Name: homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion
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1. INS-1E 细胞: 维护和电镀
<ol><li>在加湿37˚C/5% CO2孵化器中维护 INS-1E 细胞, 并用 RPMI 1640 培养基补充 5% (v/v) 热灭活胎牛血清, 2 毫米 l-谷氨酰胺, 10 毫米 HEPES, 1 毫米丙酮酸钠, 100 U/毫升青霉素/链霉素溶液, 50 µM β巯基乙醇。培养细胞在10毫升完成 RPMI 1640 中等 (每板), 直到他们到达 80-90% 汇合, 当他们可以是 trypsinized 和传代或用于胰岛素分泌试验。</li>
	<li>
1 天:吸入介质, 用5毫升?...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Dopaminergic+regulation+of+insulin+secretion+from+the+pancreatic+islet.">Minireview: Dopaminergic regulation of insulin secretion from the pancreatic islet.</a> Mol Endocrinol. 27 (8), 1198-1207 (2013).</li><li>Rubi, B., 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=Dopamine+D2-like+receptors+are+expressed+in+pancreatic+beta+cells+and+mediate+inhibition+of+insulin+secretion.">Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion.</a> J Biol Chem. 280 (44), 36824-36832 (2005).</li><li>Garcia-Tornadu, 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=Disruption+of+the+dopamine+d2+receptor+impairs+insulin+secretion+and+causes+glucose+intolerance.">Disruption of the dopamine d2 receptor impairs insulin secretion and causes glucose intolerance.</a> Endocrinology. 151 (4), 1441-1450 (2010).</li><li>Garcia-Tornadu, 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=New+insights+into+the+endocrine+and+metabolic+roles+of+dopamine+D2+receptors+gained+from+the+Drd2+mouse.">New insights into the endocrine and metabolic roles of dopamine D2 receptors gained from the Drd2 mouse.</a> Neuroendocrinology. 92 (4), 207-214 (2010).</li><li>Lopez Vicchi, 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=Dopaminergic+drugs+in+type+2+diabetes+and+glucose+homeostasis.">Dopaminergic drugs in type 2 diabetes and glucose homeostasis.</a> Pharmacol Res. 109, 74-80 (2016).</li><li>Kalra, S., Kalra, B., Agrawal, N., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine:+The+forgotten+felon+in+type+2+diabetes.">Dopamine: The forgotten felon in type 2 diabetes.</a> Recent Pat Endocr Metab Immune Drug Discov. 5 (1), 61-65 (2011).</li><li>Mahajan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromocriptine+mesylate:+FDA-approved+novel+treatment+for+type-2+diabetes.">Bromocriptine mesylate: FDA-approved novel treatment for type-2 diabetes.</a> Indian J Pharmacol. 41 (4), 197-198 (2009).</li><li>Caicedo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+and+autocrine+interactions+in+the+human+islet:+more+than+meets+the+eye.">Paracrine and autocrine interactions in the human islet: more than meets the eye.</a> Semin Cell Dev Biol. 24 (1), 11-21 (2013).</li><li>Ballon, J. S., Pajvani, U., Freyberg, Z., Leibel, R. L., Lieberman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathophysiology+of+metabolic+effects+of+antipsychotic+medications.">Molecular pathophysiology of metabolic effects of antipsychotic medications.</a> Trends Endocrinol Metab. , (2014).</li><li>Freyberg, Z., Aslanoglou, D., Shah, R., Ballon, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+and+antipsychotic+drug-induced+metabolic+dysfunction+in+schizophrenia.">Intrinsic and antipsychotic drug-induced metabolic dysfunction in schizophrenia.</a> Front Neurosci. 11, 432 (2017).</li><li>de Leeuw van Weenen, E. 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这里描述的 HTRF 胰岛素检测提供了一个快速, 有效的系统来衡量胰岛素分泌从一个培养细胞的系统。在其最重要的优势之一, 该检测提供了低背景信号, 由于高信噪比。此外, 我们已确认 HTRF 信号在较长时间内稳定 (> 24 小时)7。然而, 由于胰岛素结合单克隆抗体在检测后快速达到束缚饱和度, 抗体结合的动力学可能会受到环境温度或胰岛素分泌水平的变化的影响。因此, 我们在多个?...

