Name: imaging calcium dynamics in subpopulations of mouse pancreatic islet cells
Uploaded: 2019-11-26T12:00:00
Description: Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

AH是英国糖尿病博士生的获得者，EV得到了OXION-威康信托培训计划的支持，AIT获得了牛津生物医学研究理事会博士后奖学金。

此处描述的所有方法都是根据《联合王国动物（科学程序）法》（1986年）和牛津大学道德准则制定的。
1. 分离小鼠胰腺胰岛
<ol><li>准备培养基和隔离解决方案。<ol>
<li>组成培养基：RMPI1640（见材料表），辅以10%的胎儿小牛血清，100单位/mL青霉素，100μg/mL链霉素。将培养基放入两个60毫米塑料培养皿中（无需用任何粘合剂处理），并将培养皿保存在培养箱中（37°C...

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协议中有三个阶段对整体成功至关重要。成功将Liberase酶注入胆管，不仅决定分离程序的定量成功，而且影响分离胰岛的质量。未充气的胰腺可能导致在孤立的胰岛缺乏一些重要的代谢反应。其次，染料/传感器的表达的加载定义了延时记录的信号噪声比。在过载的小岛中，信号不存在或衰减。最后，成功和密集地定位成像室内的组织是有意义的、可分析的实验的决定性时刻。定位不当或移动的组?...

