Name: confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets
Uploaded: 2017-09-13T13:00:00.000+00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

作者感谢马西娅 Boulina 从还原铁成像核心设施帮助与显微镜。这项工作得到了 NIH 赠款 1K01DK111757-01 (JA)、F31668418 (MM)、R01 DK111538、R33 ES025673 和 R56 DK084321 (AC) 的支持。

迈阿密大学动物伦理委员会批准了所有的实验. 
 1. 完整的孤立人或小鼠胰岛的病毒感染 <ol> <li> 胰岛文化: 准备胰岛培养基: 干诺医学研究实验室 (CMRL) 1066、10% (v/v) FBS 和2毫米 l-谷氨酰胺。
	<ol> <li> 人类胰岛是从综合胰岛分布计划 (NIDDK, NIH) 获得的。抵达后, 转移胰岛 (约500小岛), 以35毫米 non-tissue 培养皿2毫升 CMRL 培养基37和 #176; C, 5%/95% CO 2 /O 2 , 在病毒感染前24小时. </li>
		<li> 鼠标胰岛可根据以前建立的协议进行隔离 16 。继隔离, 文化〜200胰岛等效35毫米 non-tissue 培养皿2毫升 CMRL 培养基在37和 #176; C, 5%/95% CO 2 /O 2 在病毒感染前24小时. 
		注意: 避免使用 GFP 或 YFP 在胰岛上表达的转基因小鼠, 以避免与 NPY-pHluorin 的荧光重叠. </li>
	</ol> </li> <li> 病毒准备 注意: NPY-pHluorin 融合被克隆到 pcDNA3 载体 10 和亚为腺病毒生产的腺病毒载体 [腺病毒血清型 5 (DE1/E3)] 由重组腺病毒制造公司。该病毒是 aliquoted 和存储在-80 和 #176; C。该病毒的股票是由公司提供的滴 10 12 到 10 13 病毒粒子 (〜 3 x 10 10 -3 x 10 11 PFU)。
	<ol> <li> 对于 体外 胰岛感染, 使用 10 6 PFU/mL, 导致大约2的感染 (莫伊) 的近似多样性 (请参阅 讨论 了解详细信息) </li> </ol> </li>
	<li> 胰岛病毒感染 
	警告: 与腺一起工作需要生物安全等级 2 (BSL2) 程序和认证。与机构生物安全干事核对有关 BSL2 程序的指导和培训。
	如上所述, <ol> <li> 准备人/鼠标小岛. </li>
		<li> 在2毫升的 CMRL 培养基中添加 5-10 和 #181; 每35毫米的培养皿中含有人/小鼠胰岛 (10% FBS 和2毫米 l-谷氨酰胺). 
		注: 根据公司数据表提供的病毒效价调整所使用的计算机病毒的音量. </li>
		<li> 在37和 #176 的病毒介质中培养胰岛; C/5%co 2 24 h. </li>
		<li> 24 小时后, 吸入含有病毒的培养基, 用2毫升 CMRL 培养基取代 (10% FBS 和2毫米 l-谷氨酰胺). </li>
		<li> 在37和 #176 中培养小岛 4-6 天; C/5%co 2 , 每3天替换一次媒体. </li>
		<li> 经过 4-6 天的培养, 预计约30% 的胰岛细胞被感染。然后, 胰岛可用于活体成像实验. </li>
	</ol> </li> </ol> 2。感染胰岛的共聚焦成像 注意: 请参阅用于共焦成像所需材料和设备的 材料表 。
<ol> <li> 试剂制备和实验设置 <ol> <li> 准备胞外溶液: 加125毫米氯化钠, 5.9 毫米氯化钾, 2.56 毫米 CaCl 2 , 1 毫米氯化镁 2 , 25 毫米 HEPES, 0.1% BSA, pH 7.4, 无菌过滤. 
		注: 此缓冲器通常是在无葡萄糖的情况下准备的, 可存储在4和 #176; C 为1月。在实验当天添加葡萄糖以达到所需的最终浓度。
		<ol> <li> 准备基础葡萄糖 (3 mM) 培养基: 添加75和 #181; L 2 米葡萄糖库存到50毫升的胞外溶液. </li>
			<li> 血糖 (16 mM) 介质: 添加400和 #181; L 2 米葡萄糖库存到50毫升胞外溶液 </li> </ol>
		</li> <li> 在含有3毫米葡萄糖的胞外溶液中稀释任何额外的刺激 (如 例如, 氯化钾或三磷酸腺苷 (ATP))。
		<li> 在开始实验前, 预处理片与聚 d-赖氨酸, 加入30和 #181; L 的聚 d-赖氨酸溶液 (1 毫克/毫升) 到片1小时, 并彻底冲洗它与 h 2 O. 
		注: 聚赖氨酸涂层片可贮存在室温下长达6月. </li>
		<li> 在实验前至少1小时, 使用1毫升吸管, 将胰岛转移到含有 3 mm 葡萄糖的胞外溶液的35毫米培养皿中。保持胰岛在37和 #176; C 和 5% CO 2 . 
