Name: measuring relative insulin secretion using a co-secreted luciferase surrogate
Uploaded: 2019-06-25T15:00:00
Description: Watch this Scientific Journal Video about Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate at JoVE.com

作者感谢科布实验室的所有现任和前任成员所做的宝贵工作和讨论,感谢迪奥内·沃提供行政援助。Michael Kalwat 得到青少年糖尿病研究基金会 SRA-2019-702-Q-R 的支持。这项工作是通过NIH R37 DK34128和韦尔奇基金会授予I1243梅兰妮·科布。这项工作的早期部分也得到了NIH F32 DK100113对迈克尔·卡尔瓦特的支持。

1. 试剂、介质和缓冲液的制备(表1)
<ol><li>用以下添加剂制备500 mL高葡萄糖(4.5克/升)Dulbeco改性鹰培养基(DMEM)的完整介质:15%胎儿牛血清(FBS)、100单位/mL青霉素、100微克/mL链霉素、292微克/mL L-谷氨酰胺和50μMβ-麦卡托托乙醇。 注:本例中稳定的细胞系在G418抗生素的250μg/mL中保持。</li>
	<li>通过制造含有 5 mM KCl、120 mM NaCl、15 mM HEPES (pH 7.4)、24 mM NaHCO 3、1mM MgCl2、2 mM CaCl2和 1 mg/mL ...

<ol>
	<li>Kalwat, M. A., 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=Insulin+promoter-driven+Gaussia+luciferase-based+insulin+secretion+biosensor+assay+for+discovery+of+beta-cell+glucose-sensing+pathways.">Insulin promoter-driven Gaussia luciferase-based insulin secretion biosensor assay for discovery of beta-cell glucose-sensing pathways.</a> ACS Sensors. 1 (10), 1208-1212 (2016).</li><li>Burns, S. M., 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=High-throughput+luminescent+reporter+of+insulin+secretion+for+discovering+regulators+of+pancreatic+Beta-cell+function.">High-throughput luminescent reporter of insulin secretion for discovering regulators of pancreatic Beta-cell function.</a> Cell Metabolism. 21 (1), 126-137 (2015).</li><li>Rajan, 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=In+vitro+processing+and+secretion+of+mutant+insulin+proteins+that+cause+permanent+neonatal+diabetes.">In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes.</a> American Journal of Physiology - Endocrinology and Metabolism. 298 (3), E403-E410 (2010).</li><li>Watkins, 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=Imaging+Secretory+Vesicles+by+Fluorescent+Protein+Insertion+in+Propeptide+Rather+Than+Mature+Secreted+Peptide.">Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide.</a> Traffic. 3 (7), 461-471 (2002).</li><li>Welsh, J. 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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=Connexin-36+Gap+Junctions+Regulate+In+Vivo+First-+and+Second-Phase+Insulin+Secretion+Dynamics+and+Glucose+Tolerance+in+the+Conscious.">Connexin-36 Gap Junctions Regulate In Vivo First- and Second-Phase Insulin Secretion Dynamics and Glucose Tolerance in the Conscious.</a> Diabetes. 61 (7), 1700-1707 (2012).</li><li>Benninger, R. K. P., Head, W. S., Zhang, M., Satin, L. S., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+and+other+mechanisms+of+cell-cell+communication+regulate+basal+insulin+secretion+in+the+pancreatic+islet.">Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.</a> The Journal of Physiology. 589 (22), 5453-5466 (2011).</li><li>Konstantinova, I., 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=EphA-Ephrin-A-mediated+beta+cell+communication+regulates+insulin+secretion+from+pancreatic+islets.">EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets.</a> Cell. 129 (2), 359-370 (2007).</li><li>Jaques, F., 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=Dual+effect+of+cell-cell+contact+disruption+on+cytosolic+calcium+and+insulin+secretion.">Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion.</a> Endocrinology. 149 (5), 2494-2505 (2008).</li><li>Calabrese, A., 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=Connexin+36+Controls+Synchronization+of+Ca2++Oscillations+and+Insulin+Secretion+in+MIN6+Cells.">Connexin 36 Controls Synchronization of Ca2+ Oscillations and Insulin Secretion in MIN6 Cells.</a> Diabetes. 52 (2), 417-424 (2003).</li><li>Bielefeld-Sevigny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AlphaLISA+immunoassay+platform-+the+&amp;#34;no-wash&amp;#34;+high-throughput+alternative+to+ELISA.">AlphaLISA immunoassay platform- the &amp;#34;no-wash&amp;#34; high-throughput alternative to ELISA.</a> Assay and Drug Development Technologies. 7 (1), 90-92 (2009).</li><li>Aslanoglou, D., George, E. 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在这里,我们提出一种方法,快速评估来自MIN6+细胞的葡萄糖刺激胰岛素分泌反应。为了在测定中获得最佳反应,重要的是将MIN6细胞播种在适当的密度,并允许它们成为85-95%的汇合物。这改善β细胞对葡萄糖的反应,因为改善的细胞-细胞接触和同步,这发生在主小岛17,18,19,20,21以及MIN6</sup...