该方法的目的是在胰腺胰岛细胞的轻微亚群中实时成像细胞钙浓度（[Ca2]+cyt）的实时变化。这允许揭示这些细胞中激素分泌的机制，揭示不同细胞类型之间的串扰的细节，并有可能在小岛信号的大图中引入一个总体维度。
胰岛由几种细胞类型组成。除了更知名的胰岛素分泌β细胞外，至少有两个亚群在调节血糖3方面也至关重要。β-细胞（约占胰岛细胞的17%）在血糖变得过低时分泌胰高血糖素，这表示葡萄糖从肝脏的储存库释放到血液中。过量的胰高血糖素水平（高血糖素）和糖高血糖素释放控制受损伴随（并且，技术上，可以促进）胰岛素敏感性受损的糖尿病前期状况4。• 细胞（约2%）分泌躯体他汀，以响应葡萄糖升高。这种无处不在的肽类激素很可能在胰岛内的β-和β细胞附近存在高浓度，对胰高血糖素和胰岛素分泌有很强的Gi受体介导衰减作用。
β-细胞和β-细胞与其近亲β-细胞共享大部分葡萄糖传感机械。所有三种细胞类型都配备了ATP敏感K+通道，精密的代谢传感器5，控制这些兴奋细胞的血浆膜电位。同时，胰岛素、索马他汀和胰高血糖素的分泌受到葡萄糖的不同调节。因此，在小岛细胞的两个次要亚种群中成像Ca2+动力学可以提供血糖和小岛分泌输出之间的串扰。
使用贴片夹电生理学监测β-和β-细胞兴奋性的早期尝试很快在单个β和β细胞中进行了Ca2+成像。这些实验中细胞的身份通过抗胰高血糖素或抗球瘤素抗体的后染色得到验证。这些努力经常受到发现，小岛细胞的行为非常不同，在小岛内和作为单细胞的阻碍。虽然β-细胞似乎是小岛排列的主要恩人（由于其绝大多数是它们强大的电耦合的基础），但令人惊讶的是，主要差异是在β-细胞中发现的。在完整的小岛内，这些细胞在低血糖下持续持续地被激活，这仅对大约7%的单分散β细胞6有效。因此，报告完整胰岛内β-和β细胞的活性被认为是对体内条件的接近。
通常，有两种方法可以报告专门来自β-细胞或β-细胞亚种群的Ca2+动力学：（i） 通过组织特异性启动子表达基因编码的Ca2+传感器，或（ii）使用标记化合物。更优雅的前一种方法增加了真正的3D成像的实质性优势，从而研究了小岛内的细胞分布。然而，它不能应用于完整的人类小岛材料。另一个潜在的问题是启动子的"泄漏"，特别是当β-/β-细胞转位或β-细胞对高葡萄糖的反应到位时。后一种方法可用于新鲜分离的组织，包括人体样本或培养胰岛。然而，数据仅从小岛细胞的外围层收集，因为在不改变小岛结构的情况下，在更深的层中传递染料/标记分子是具有挑战性的。后一种方法的一个意外优势是与广域成像模式的兼容性，它允许将实验扩展到数十个或数百个小岛（即数千到数万个细胞）的同步成像。
钙在体内成像使用基因编码的GCaMP7（或近机8）系列传感器，这是循环渗透绿色荧光蛋白（GFP）的变体，与钙结合蛋白镇静剂及其靶序、M13片段肌苷光链激酶7、9。GCaMP在纳米摩尔Ca2+浓度范围内具有极好的信噪比，具有高2光子横截面，这使得它们成为体内工作10、11的理想选择。使用重组传感器的具有挑战性的方面是它们输送到细胞中。异质性表达需要使用病毒载体和多小时的外生培养，这经常引起人们对细胞功能可能去分化或恶化的担忧。尽管经过预先设计的鼠标模型可以表达 GCaMP 来解决这个问题，但它们通过大幅延长提前期并将工作限制在非人工模型上，增加了新的挑战。对细胞内pH值变化的极高灵敏度是基于蛋白质的传感器12的另一个不利因素，然而，对于感知振荡信号（如Ca2+）来说，问题就不那么严重了。
可捕获染料（如绿色荧光Fluo4）的优点是，它们可以在一小时内被装入新鲜分离的组织。可以预见的是，可捕获染料的信号噪比较低，光稳定性（远）低于其重组染料。我们不能证实13日关于可染色染料14毒性的报告，然而，染料超载是一个常见的问题。
基于圆形排列的红色重组 Ca2+传感器自 201115年以来发展迅速，最近的发展带来了与 GCaMPs16在组织成像方面的激烈竞争，因为红光的穿透深度较高。市售的红色可捕获染料可以可靠地用于单细胞成像，但在组织水平上，无法与绿色模拟物很好地竞争。
在失焦光成为关键问题的组织中，成像技术似乎很少选择。共聚焦系统通过取消对焦光的失焦光提供可接受的单细胞分辨率，NA 上的任何目标高于 0.3（GCaMP6）或 0.8（可染色染料）。从技术意义上讲，传统的共聚焦显微镜可用于同时成像来自数百个（GCaMP）或数十个小岛（可染色剂）的[Ca2]cyt。在组织中传感器的 3D 表达的情况下，唯一现实的共聚焦模式可能是光片显微镜。
当传感器在小岛组织内的细胞周围层中表达时，情况稍有不同。对于具有生动的细胞内表达模式的明亮重组传感器，使用具有低NA目标的广域成像模式可以提供足够的质量，并奖励研究人员在视野领域方面大幅增加，从而奖励吞吐量。广域系统提供较差的空间分辨率，因为失焦光不会被取消;因此，具有高NA（低景深）目标的成像组织信息较少，因为单细胞信号受到邻近细胞的很大污染。对于低NA（高景深）目标而言，污染要小得多。
但是，有些任务的高吞吐量和/或采样率成为其中的关键优势。β和β-细胞表现出很大的异质性，从而产生了对高样本量的需求，以揭示亚种群的贡献。广域成像快速且更灵敏，工业级大型视场系统分别以与十个或单个小岛上的共聚焦实验相同的信噪比对数百个 （GCaMP） 或数十个 （Fluo4） 小岛进行成像。这种吞吐量差异使得宽场系统有利于具有单单元分辨率的人口成像，这对于小亚种群（如 β-细胞）尤为重要。同样，尝试从 Ca2+尖峰17重建电气活动将受益于广域成像模式提供的更高采样率。同时，一些"小问题"，如胰腺β-细胞在刺激主导β细胞亚群时的活动，需要使用共聚焦系统。影响对共焦点模式决策的一个因素是来自β-细胞亚群的大量污染信号。
虽然在成像实验后使用激素特异性抗体染色来验证细胞的特性仍是一种选择，但可以使用功能标记化合物（如肾上腺素和ghrelin）来识别次要细胞亚群，这些化合物分别在β-18和β细胞19、20中选择性地刺激Ca2+动力学。
时间延时成像数据分析旨在提供除琐碎的药理学之外的信息，如人口异质性、不同信号的相关性和相互作用。按照惯例，成像数据被分析为强度与时间，并归化为初始荧光（F/F0）。由于荧光信号的漂白或自荧光或pH值的变化（通常由葡萄糖12的毫摩尔水平引起）的污染，经常需要基线校正。Ca2+数据可以以许多不同的方式进行分析，但三个主要趋势是测量计算的尖峰频率、高原分数或曲线下面积的变化。我们发现后一种方法是有利的，特别是在应用严重采样不足的共聚焦数据方面。pAUC指标的优点是它对信号频率和振幅变化的灵敏度，而计算频率需要大量的振荡21，这是很难用传统的成像实现。pAUC 分析的限制因素是其对基线变化的高灵敏度。