		注: 如果需要, 可以在这一步中标记等离子膜。对细胞膜进行标记, 添加2和 #181; M di-8-ANEPP 染料到胞外溶液与3毫米葡萄糖。在染料溶液中孵育1小时37和 #176 的小岛; C/5%co 2 。等离子体膜染料可以在 488 nm 激发, 并在 620 nm 检测到. </li>
		在开始实验之前, 用真空硅脂将片连接到成像室。将成像室固定在成像平台上。使用吸管, 安置 20-30 个小岛在聚-d-赖氨酸处理的区域片并且让小岛坚持表面为 20 min. 
		注意: 重要的是不要让片完全干燥以避免胰岛损伤. </li>
		<li> 当胰岛附着在片上时, 通过用水彻底冲洗来准备灌注系统。将每个解决方案添加到不同的通道: 3 mm 葡萄糖 (通道 1), 16 mm 葡萄糖 (通道 2), 16 毫米葡萄糖与100和 #181; m 3-异丁基 1-嘌呤 (IBMX) 和10和 #181; m 佛司可林 (渠道 3), 25 毫米氯化钾在3毫米葡萄糖 (渠道 4), 10 和 #181; m ATP 在3毫米葡萄糖 (通道 5)。从系统中卸下所有气泡, 方法是分别打开每个通道, 并让溶液流几分钟, 并确保流是一致的 (0.5 毫升/分钟) 和油管没有泄漏. </li>
		<li> 将单个内嵌解决方案加热器连接到灌注出水管, 并将流出缓冲器的温度调整到37和 #176; C. </li>
		<li> 准备吸入泵。卸下系统中的所有气泡, 并确保流是一致的, 并且油管不会泄漏. </li>
		<li> 一旦胰岛附着在片表面, 则用含有3毫米葡萄糖的胞外溶液轻轻填满成像室。避免把小岛从片的表面清洗掉. </li>
		<li> 将成像平台与胰岛放置到显微镜阶段, 并将其连接到灌注系统和抽吸泵. </li>
		<li> 打开流并不断灌注3G 胞外溶液的胰岛。该系统现已准备好进行共聚焦成像. </li>
	</ol> </li> <li> 共焦成像 <ol> <li> 在显微镜场中以较低的放大率定位小岛。一旦专注于小岛, 切换到更高的放大目标 ( 例如 , 63X 水浸泡目标 (63 x/0.9 NA)). </li>
		<li> 使用软件打开购置 ( 材料表 ) 并激活共振扫描仪模式. </li>
		<li> 选择 XYZT 成像模式并配置捕获设置, 如下所示: <ol> <li> 打开氩激光器和 488 nm 激光线, 并将激光功率调整为 50% pHluorin 励磁. </li>
			<li> 收集 505-555 nm 的排放量 </li>
			<li> 选择 512 x 512 像素的分辨率。按 #34; 活 #34; 按钮开始成像并调整增益水平 (典型增益约 600 V). </li>
			<li> 设置 z 堆栈的开始和结束: 将焦点放在胰岛的顶端, 然后选择和 #34; 开始和 #34; 然后移动到最后一个可以集中的平面, 然后选择和 #34; 结束 #34;。使用5和 #181 的 z 步大小; m。该软件将自动计算共焦平面的数量. </li>
			<li> 将每个 z 堆栈的获取时间间隔设置为接近 1.5-2 秒, 然后选择选项和 #34; 获取直到停止和 #34; 连续成像. </li>
			<li> 按 #34; 启动和 #34; 按钮到 initialize. </li>
		</ol> </li> <li> 使用各种刺激协议来诱导颗粒胞, 通过灌注所需的刺激来诱发小岛。刺激协议可以定制, 以符合预期的科学目的 (见下文). </li>
	</ol> </li> <li> 刺激协议 注意: 在每一个刺激协议中, 首先记录至少2分钟的胰岛背景活动, 在恒定灌注过程中含有3毫米葡萄糖的胞外溶液。灌注在一段时间内用兴奋剂。兴奋剂的顺序, 刺激的持续时间和录音的持续时间可以定制, 以符合预期的科学目的。在开始新的刺激之前, 一定要用含有3毫米葡萄糖的胞外溶液彻底清洗小岛。下面找到用于演示方法功能的示例激发协议。
	<ol> <li> 使用氯化铵 (NH 4 Cl) 刺激 pHluorin pH 敏感性和病毒感染效率的阳性对照 ( 图 3 ): 3 mm 葡萄糖 (2 分钟) 和 #8594; 50 mm NH 4 cl (2 分钟) 在 3 mm 葡萄糖#160; #8594; 3 毫米葡萄糖 (2 分钟) 注意: 在 NH 4 Cl 溶液中, 以摩尔的基础取代氯化钠. </li>
		<li> 通过增加葡萄糖浓度 ( 图 5 和 图 6 ) 刺激胰岛素颗粒胞: 3 毫米葡萄糖 (2 分钟) 和 #160; #8594; 16 mm 葡萄糖 (15-30 分钟) 和 #160; #8594; 3 mm 葡萄糖(2 分钟 
		注: 为了看到几个爆发的活动, 灌注的小岛连续与16G 解决方案至少15分钟. 
		注意: 为了提高分泌响应 17 的一致性, 用户可以在3G 和16G 的解决方案中添加 IBMX (100 和 #181、10和 #181; m 佛司可林) 的提高阵营的代理。这不会改变颗粒分泌的时间模式。有关详细信息, 请参见 15 和 讨论 。
	</ol>
	</li>
</ol>