这里介绍的方法允许从转基因β细胞系的胰岛素分泌快速和负担得起的96孔板格式测定。该协议的关键是胰岛素的改良版本,将自然分泌的高斯卢西酶(GLuc,#18 kDa)插入C肽(见图1),以产生胰岛素高西亚(InsGLuc)1,2。其他较大的蛋白质,如GFP(+25 kDa),已成功插入胰岛素的C-肽中,并表现出从蛋白酶-GFP到胰岛素和GFP-C-肽3、4的预期转化后处理。对于该协议中的测定,GLuc已经针对哺乳动物的表达进行了柯顿优化,并引入了两个突变来增强发光动力学5,6。治疗条件的多种组合和复制可以很容易地以96孔板格式进行测试,并在实验后立即获得分泌结果。
如前所述,如前所述,一个主要优势是这种基于荧光酶的分泌测量(<0.01美元/井)的成本较低,这与酶相关免疫吸附测定(ELISAs)的成本和技术方面相对较高。> $2/油井) 和同质时间解荧光 (HTRF) 或其他 Fürster 共振能量转移 (FRET) 为基础的抗体 (> $1/well) 测定。与通过参照标准曲线测量胰岛素浓度的基于抗体的测定相比,InsGLuc 测定测量分泌活性,作为对板上控制井的相对比较。因此,每个实验都需要包含适当的控件。这种区别是一种权衡,允许快速和廉价的测量。然而,InsGLuc分泌已被证明与ELISA1,2测量的胰岛素分泌高度相关。该技术已扩展至高通量筛选1、2 、7,并确定了胰岛素分泌的新型调制器,包括电压门状钾通道抑制剂7以及天然产物抑制剂β细胞功能,色霉素A28。InsGLuc 的使用最适合那些计划持续测试许多不同的治疗条件以测试其对胰岛素分泌的影响的研究人员。在后续实验中,有必要在亲子细胞系中重复关键发现,在小鼠或人类胰岛中以最佳方式重复关键发现,并使用基于抗体的测定测量胰岛素分泌。

