。该读板器应设置为衬底到一个井注入，等待一个固定时间 （如我们使用5秒摇步骤），并读那么好。加入底物到整个板读数之前构成显著挑战除非衬底可以被同时加入到所有孔中。如果喷射器不可用，该问题可以部分通过孵育板规避为衬底阿迪之间10分钟化和测量（从而避免了衰减曲线的陡峭部分）（图7B）。 </li><li>重复注射，等待时间，并宣读了每口井的上板的步骤。 </li></ol>

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J., Wires, E. S., Trychta, K. A., Richie, C. T., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SERCaMP:+a+carboxy-terminal+protein+modification+that+enables+monitoring+of+ER+calcium+homeostasis.">SERCaMP: a carboxy-terminal protein modification that enables monitoring of ER calcium homeostasis.</a> Mol Biol Cell. 25 (18), 2828-2839 (2014).</li><li>Henderson, M. J., Richie, C. T., Airavaara, M., Wang, Y., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesencephalic+astrocyte-derived+neurotrophic+factor+(MANF)+secretion+and+cell+surface+binding+are+modulated+by+KDEL+receptors.">Mesencephalic astrocyte-derived neurotrophic factor (MANF) secretion and cell surface binding are modulated by KDEL receptors.</a> J Biol Chem. 288 (6), 4209-4225 (2013).</li><li>Shipman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic+acid+(HEPES)+as+a+tissue+culture+buffer.">Evaluation of 4-(2-hydroxyethyl)-1-piperazine&#235;thanesulfonic acid (HEPES) as a tissue culture buffer.</a> Proc Soc Exp Biol Med. 130 (1), 305-310 (1969).</li><li>Zigler, J. S., Lepe-Zuniga, J. L., Vistica, B., Gery, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+cytotoxic+effects+of+light-exposed+HEPES-containing+culture+medium.">Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium.</a> In Vitro Cell Dev Biol. 21 (5), 282-287 (1985).</li><li>Howard, D. B., Powers, K., Wang, Y., Harvey, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropism+and+toxicity+of+adeno-associated+viral+vector+serotypes+1,+2,+5,+6,+7,+8,+and+9+in+rat+neurons+and+glia+in+vitro.">Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro.</a> Virology. 372 (1), 24-34 (2008).</li><li>Smeets, E. F., Heemskerk, J. W., Comfurius, P., Bevers, E. M., Zwaal, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thapsigargin+amplifies+the+platelet+procoagulant+response+caused+by+thrombin.">Thapsigargin amplifies the platelet procoagulant response caused by thrombin.</a> Thromb Haemost. 70 (6), 1024-1029 (1993).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat Protoc. 4 (4), 582-591 (2009).</li><li>Sobrevals, L., 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=AAV+vectors+transduce+hepatocytes+in+vivo+as+efficiently+in+cirrhotic+as+in+healthy+rat+livers.">AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers.</a> Gene Ther. 19 (4), 411-417 (2012).</li><li>Wang, Z., 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=Rapid+and+highly+efficient+transduction+by+double-stranded+adeno-associated+virus+vectors+in+vitro+and+in+vivo.">Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo.</a> Gene Ther. 10 (26), 2105-2111 (2003).</li><li>Seppen, J., 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=Adeno-associated+virus+vector+serotypes+mediate+sustained+correction+of+bilirubin+UDP+glucuronosyltransferase+deficiency+in+rats.">Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.</a> Mol Ther. 13 (6), 1085-1092 (2006).</li><li>Hareendran, S., 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=Adeno-associated+virus+(AAV)+vectors+in+gene+therapy:+immune+challenges+and+strategies+to+circumvent+them.">Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them.</a> Rev Med Virol. 23 (6), 399-413 (2013).</li></ol>

The authors have nothing to disclose.

这个协议强调了在体外和 GLUC-SERCaMP的体内效用并监控ER钙耗竭。虽然蛋白修饰产生SERCaMP似乎推广到其他记者蛋白12，我们选择了Gaussia荧光素酶是因为其强健（200-1000倍以上）生物发光相对于其他的荧光素酶18。我们证明跨越递送至原代大鼠皮质神经元，SH-SY5Y细胞，和大鼠肝100倍的剂量范围GLUC-SERCaMP病毒的检测的毒胡萝卜素诱导的GLUC-SERCaMP释放（图3...