<ol>
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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57 (8), 1655-1663 (2014).</li></ol>

作者宣布, 他们没有竞争的金融利益。

这份手稿描述了一种技术, 可以用来可视化胞胰岛素颗粒的β细胞在完整的胰岛的共聚焦显微镜。它使用 NPY-pHluorin 作为荧光记者克隆成腺病毒, 以确保高转染效率。
虽然该方法在我们手中是高效的, 但它可能需要一些修改, 主要取决于两个参数: 1) 胰岛的质量和 2) 的病毒库存的效价优化感染条件。在胰岛病毒感染之前, 允许胰岛准备在37° c 过夜时从隔离和运输压力中恢复。检?...

胰岛素是由胰岛的β细胞产生的, 它是葡萄糖代谢的关键调节器1。死亡或功能障碍的β细胞扰乱葡萄糖稳态和导致糖尿病2。胰岛素是包装在密集核心颗粒, 释放在一个 Ca2相关的方式3。阐明胰岛素颗粒胞是如何调节的, 是充分了解什么决定胰岛素分泌和开辟新的途径, 以确定治疗糖尿病的新型治疗靶点的关键。
胰岛素胞已被广泛研究使用电生理学方法, 如膜电容测量, 和显微方法结合荧光分子。膜电容测量具有良好的时间分辨率和允许单细胞记录。然而, 电容的变化反映了细胞的净表面变化, 不捕获单个融合事件或区分胰岛素颗粒融合与其他胰岛素分泌泡3。显微方法, 如双光子或全内反射荧光 (TIRF) 显微镜结合荧光探针和囊泡货物蛋白, 提供了额外的细节。这些技术捕获单个 exocytotic 事件以及前 post-exocytotic 阶段, 并可用于研究3单元人口中的 exocytotic 模式。
荧光记者可分为三种类型: 1) 胞外, 2) 细胞质, 或 3) 水泡。1) 细胞外的记者是极地示踪剂 (如, 糖, 磺 B (局), 路西法黄, pyranine), 可以通过细胞外环境介绍4。使用极性示踪剂可以对细胞中的融合孔进行调查, 并捕获各种胞间结构, 如血管。然而, 他们不报告囊泡货物行为。2) 细胞质记者是荧光探针耦合到膜相关蛋白, 面对细胞质和参与对接和胞。例子包括可溶性的N-ethylmaleimide 敏感因子附着蛋白受体 (网罗) 家族, 已成功地用于研究神经递质释放5神经科学。这种蛋白质有多个结合的伙伴, 而不是胰岛素颗粒的具体。3) 水泡记者是荧光探针融合到水泡货物蛋白质, 允许调查货物特定的泡状行为。胰岛素颗粒特定的货物蛋白包括胰岛素, c 肽, 胰岛淀粉样多肽, 和 NPY 等6,7。NPY 只是存在于胰岛素含有颗粒, 并与胰岛素 co-released, 使它成为一个优秀的合作伙伴的荧光记者8。
以前使用不同的荧光蛋白与 NPY 的融合来研究神经内分泌细胞中胞的各个方面, 如特定的结合异构体的要求9,10以及时效的释放取决于肌动蛋白骨架和肌球蛋白 II11,12。在这项研究中, 我们选择了 pHluorin 作为荧光记者, 这是一个修饰 GFP, 是荧光在酸性 ph 内致密核心颗粒, 但成为明亮的荧光后, 暴露于中性细胞外 ph 值13。成熟的胰岛素颗粒的酸性 pH 值低于5.5。一旦颗粒与等离子体膜融合并打开, 其货物就暴露在中性胞外 ph 值为 7.4, 允许使用 ph 敏感蛋白 pHluorin 作为记者7,14。
鉴于 pHluorin 的 pH 敏感性和胰岛素颗粒中 npy 的选择性表达, npy-pHluorin 融合结构可用于研究胰岛素颗粒胞的各种性质。病毒传递的融合结构确保高转染效率和工作在初级β细胞或细胞系, 以及在孤立的小岛。这种方法也可以作为研究胞在任何其他细胞类型与 NPY 包含囊泡的指导。它也可以与任何转基因小鼠模型结合, 研究某些条件 (knockdowns, 过度表达,等) 对胞的影响。这项技术以前被用来描述的空间和时间模式的胰岛素颗粒分泌的β细胞在人胰岛的人口15。