<table>
	<tbody>
		<tr>
			<td>Cell culture materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rIns-GLuc stable MIN6 cells</td>
			<td></td>
			<td></td>
			<td>Parental MIN6 cell line stably expressing pcDNA3.1+rInsp-Ins-eGLuc and maintained in 250 ug/ml G418</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>4.5 g/L glucose media</td>
		</tr>
		<tr>
			<td>fetal bovine serum, heat-inactivated</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fisher Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercaptoethanol</td>
			<td>Thermo-Fisher Scientific</td>
			<td>BP 176-100</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>Thermo-Fisher Scientific</td>
			<td>BP379-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T3924-500</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Gold Biotechnology</td>
			<td>G418-10</td>
			<td>Stock solution 250 mg/mL in water. Freeze aliquots at -20C.</td>
		</tr>
		<tr>
			<td>T75 tissue culture flasks</td>
			<td>Fisher Scientific</td>
			<td>07-202-000</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well tissue culture plates</td>
			<td>Celltreat</td>
			<td>229196</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoirs (50 mL)</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secretion assay reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (RIA grade)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>50-146-952</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Thermo-Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo-Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes, pH 7.4</td>
			<td>Thermo-Fisher Scientific</td>
			<td>50-213-365</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Thermo-Fisher Scientific</td>
			<td>15568414</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Thermo-Fisher Scientific</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optional drugs for stimulation experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diazoxide</td>
			<td>Sigma</td>
			<td>D9035</td>
			<td>Stock solution: 50 mM in 0.1N NaOH. Add equal amount of 0.1N HCl to any buffer where diazoxide is added.</td>
		</tr>
		<tr>
			<td>epinephrine (bitartrate salt)</td>
			<td>Sigma</td>
			<td>E4375</td>
			<td>Stock solution: 5 mM in water</td>
		</tr>
		<tr>
			<td>PMA (phorbol 12-myristate)</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td>Stock solution: 100 &#181;M in DMSO</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Guassia assay materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate (Na2HPO4)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo-Fisher Scientific</td>
			<td>G334</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bromide</td>
			<td>Thermo-Fisher Scientific</td>
			<td>AC44680-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo-Fisher Scientific</td>
			<td>AC44608-5000</td>
			<td>Stock solution: 0.5 M pH 8</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>RPI</td>
			<td>T60040-1000.0</td>
			<td>Stock solution: 1 M pH 8</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Fisher Scientific</td>
			<td>AAA1775922</td>
			<td>US Patent US7718389 suggested ascorbate can increase coelenterazine stability.</td>
		</tr>
		<tr>
			<td>Na2SO3</td>
			<td>Sigma</td>
			<td>S0505-250G</td>
			<td>US Patent US8367357 suggested sulfite may decrease background due to BSA</td>
		</tr>
		<tr>
			<td>Coelenterazine (native)</td>
			<td>Nanolight / Prolume</td>
			<td>3035MG</td>
			<td>Stock solution: 1 mg/ml in acidified MeOH (2.36 mM)</td>
		</tr>
		<tr>
			<td>OptiPlate-96, White Opaque 96-well Microplate</td>
			<td>Perkin Elmer</td>
			<td>6005290</td>
			<td>Any opaque white 96 well plate should be sufficient. Clear bottom plates will also work, however some signal will be lost.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy H1 Hybrid plate reader or equivalent</td>
			<td>BioTek</td>
			<td>8041000</td>
			<td>A plate reader with luminescence detection and 96-well plate capabilities is required.</td>
		</tr>
		<tr>
			<td>8-channel VOYAGER Pipette (50-1250 &#181;L)</td>
			<td>Integra</td>
			<td>4724</td>
			<td>An automated multichannel pipette is extremely useful for rapid addition of luciferase reagents and plating cells in 96 well format</td>
		</tr>
		<tr>
			<td>8-channel 200 &#181;L pipette</td>
			<td>Transferpette S 20-200 &#181;L</td>
			<td>2703710</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring relative insulin secretion using a co-secreted luciferase surrogate

对分泌胰岛素样品后收集进行基于抗体的检测通常需要几个小时到一天的测定时间,并且可能非常昂贵,具体取决于特定的测定。分泌的荧光素酶测定可加快结果,大大降低每个样品的测定成本。在这里,我们提出了一种相对使用不足的方法,通过使用高西亚荧光素酶基因插入C肽中,测量胰腺β细胞的胰岛素分泌活性。在蛋白酶处理蛋白酶胰岛素过程中,将C-肽切除释放胰岛素分泌囊泡内的荧光素酶,与胰岛素共同分泌。由于荧光素酶测定的速度,在样品收集后几分钟内即可得到结果。测定的一个限制是,它是胰岛素分泌的相对测量,而不是绝对定量。但是,此协议经济、可扩展,可以使用大多数标准发光板读取器执行。模拟和数字多通道移液器有助于多个步骤的测定。可以同时测试许多不同的实验变异。一旦确定了一组聚焦条件,应直接使用具有标准曲线的基于抗体的测定法测量胰岛素浓度,以确认荧光素酶测定结果。

为了测量在控制条件下的测定性能,可以完成一个简单的葡萄糖剂量反应曲线或使用二氧化二氮范式的刺激。在前者的情况下,在无葡萄糖条件下预孵化细胞1小时,然后以葡萄糖浓度增加治疗1小时,在5 mM及以下的分泌活性应极小,而观察到8以上增加的分泌mM 葡萄糖 (图 2)35 mM KCl 的刺激也可用作刺激分泌的阳性控制。在刺激期间纳入分泌调节药物应给予分?...