内质网（ER）中的许多细胞的能力的功能，包括蛋白质折叠，蛋白质分泌，脂质稳态，和细胞内信号1。中央对正常的雌激素受体 ​​的功能是保持管腔钙离子浓度在约5000倍于细胞质2-4找到。这种能量密集型过程由萨尔科/内质网钙ATP酶（SERCA），一个泵，移动钙离子进入内质网调节。从ER钙的流出主要由兰尼（碱受体）和肌醇三磷酸（IP3受体）受体介导的。由于许多ER过程是依赖于钙，破坏商店会导致内质网应激和最终的细胞死亡。 ER钙失调已经在疾病，包括心肌病，糖尿病，阿尔茨海默氏观察到的，和帕金森氏5。由于这些疾病的进步性，已经挑战划定因果重发病机制和改建在ER钙库之间lationship。多种技术已经允许在我们的ER钙动力学，包括染料和遗传编码钙指标（GECIs）的理解显著进展。低亲和性钙染料，当结合到钙离子在荧光其中增加，可以装载到细胞以检查亚细胞区室的高浓度的钙6。 GECIs，如D1ER和捕手允许与亚细胞定位7-9的更精确的控制监测钙的波动。近日，又有类GECIs称为钙测定细胞器包埋蛋白质指标（CEPIA）已被描述10。第三种方法结合遗传学和小分子化学是靶向酯酶染料加载（TED），其利用遗传编码羧酸酯酶（靶向ER）与基于酯的钙染料11。 虽然AFORementioned方法都有内在的优势和弱点，他们可以通过荧光急性测量提供了有价值的见解ER钙的动态。然而，它们不是最佳的为经常需要调查疾病进展的纵向研究。以制定一个方法过度延长的时期以监测钙动力学的目标，我们确定并开发出一种蛋白修饰以创建分泌的ER钙监测蛋白（SERCaMPs）12。 SERCaMP规避与其他方法相关的，通过提供一种微创方法反复询问ER钙库一些局限性。我们已经证实了羧基末端肽ASARTDL（丙氨酸 - 丝氨酸 - 丙氨酸 - 精氨酸 - 苏氨酸 - 天冬氨酸 - 亮氨酸）足以促进ER滞留;然而，导致减少在ER钙的条件下，肽序列是不再能保留ER localization和蛋白质分泌13。所述SERCaMP技术的基础是ASARTDL到一个分泌蛋白的羧基末端（例如Gaussia萤光素酶，或GLUC），使得分泌受ER钙耗竭触发，从而产生的ER钙失调12健壮记者的附属物。 GLUC-SERCaMP通过转基因方法中的表达使得生物流体包括细胞培养基和等离子体对作为内质网钙稳态的指标的变化GLUC活性进行分析。该方法具有渐进的改变在ER钙库在体外和体内的纵向研究应用。以下协议被写为用于使用GLUC基于SERCaMP研究ER钙稳态的概要，但协议可以作为替代记者SERCaMPs的指南。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>1.5mL tubes</td>
			<td>Fisher&#160;</td>
			<td>02-682-550</td>
			<td></td>
		</tr>
		<tr>
			<td>10% NP-40 solution&#160;</td>
			<td>Pierce</td>
			<td>28324</td>
			<td>for intracellular GLuc assays</td>
		</tr>
		<tr>
			<td>1mL luer-lok syringes</td>
			<td>Fisher</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>200uL filter tips</td>
			<td>Rainin</td>
			<td>RT-L200F</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 surgical sutures</td>
			<td>Fisher</td>
			<td>NC9598192</td>
			<td></td>
		</tr>
		<tr>
			<td>30g needles</td>
			<td>Fisher Scientific</td>
			<td>14-821-13A&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microplate sealing sheets</td>
			<td>Thermo</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads</td>
			<td>Fisher</td>
			<td>22-246-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Auto Flow System</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-AF9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal recovery chamber</td>
			<td>Lyon Vet</td>
			<td>ICU-912-004</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher</td>
			<td>NC9386574</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine growth serum</td>
			<td>Thermo</td>
			<td>SH30541.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine, Native</td>
			<td>Regis Technologies</td>
			<td>1-361204-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting needles 3/8 circle sutures</td>
			<td>WPI</td>
			<td>501803</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital ultrasconic cleaner</td>
			<td>Fisher Scientific</td>
			<td>FS60D</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose, GlutaMAX, pyruvate</td>
			<td>Life Technologies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA mass ladder</td>
			<td>Life Technologies</td>
			<td>10496-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase (recombinant protein)</td>
			<td>Nanolight</td>
			<td>321-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Gaussia luciferase antibody (for WB, ICC, or IHC)</td>
			<td>New England Biolabs</td>
			<td>E8023S</td>
			<td>1:2000 (WB)</td>
		</tr>
		<tr>
			<td>Germinator 500</td>
			<td>CellPoint Scientific</td>
			<td>DS-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Gluc assay plates (96 well, opaque)</td>
			<td>Fisher</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Life Technologies</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Allmedtech</td>
			<td>63323-276-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Butler Schein</td>
			<td>29404</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>995-2949</td>
			<td></td>
		</tr>
		<tr>
			<td>Kwik Stop Styptic powder</td>
			<td>Butler Schein</td>
			<td>5867</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma</td>
			<td>G8540</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>a452-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge 22R Centrifuge</td>
			<td>Bekman Colter</td>
			<td>368831</td>
			<td></td>
		</tr>
		<tr>
			<td>Neosporin</td>
			<td>Fisher</td>
			<td>19-898-143</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stereoscope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin Gel and PCR Cleanup</td>
			<td>Machery-Nagel</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet</td>
			<td>Rainin</td>
			<td>L-200XLS+</td>
			<td></td>
		</tr>
		<tr>
			<td>p24 Lenti-X rapid titer kit</td>
			<td>Clontech</td>
			<td>632200</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR film seal</td>
			<td>Fisher</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>ReFresh Charcoal Filter canister</td>
			<td>E-Z Anesthesia</td>
			<td>EZ-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades, #10</td>
			<td>Fine Science tools Inc</td>
			<td>10010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>SD rats 150-200g</td>
			<td>Charles River</td>
			<td>Rats</td>
			<td>rats ordered at 150-200g.&#160; Surgery 5 days after arrival</td>
		</tr>
		<tr>
			<td>Small animal ear tags</td>
			<td>National Band and Tag co</td>
			<td>1005-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical drapes</td>
			<td>Braintree Scientific</td>
			<td>SP-MPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy 2 plate reader</td>
			<td>BioTek</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma</td>
			<td>T9033</td>
			<td>harmful to human health</td>
		</tr>
		<tr>
			<td>Virapower lentiviral packaging mix</td>
			<td>Life Technologies</td>
			<td>K4975-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Xfect Transfection reagent</td>
			<td>Clontech</td>
			<td>631318</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Valley Vet</td>
			<td>468RX</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring endoplasmic reticulum calcium homeostasis using a gaussia luciferase sercamp