对能量代谢的调节由一种主要的合成代谢荷尔蒙--胰岛素--进行微调。胰岛素是由胰β细胞合成和释放, 以响应增加的胞外葡萄糖水平。释放的胰岛素触发葡萄糖的摄取胰岛素敏感组织1,2。生理上, 这与饭后葡萄糖浓度升高有关, 其次是胰岛素分泌调节葡萄糖摄取。在葡萄糖稳态紊乱导致代谢损害达到高潮的胰岛素抵抗, 最终在2型糖尿病的发病2,3,4。
虽然胰岛素分泌已被广泛研究, 但其调控机制仍不清楚。一个重要的调查领域是通过 beta 细胞5,6,7,8, 识别出新的胰岛素分泌调节剂。这些研究需要更好地了解葡萄糖刺激与胰岛素分泌的耦合关系。因此, 准确监测和量化葡萄糖刺激胰岛素分泌 (GSIS) 水平的能力是必不可少的。然而, 迄今为止, 只有有限数量的方法可以使用胰岛素分泌细胞系和/或胰岛来量化 GSIS。一种是放射免疫 (RIA), 它利用放射性同位素标记的胰岛素和抗体。这一方法的主要局限性包括放射性材料的处理和处置造成的安全问题。此外, 这种方法是劳动密集型的, 涉及多长时间的洗涤和孵化步骤。酶联免疫吸附试验 (ELISA) 是另一种昂贵的和劳动密集型的方法, 利用抗体进行胰岛素检测。抗体亲和性和胰岛素识别效率的变化是这种方法的限制因素, 会影响结果的重现性。ELISA 和 RIA 都不是为高通量实验设计的。AlphaScreen 是一种用于检测和测量胰岛素分泌水平的均质检测方法。AlphaScreen 技术的基础是将环境氧转化为激发氧的单态态, 这种状态可以与化学发光物种发生反应, 从而产生化学发光。由于该检测是同源的, 许多与 RIA 和 ELISA 相关的洗涤步骤被消除。然而, 由于反应的性质, 信号的不稳定性是一个限制因素, 可能会影响检测的读数。(Heyduk 和同事9开发的 TR 钳, 是基于两个单独的抗体对胰岛素分子的不同表位进行绑定的另一种均匀的胰岛素测量方法。抗体每个化学上链接到双链 dna 与短互补单链 dna 悬。抗体与胰岛素的结合使它们聚集在一起, 导致双链 DNA 双工。每种抗体也与各自的捐献者或受体荧光相关联, DNA 双工的联想汇集这些显影产生 Förster 共振能量转移 (烦恼)。然而, 一个潜在的限制是 TR 钳, 取决于烦恼本身。在焦虑反应过程中, 无法迅速消散背景荧光, 可能导致实验中的背景荧光和低信噪比的水平较高。因此, 对于以高通量方式量化 GSIS 的可靠、稳健和经济高效的检测, 仍然需要存在。
生物物理学的最新进展最终导致了一种均匀时间分辨荧光能量共振转移 (HTRF) 的研究。具体来说, 虽然分析中的能量转移可以被描述为基于焦虑的, 更准确地说, HTRF 依赖于发光能量共振转移 (LRET)10 , 这是能量在供体和受体之间的非辐射传递。物种11,12,13。这一区别是重要的, 因为荧光或淬火基于焦虑交互的时间与 LRET 有很大的不同, 尽管同样类型的浇口可以用于烦恼和 LRET。此外, 稀土稀土 cryptate 化合物, 如铕或铽 cryptate 在 HTRF 中的使用产生长荧光半衰期12,14。这提供了在捐助者励磁与接受方 (即,时间分辨化验) 之间的排放量之间引入时间延迟 (µsec) 的独特优势。这个时间延迟允许在测量受体发射荧光之前有足够的时间让背景荧光消散。因此, 读数是没有非特定的荧光, 因此, 高信噪比达到 (图 1)。此外, HTRF 的均一性消除了清洗未绑定物种的洗涤步骤的需要, 使得该检测比 ELISA 法或基于 RIA 的方法要快得多。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg"> 图 1: HTRF 胰岛素检测机制的示意图.两个独立产生的单克隆抗体专门识别和绑定到胰岛素在不同的地点。这些抗体是共轭的铕 cryptate 捐赠者或 XL665 受体。捐赠者的激发在337毫微米导致放射在620毫微米。由此产生的能量转移导致 XL665 以更长的波长发出, 665 nm。<a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在这里, 我们提供了一个详细的协议, 以使用基于 HTRF 的方法来确定 GSIS 的水平, 从 INS-1E 细胞, 一个建立良好的胰岛素分泌β细胞衍生的大鼠胰岛素瘤细胞系15。此外, 该检测可用于鉴定胰岛素分泌分子调控剂的药理学特征。我们应用这种 HTRF 的胰岛素检测, 以检查多巴胺 D2样受体调节 GSIS。越来越多的研究表明, 神经递质多巴胺是 GSIS8,16,17,18,19,20的重要调节器,21,22. 多巴胺以阴性分泌/分泌方式影响 GSIS, 通过对多巴胺 D2样受体 (d2, D3, d4受体) 的反应, 在β胰腺细胞的表面表达8,16,19. 利用这一方法, 我们确认多巴胺作为 GSIS 的负调节因子的作用, 并证明多巴胺 D2样受体激动剂溴隐亭和 quinpirole 也能减少 GSIS。

