Name: homogeneous time-resolved förster 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毫升预热的 PBS 冲洗细胞一次。加入0.5 毫升胰蛋白酶 (0.025%) 稀释1:1 在0.5 毫升 PBS 胰蛋白酶处理细胞和孵育 3-4 分钟在37˚C。停用胰蛋白酶, 加入9毫升完整的培养基, 将细胞转移到15毫升离心管吹打。</li>
	<li>颗粒细胞通过离心和重新悬浮细胞颗粒在大约5毫升新鲜的媒介。</li>
	<li>取10µL 的重悬浮细胞, 并与10µL 台盼蓝生命染料混合, 检查细胞的活力。使用 hemocytometer 对10µL 中的活细胞和死体细胞进行计数。生存能力水平应高于90%。将新鲜培养基中的重悬浮细胞稀释至每毫升100万细胞。</li>
	<li>种子0.5 毫升 INS-1E 细胞每井在聚 l-赖氨酸预涂覆的24井板, 在密度50万细胞/好。</li>
	<li>
2 天:去除介质 18-24 小时后电镀和添加500µL/井新鲜 RPMI 1640 媒体。将细胞孵化为另外24小时, 使细胞从先前的传代步骤中完全恢复。这个额外的时间允许细胞在组织培养板上传播。</li>
</ol>2. 胰岛素分泌物测定 (3 天)
<ol><li>准备 KRB 缓冲器: 132.2 毫米氯化钠, 3.6 毫米氯化钾, 5 毫米 NaHCO3, 0.5 毫米 NaH2PO4, 0.5 毫米氯化镁2, 1.5 毫米 CaCl2, 0.001 克/毫升牛血清白蛋白 (BSA), pH 7.4。</li>
	<li>从细胞中吸取介质, 用预热过的 PBS 冲洗两次。</li>
	<li>对于葡萄糖饥饿步骤, 在37˚C/5% CO2中添加450µL/井 KRB (含 BSA), 无葡萄糖1小时。</li>
	<li>在葡萄糖饥饿的步骤, 准备稀释的药物 (s) 在 KRB 含有200毫米葡萄糖 (10x 浓度)。<ol>
<li>准备药物在10x 最后的集中在 KRB 补充与200毫米葡萄糖 (也10x 最后的化验葡萄糖集中)。如果药物库存 (加上10x 葡萄糖) 在亚砜, 确保亚砜的百分比保持一致的整个化验 (理想的最终百分比小于0.1% 亚砜)。</li>
		<li>对于多巴胺和 quinpirole 治疗, 使用最后的检测药物浓度范围为100µM 到 100 pM (从最高到最低浓度), 最后一点的剂量反应不含药物。对于溴隐亭, 使用最后的检测浓度范围为10µM 到下午10点, 最后一点的剂量反应是无药控制。</li>
	</ol>
</li>
	<li>葡萄糖饥饿后, 加入药物序列稀释的化验, 以产生剂量反应。</li>
	<li>添加50µL/井每系列稀释到相应的水井 (总化验量500µL)。</li>
	<li>对于葡萄糖刺激步骤, 孵化细胞与各自药物系列稀释 (在20毫米葡萄糖存在) 90 分钟在37˚C/5% CO2.包括一套控制井: (1) 在没有任何额外的药物和 (2) 不受药物或葡萄糖刺激的细胞 (提供基本分泌速率) 的情况下, 单独刺激20毫米葡萄糖。</li>
	<li>在刺激步骤之后, 小心地取出上清液 (直接使用或存储在4˚C)。 注: 此时可引入额外的5分钟温和离心步骤 (600 x g, 1 分钟), 以去除检测上清液中的剩余细胞。</li>
</ol>3. HTRF 测定胰岛素分泌物
<ol><li>稀释试验上清液1:10 在 KRB (不 BSA), 优选在清楚的96井板材。</li>
	<li>准备胰岛素标准曲线的 HTRF 胰岛素检测 (表 1)。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>标准库存解决方案 500 ng/毫升</td>
			<td>串行稀释</td>
			<td>工作 [胰岛素] ng/毫升</td>
		</tr>
<tr>
<td>性病7</td>
			<td>30µl 库存 + 140 µl KRB</td>
			<td>150</td>
		</tr>