<table><tbody><tr><td>Upright laser-scanning confocal microscope</td><td>Leica Microsystems, Wetzlar, Germany</td><td>TCS-SP5</td><td>includes LAS AF, the image acquisition software</td></tr><tr><td>Imaging chamber</td><td>Warner instruments</td><td>RC-26</td><td></td></tr><tr><td>Imaging chamber platform</td><td>Warner instruments</td><td>PH-1</td><td></td></tr><tr><td>22 x 40 glass coverslips</td><td>Daiggerbrand</td><td>G15972H</td><td></td></tr><tr><td>Vacuum silicone grease</td><td>Sigma</td><td>Z273554-1EA</td><td></td></tr><tr><td>Multichannel perfusion system</td><td>Warner instruments</td><td>VC-8</td><td></td></tr><tr><td>Single inline solution heater</td><td>Warner instruments</td><td>SH-27B</td><td></td></tr><tr><td>Temperature controller</td><td>Warner instruments</td><td>TC-324C</td><td></td></tr><tr><td>Peristaltic Suction pump</td><td>Pharmacia</td><td>P-1</td><td></td></tr><tr><td>35 mm Petri dish, non-tissue culture treated</td><td>VWR</td><td>10861-586</td><td></td></tr><tr><td>CMRL Medium, no glutamine</td><td>ThermoFisher</td><td>11530037</td><td></td></tr><tr><td>FBS, heat inactivated</td><td>ThermoFisher</td><td>16140071</td><td></td></tr><tr><td>L-Glutamine 200 mM</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>5 M NaCl solution</td><td>Sigma</td><td>S5150</td><td></td></tr><tr><td>3 M KCl solution</td><td>Sigma</td><td>60135</td><td></td></tr><tr><td>1 M CaCl&lt;sub&gt;2 &lt;/sub&gt;solution</td><td>Sigma</td><td>21115</td><td></td></tr><tr><td>1 M MgCl&lt;sub&gt;2 &lt;/sub&gt;solution</td><td>Sigma</td><td>M1028</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>1 M HEPES solution</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>Vacuum filter</td><td>VWR</td><td>431098</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma-aldrich</td><td>P6407</td><td></td></tr><tr><td>Di-8-ANNEP</td><td>ThermoFisher</td><td>D3167</td><td></td></tr><tr><td>3-isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I5879</td><td></td></tr><tr><td>Forskolin</td><td>Sigma</td><td>F3917</td><td></td></tr></tbody></table>