内质网（ER）中含有的细胞内钙的最高水平，其浓度大约5000倍大于细胞质水平。严格控制ER钙是必不可少的蛋白质折叠，修改和贩运。扰动ER钙可导致解折叠蛋白质效应，一个三脚ER应激反应机制的激活，和在各种疾病有助于发病机理。在疾病发作和进展并监控ER钙改变的能力是在实践中在原则上重要，但具有挑战性。用于监测的ER钙目前可用的方法，如钙依赖性荧光染料和蛋白质，都提供了深入了解细胞中的ER钙动力学，但是这些工具不适合用于体内研究 。我们的实验室已经证明，一个修改Gaussia萤光素酶的羧基末端赋予记者的分泌响应于ER钙枯竭。该方法使用荧光素酶基，分泌的ER钙监测蛋白（SERCaMP），用于在体外和体内应用如本文所述。该视频强调了ER钙纵向监测肝注射，药理学处理GLUC-SERCaMP，血液采集和加工，检测参数。

该GLUC-SERCaMP方法通过取样外液允许对ER钙稳态评估。多个控件可以包括在实验设计，以提高结果的解释。首先，使用组成型分泌记者的（例如未经GLUC C-末端ASARTDL或“GLUC-否标签”）可以被用来评估对分泌途径（全球细胞分泌）和转基因表达实验处理的影响。例如，增加在两个GLUC-SERCaMP和GLUC-否标签的胞外水平将被认为是一个不明确的结果。可替换地，在增加GLUC-SERCaMP分泌与对应缺乏GLUC-否标...

Watch this Scientific Journal Video about Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP at JoVE.com

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

monitoring-endoplasmic-reticulum-calcium-homeostasis-using-gaussia

Research

JoVE Journal

Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

13.0K 次观看.  National Institutes of Health. Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

视频: Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

使用自制双辉光萤光素酶检测的高通量筛选功能

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

转染RAW264.7细胞用荧光素酶报告基因

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

基于FRET钙内波动培养平滑肌细胞使用的肌质网的测量共焦成像

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...