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well white half area plate</td>
			<td style="width:104px;">Greiner Bio-One</td>
			<td style="width:139px;">82050-042</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">384-well white low-volume, round-bottom plate</td>
			<td style="width:104px;">Corning</td>
			<td>15100157</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insulin High Range Kit</td>
			<td style="width:104px;">Cisbio Bioassays</td>
			<td style="width:139px;">62IN1PEH&#160;</td>
			<td>10,000 test HTRF-based insulin assay kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PHERAstar FSX plate reader</td>
			<td style="width:104px;">BMG Labtech</td>
			<td>FSX</td>
			<td>Plate reader adapted for HTRF-based assay readings</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plate sealers</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:139px;">DY992</td>
			<td>To seal plate while antibodies incubate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer</td>
			<td style="width:104px;">Hausser Scientific</td>
			<td style="width:139px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI Medium 1640 (1x)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11875-093</td>
			<td>[+] L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 0.05%&#160;</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">25-052-CI</td>
			<td>0.53 mM EDTA, (-) sodium bicarbonate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DPBS (Dulbecco&#39;s phosphate buffered saline)</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">21-031-CV</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">35-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEPES</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">156-30-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium pyruvate</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">11360070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin/Streptomycin solution 100x</td>
			<td style="width:104px;">Corning</td>
			<td style="width:139px;">30-002 CT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">M1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue Stain (0.4%)</td>
			<td style="width:104px;">Gibco&#160;</td>
			<td style="width:139px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dopamine hydrochloride</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">8502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromocriptine mesylate</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(-)-Quinpirole hydrochloride</td>
			<td style="width:104px;">Tocris</td>
			<td style="width:139px;">1061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:139px;">12659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Poly-L-Lysine</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">P4832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">G7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO (Dimethyl sulfoxide)</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:139px;">276855</td>
			<td></td>
		</tr>
	</tbody>
</table>

homogeneous time-resolved f&#246;rster resonance energy transfer-based assay for detection of insulin secretion

胰岛素分泌的检测对于阐明调控分泌物的机制以及代谢研究至关重要。虽然许多胰岛素检测已经存在数十年, 最近出现的均匀时间分辨 Förster 共振能量转移 (HTRF) 技术已经大大简化了这些测量。这是一种快速、经济高效、重现性强的光学检测方法, 依赖于具有长持久辐射的明亮显影的抗体, 这有助于时间分辨 Förster 共振能量转移。此外, HTRF 胰岛素检测也适合于高通量筛查技术的发展。在这里, 我们使用 HTRF 检测胰岛素分泌的 INS-1E 细胞, 一个大鼠胰岛素瘤衍生细胞系。这使我们能够估计胰岛素的基本水平及其对葡萄糖刺激反应的变化。此外, 我们使用这个胰岛素检测系统来确认多巴胺作为葡萄糖刺激胰岛素分泌的负调节因子 (GSIS) 的作用。类似地, 其他多巴胺 D2样受体激动剂, quinpirole 和溴隐亭, 以浓度依赖性的方式减少 GSIS。我们的研究结果突出了 HTRF 胰岛素检测格式在确定 GSIS 中多种药物的作用及其药理学特征方面的效用。

我们通过使用纯化的人胰岛素标准的预定义浓度 (图 2) 生成胰岛素标准曲线来验证我们的胰岛素 HTRF 检测。标准曲线的生成允许我们推断比例荧光读数, 从而确定分泌的胰岛素水平, 以应对药物治疗 (图 2)。曲线拟合的板内变化最小 (R2 = 0.99996 5.0 x 10-5) interplate 变化 (R2 = 0.947 @ 5.5 x 10-5</sup...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃƒÂ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

Homogeneous Time-resolved FÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

在这里, 我们提出的均匀时间解决烦恼 (HTRF) 作为一种有效的方法来快速检测胰岛素分泌的细胞。

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Research

JoVE Journal

Medicine

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Production and Detection of Reactive Oxygen Species ROS in Cancers

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

科研

医学

Improving IV Insulin Administration in a Community Hospital

提高在社区医院静脉注射胰岛素政府

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

小鼠后肢联合活体显微镜和超声造影研究胰岛素诱导的血管舒张和肌肉灌注

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Cardiac Magnetic Resonance Imaging at 7 Tesla

7号特斯拉的心脏磁共振成像

CMR at an ultra-high field (magnetic field strength B0&#160;&#8805; 7 Tesla) benefits from the signal-to-noise ratio (SNR) advantage inherent at higher ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

持续药物输注系统对小鼠脑内下丘脑胰岛素信号的研究

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

危重病人全血内毒素血症检测的内毒素活性测定

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

利用隔离的完美大鼠小肠进行肠道激素分泌的机制

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

心脏磁共振评估疑似心脏血栓:常规和新兴技术

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...