confocal imaging of neuropeptide y-phluorin: a technique to visualize insulin granule exocytosis in intact murine and human islets

胰岛素分泌在正常的生理条件下和疾病中起着核心作用。目前研究胰岛素颗粒胞的方法是使用电生理学或显微镜结合荧光记者的表达。然而, 大多数这些技术已被优化的克隆细胞系或需要游离胰岛。相比之下, 这里提出的方法可以实时显示胰岛素颗粒胞在完整的胰岛。在本议定书中, 我们首先描述的病毒感染的孤立胰岛与腺病毒, 编码 pH 敏感的绿色荧光蛋白 (GFP), pHluorin, 耦合到神经肽 Y (NPY)。其次, 我们描述了病毒感染后五天胰岛的共聚焦成像, 以及如何监测胰岛素颗粒的分泌。简要地, 被感染的小岛被放置在一个片在一个成像室和图像下的直立激光扫描共聚焦显微镜, 而不断灌注的胞外溶液含有各种刺激。共聚焦图像跨越50µm 的胰岛是获得作为延时录音使用的快速共振扫描仪。胰岛素颗粒与等离子体膜的融合可以随着时间的推移而随之而来。这个过程也允许测试一个电池的刺激在一个单一的实验, 是兼容的鼠标和人类的胰岛, 并可以结合各种染料的功能成像 (如, 膜电位或胞浆钙染料)。

该技术的整个工作流显示在图 1中。简单地, 老鼠或人的小岛可以感染腺病毒编码 NPY pHluorin 和影像, 在几天的文化, 在共聚焦显微镜下。当颗粒与等离子体膜和开放的保险丝, 荧光的增加观察和可以量化 (图 1)。为了确定 NPY-pHluorin 是否确实是一个合适的工具, 以监测胰岛素颗粒动力学, 感染的胰岛 immunostained 与抗体抗 GFP 和不同胰...

Watch this Scientific Journal Video about Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets at JoVE.com

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

我们描述了一个在完整的胰岛使用 pHluorin, pH 敏感的绿色荧光蛋白可视化的胞的协议。孤立的胰岛感染腺病毒编码 pHluorin 耦合到囊泡货物神经肽 Y。这使得检测胰岛素颗粒融合事件的共聚焦显微镜。

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

confocal-imaging-neuropeptide-y-phluorin-technique-to-visualize

Research

JoVE Journal

Biology

9.1K 次观看.  University of Miami. 我们描述了一个在完整的胰岛使用 pHluorin, pH 敏感的绿色荧光蛋白可视化的胞的协议。孤立的胰岛感染腺病毒编码 pHluorin 耦合到囊泡货物神经肽 Y。这使得检测胰岛素颗粒融合事件的共聚焦显微镜。

视频: Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

科研

医学

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

人类胰岛神经网络的高分辨率3D 成像

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

生物工程

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

免疫与感染

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

胰岛嵌段石蜡切片

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

生物学

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

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, ...

切片人胰腺用于胰岛功能研究

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

作者聚焦:使用创新的胰腺切片平台推进 1 型糖尿病研究

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

生成人胰腺组织切片以研究胰腺内分泌和外分泌生理学

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

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观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

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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

生成人胰腺组织切片以研究胰腺内分泌和外分泌生理学