<tr>
<td>性病6</td>
			<td>30µl 性病 7 + 45 µl KRB</td>
			<td>60</td>
		</tr>
<tr>
<td>性病5</td>
			<td>30µl 性病 6 + 45 µl KRB</td>
			<td>24</td>
		</tr>
<tr>
<td>性病4</td>
			<td>30µl 性病 5 + 45 µl KRB</td>
			<td>9。6</td>
		</tr>
<tr>
<td>性病3</td>
			<td>30µl 性病 4 + 45 µl KRB</td>
			<td>3.84</td>
		</tr>
<tr>
<td>性病2</td>
			<td>30µl 性病 3 + 45 µl KRB</td>
			<td>1.54</td>
		</tr>
<tr>
<td>性病1</td>
			<td>30µl 性病 2 + 45 µl KRB</td>
			<td>0.61</td>
		</tr>
<tr>
<td>性病0</td>
			<td>45µl KRB</td>
			<td>0</td>
		</tr>
<tr>
<td colspan="3">注: 性病股为500毫升</td>
		</tr>
</tbody></table>表1。串行稀释, 使胰岛素标准曲线。
<ol start="3"><li>将标准曲线样品和稀释后的测定上清液到 HTRF 板上。用 HTRF 测定分泌胰岛素的方法可以在96井或384井板中进行, 同时要记住化验量必须重新调整。使用10µL/井样品在96井白色半区域板材或5µL/井在384井白色低容量, 圆底板 (参见材料表)。</li>
	<li>在1:2 捐赠者 (cryptate)/受体 (XL-665) 比率中准备抗体组合, 检测缓冲区 (参见材料表)。 注: 有关 HTRF 化验的进一步具体细节可从制造商处获得。</li>
	<li>将抗体组合添加到30µL/井 (96 井板化验格式) 或15µL/井 (用于384井板化验格式) 的化验。</li>
	<li>把盘子封上, 室温下孵化。</li>
	<li>阅读板后2小时, 4 小时, 和/或夜间孵化与抗体使用的板读取器和适当的 HTRF 光学模块 (337 665 620 nm) (见材料表和制造商的说明)。将集成开始时间设置为60µs, 并在400µs 中集成. 使用200闪烁每井。 注: 这些参数是基于使用我们的特定读者。读数在620毫微米和665毫微米可能变化在不同的仪器之间。这是我们建议使用665/620 比率的原因之一。在计算这一比例时, 读者与读者之间的任何潜在差异都将被规范化, 并提供一致的价值, 而不管用来测量 HTRF 的仪器是什么。</li>
</ol>4. 数据分析和规范化
<ol><li>通过外推比例荧光读数 (665 nm/620 nm) 到二阶二次多项式曲线 (图 2) 计算出检测井的胰岛素浓度。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg"> 图 2: 胰岛素标准曲线.采用已知浓度的人胰岛素储备, 生成胰岛素标准曲线。结果 HTRF 比率 (665 毫微米/620 毫微米) 被绘了反对胰岛素浓度。数据最适合于第二阶二次多项式曲线 (R2 = 0.99996)。这是一个具有代表性的标准曲线。误差线 = SEM.<a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="2"><li>以推断的数据作为胰岛素分泌的 ng/毫升, 规范化 (胰岛素分泌反应增加配体浓度) 到% 最大胰岛素分泌物检测井的平均值 (20 毫米葡萄糖单独情况)。</li>
	<li>从单个实验中使用曲线拟合 (R2) 计算板内变化。在实验中, 从实验内的重复项中派生出单个 R2值, 允许计算曲线拟合的平均值的标准误差。</li>
	<li>为确定 interplate 变化, 请使用来自至少三个单独实验的数据来计算集体曲线 R2值的平均值的标准误差。</li>
</ol>