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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">40x/1.3 objective</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:216px;">Axiovert 200 microscope</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">emission</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">Excitation</td>
			<td style="width:277px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
			<td style="width:277px;">Sigma-&#1040;ldrich</td>
			<td style="width:119px;">F7524-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluo4</td>
			<td style="width:277px;">ThermoFisher (Life Technologies)</td>
			<td style="width:119px;">&#160; F14201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GCaMP6f, in (human type 5) adenoviral vector</td>
			<td style="width:277px;">Vector Biolabs</td>
			<td style="width:119px;">1910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hanks&#39; solution&#160;</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase</td>
			<td style="width:277px;">Sigma-&#1040;ldrich</td>
			<td style="width:119px;">5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">penicillin/streptomycin</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI medium</td>
			<td style="width:277px;">ThermoFisher (GibCo, Life Technologies)</td>
			<td style="width:119px;">61870044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss LSM510-META confocal system</td>
			<td style="width:277px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
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imaging calcium dynamics in subpopulations of mouse pancreatic islet cells

胰腺胰岛激素调节血糖平衡。血糖的变化导致胰腺胰岛细胞中细胞钙的振荡，引发三种主要激素的分泌：胰岛素（来自β细胞）、胰高血糖素（β细胞）和体素（β细胞）。β-细胞，构成大多数小岛细胞，并相互电耦合，作为一个单一实体对葡萄糖刺激作出反应。次要亚群、β细胞和β细胞的兴奋性（约占20%（30%）4%（10%）总啮齿动物1（人类2）的岛细胞数，分别不太可预测，因此特别感兴趣。
钙传感器被输送到隔离小岛内的细胞周围层。然后，胰岛或一组胰岛被固定，并使用荧光显微镜进行成像。成像模式的选择是更高的吞吐量（广域）和更好的空间分辨率（共聚焦）。按照惯例，激光扫描共聚焦显微镜用于成像组织，因为它提供了相邻细胞之间的信号的最佳分离。如果将β-细胞的主要种群中的污染信号降至最低，也可以使用宽场系统。
一旦钙动力学对特定刺激的反应被记录下来，数据以数字形式表示为荧光强度与时间，归化为初始荧光和基线校正，以消除与漂白相关的影响。荧光。计算曲线 （pAUC） 下的尖峰频率或部分面积的变化与时间的变化，以量化观察到的效果。pAUC 更敏感、更坚固，而尖峰频率则提供了有关钙增加机制的更多信息。
小细胞亚群可以通过对标记化合物（如肾上腺素和ghrelin）的功能反应来识别，这些化合物诱导特定细胞群中细胞钙的变化。

胰岛与可捕获的染料的负荷相当好（图1A），除非膜的脂质成分受到影响（例如，长期接触脂肪酸）。人类腺病毒类型5（Ad5）载体也针对所有小岛细胞（图1B）。当在同一单元中表示多个重组传感器时，可能会出现问题。此外，使用上述技术，胰岛通常非常固定，可提供出色的稳定性和解决方案访问。
在β...