Watch this Scientific Journal Video about Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate at JoVE.com

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

该协议描述了如何使用胰岛素相关的高辛比酶作为β细胞胰岛素分泌的代理,在中等通量下执行快速低成本荧光素酶测定。大多数发光板读取器和多通道移液器都可以进行测定。

使用共同分泌的Luciferase代理物测量相对胰岛素分泌

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

该协议描述了从β细胞系检测胰岛素分泌的替代快速方法，以加快药物发现和表征。这种方法很有用，因为可以同时测试多种治疗方法，结果在实验后立即提供，而且这种检测方法比其他方法更实惠。该方法用于胰岛素分泌新小分子调节剂的筛选，可以识别未来糖尿病治疗的铅化合物。增加对β细胞功能的了解有助于对蛋白质或神经递质分泌的一般知识的加深。此胰岛素分泌处理程序已建立，以在其他β细胞线和人类胰岛中使用扁病毒系统。对于熟悉细胞培养和操作多通道移液器的个人来说，这种检测应该非常简单。在计划任何实验之前，验证您的父母或稳定的β细胞系是否使用胰岛素ELISA对葡萄糖有适当的反应。首先根据手稿说明制备克雷布斯-林格碳酸氢盐缓冲液或KRBH和高西亚荧光素工作溶液。使用标准细胞培养技术在T75烧瓶中培养MIN6细胞。准备InsGLuc MIN6细胞电镀，用PBS两次清洗细胞的汇合瓶，并加入两毫升的三辛。在37摄氏度下孵育细胞约5分钟，或直到细胞与烧瓶分离。确定细胞浓度，并在完整介质中稀释细胞至每毫升100万个细胞，相当于96井板每100微升的10万个细胞。细胞应在三到四天后充分汇合以进行测定。在测定当天，为实验准备足够的无葡萄糖KRBH，并设置一个储液罐。将介质从 96 井板中清除，快速将其倒置到实验室水槽上，然后牢固地印在一叠纸巾上，以去除多余的介质。每井用100微升无葡萄糖KRBH清洗盘子两次，如果进行药物治疗，请根据手稿说明将药物添加到KRBH中。然后，在每井中加入100微升无葡萄糖KRBH，并在37摄氏度下孵育一小时。孵育后，将缓冲液去掉，并在纸巾上印上盘子。每井添加 100 微升无葡萄糖 KRBH，洗去任何累积的背景，然后将板冲走。添加控制和刺激条件，或不进行药物治疗，每孔100微升，并在37摄氏度下孵育板一小时。在每个板块上包括基底和刺激控制很重要，以确定给定实验中细胞的响应能力。如果细胞随着时间的推移而失去反应，从新鲜的液氮中重新培养它们。使用多通道移液器仔细收集 50 微升的上清液，将其转移到不透明的白色 96 和测定板中，并在长椅上保持样品室温，直到准备好进行荧光素测定。通过将所需数量的CTZ溶液移液到GLuc测定缓冲液中，准备高西亚荧光素酶测定工作溶液，注意防止CTZ变暖。使用多通道移液器，使用 KRBH 超级纳特器快速将 GLuc 检测工作溶液添加到板上。如果井两侧有任何水滴，请将板在桌面中短暂旋转，摆动铲斗离心机。然后将板放入板读卡器中，用 0.1 秒的集成时间读取发光。通过测量胰岛素分泌，作为对葡萄糖浓度增加的反应，该检测在控制条件下进行了测试。在五毫摩尔葡萄糖以下观察到很少的分泌活性，在八毫摩尔以上观察到的分泌增加。当受到二氧化氮范式的挑战时，细胞表现出预期的分泌反应。如果没有细胞外氯化钾，Diazoxide 治疗会阻断胰岛素分泌。当氯化钾存在时，分泌物可以通过进一步添加葡萄糖来放大。还证明，InsGLuc MIN6细胞对氯化钾、二氧化二氮、PMA和肾上腺素等分泌调节化合物的反应如预期。按照这个程序，关键的结果应该通过分析KRBH超级纳在抗体为基础的ELISA或HTRF测定，允许确定胰岛素浓度确认。Broad 研究所的一个小组做了许多开创性的工作，为我在研究中越来越多地使用这种分析方法铺平了道路。多个研究小组，包括我们和布罗德小组，正在糖尿病领域的药物发现工作中采用这种方法。

Research

JoVE Journal

Biology

Measuring Exocytosis in Neurons Using FM Labeling

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

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 ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

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

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 ...

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

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 ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

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 ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

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 ...