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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=The+dopamine+receptor+D2+agonist+bromocriptine+inhibits+glucose-stimulated+insulin+secretion+by+direct+activation+of+the+alpha2-adrenergic+receptors+in+beta+cells.">The dopamine receptor D2 agonist bromocriptine inhibits glucose-stimulated insulin secretion by direct activation of the alpha2-adrenergic receptors in beta cells.</a> Biochem Pharmacol. 79 (12), 1827-1836 (2010).</li></ol>

我们感谢尼古拉-皮埃尔 (Cisbio 生物鉴定) 的有益建议和皮埃尔 Maechler (日内瓦大学) 慷慨提供 INS-1E 细胞。这项工作得到了国防部的资助 (PR141292 授予中福), 以及匹兹堡基金会 (中福) 的 Emmerling 基金。

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><tbody><tr><td>96-well white half area plate</td><td>Greiner Bio-One</td><td>82050-042</td><td></td></tr><tr><td>384-well white low-volume, round-bottom plate</td><td>Corning</td><td>15100157</td><td></td></tr><tr><td>Insulin High Range Kit</td><td>Cisbio Bioassays</td><td>62IN1PEH&amp;nbsp;</td><td>10,000 test HTRF-based insulin assay kit</td></tr><tr><td>PHERAstar FSX plate reader</td><td>BMG Labtech</td><td>FSX</td><td>Plate reader adapted for HTRF-based assay readings</td></tr><tr><td>Plate sealers</td><td>Fisher Scientific</td><td>DY992</td><td>To seal plate while antibodies incubate</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>3100</td><td></td></tr><tr><td>RPMI Medium 1640 (1x)</td><td>Gibco&amp;nbsp;</td><td>11875-093</td><td>[+] L-glutamine</td></tr><tr><td>Trypsin 0.05%&amp;nbsp;</td><td>Corning</td><td>25-052-CI</td><td>0.53 mM EDTA, (-) sodium bicarbonate</td></tr><tr><td>DPBS (Dulbecco&#039;s phosphate buffered saline)</td><td>Corning</td><td>21-031-CV</td><td>Without calcium and magnesium</td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>HEPES</td><td>Gibco&amp;nbsp;</td><td>156-30-080</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco&amp;nbsp;</td><td>11360070</td><td></td></tr><tr><td>Penicillin/Streptomycin solution 100x</td><td>Corning</td><td>30-002 CT</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M1348</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco&amp;nbsp;</td><td>15250-061</td><td></td></tr><tr><td>Dopamine hydrochloride</td><td>Sigma</td><td>8502</td><td></td></tr><tr><td>Bromocriptine mesylate</td><td>Tocris</td><td>427</td><td></td></tr><tr><td>(-)-Quinpirole hydrochloride</td><td>Tocris</td><td>1061</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Calbiochem</td><td>12659</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P4832</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>DMSO (Dimethyl sulfoxide)</td><td>Sigma</td><td>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 FÃ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

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

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。

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

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

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.2K 次观看.  University of Pittsburgh. 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。

视频: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

高度敏感和快速荧光检测用便携式分析仪FRET

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

科研

生物化学

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

生物工程

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

时间分辨Förster共振能量转移测定法，用于测量人细胞中的内源性磷酸化STAT蛋白

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

人类多能干细胞分化为产生胰岛素的胰岛簇

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

发育生物学

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

人胰岛自身抗体的电化学发光测定

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

免疫与感染

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

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 analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

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 ...

生物学

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

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, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

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, ...

人假胰岛的细胞内信号转导事件和激素分泌

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

作者聚焦：使用 Pseudoislet 方案研究胰岛异常和功能 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

用于同步评估荧光生物传感器动力学和激素分泌谱的人假胰岛系统

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

均匀时间分辨 Förster 共振能量转移法检测胰岛素分泌

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高度敏感和快速荧光检测用便携式分析仪FRET

自动声胶肽受体激动剂的效价测定测定的连续稀释

FIBS功能的无创性代谢轮廓

时间分辨Förster共振能量转移测定法，用于测量人细胞中的内源性磷酸化STAT蛋白

人类多能干细胞分化为产生胰岛素的胰岛簇

人胰岛自身抗体的电化学发光测定

一个定量测定胰岛素表达细胞集落形成先觉

使用共同分泌的Luciferase代理物测量相对胰岛素分泌

用于同步评估荧光生物传感器动力学和激素分泌谱的人假胰岛系统

用于同步评估荧光生物传感器动力学和激素分泌谱的人假胰岛系统