Watch this Scientific Journal Video about Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells at JoVE.com

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

在这里，我们提出了一个协议，用于在异质细胞群（如胰腺胰岛细胞）中成像和量化钙动力学。荧光光被输送到小岛内细胞的外围层，然后固定和成像，并每细胞对荧光强度的动态进行分析。

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 ...

该协议允许兰格汉斯胰岛内细胞小亚细胞的快速分离功能。该技术的优点包括获取数据的实时性和高统计能力，因此具有很高的可重复性。要固定小岛进行成像，请为倒置显微镜组装成像室，并在室内放置玻璃盖滑。确保玻璃室界面防水，并确认盖滑在显微镜目标的范围内。接下来，切割20个20毫米方的细而粗网格，并使用45至50微米的厚胶带在一块细网上创建两个隔开墙。将粗网和重量浸入35毫米的培养皿中，将细网置于解剖显微镜下。将细网与空间墙倒置，隔开器朝上。并使用 p20 移液器在两个分空间之间传输多个小岛。使用制表师的钳子，将网格倒置在倒置显微镜的成像室内，使空间器朝下直接位于室盖滑上，将空间和网格之间的小岛困在盖滑的中间。注意网格水分充足，不含有过多的溶液，从而引发样品的横向运动。然后将粗网格和重量放在腔室中细网格的顶部，并将成像溶液添加到腔室中。一旦小岛固定，选择倒置显微镜上的成像模式和目标，将带小孔的腔室置于显微镜的温度控制阶段。设置灌注后，将流入位置低于腔室内的流出，并设置流出通量大于流入流量。确保流出与溶液的接触最小，从而在多个连续的小液滴中去除溶液，避免连续去除溶液的长时间间隔。接下来，使用含有三毫摩尔葡萄糖的成像溶液开始灌注，并选择光路和滤光片进行成像绿色荧光。然后运行实时成像以设置成像参数并调整视图以捕获感兴趣的小岛。要优化图像的声噪比，请调整激发光强度、曝光时间和装箱，确保设置允许以尽可能低的光强和曝光度对小岛内的每个细胞进行独特的可视化。然后以 0.1 到 5 赫兹对小岛进行图像，在捕获时检查获取数据的质量。当α细胞活性可检测到时，尽可能使用采集软件中信号动力学的在线图表，并将肾上腺素或谷氨酸加入沐浴溶液两到五分钟。将观察到钙的快速跳跃，然后减缓或取消α细胞振荡。接下来，添加 ghrelin，最近报告有选择性地激活三角洲细胞。钙的可逆性快速增加将在小的小细胞亚细胞中观察到。然后加入10毫摩尔葡萄糖。将观察到β细胞亚细胞亚细胞的协同振荡反应。另请注意以前被肾上腺素或谷氨酸和格列林激活的细胞的反应。保存图像后，将数据导入相应的数据分析软件程序，并使原始荧光强度数据与荧光的初始值标准化。如果细胞到细胞变异荧光强度数据集仍然很大，则为应用控制溶液的时间范围定义一个控制区域。如果控制区域具有清晰的非振荡信号，则假定每次应用控制溶液后，荧光强度将返回到基线。要更正每个单元格的时间推移数据，请将数据拆分为由添加控制解的点分隔的段，并应用于每个段的线性校正。如果控制范围具有明显的振荡或存在其他因素，请使用尖峰检测算法。要测量钙尖峰的频率和对添加激动剂和拮抗剂的反应，请将记录拆分为相等的时间间隔，计算间隔内的峰值，并规范化到间隔持续时间以计算部分频率的时间过程。或者，设置阈值并计算每个间隔的高原分数。分数指示单元格在兴奋状态下所花费的时间间隔内的时间百分比。或计算每个间隔的曲线下的部分区域。除非膜的脂质成分受到影响，否则小岛与热带染料的负荷相当好。人类腺病毒5型载体也成功地瞄准了小岛细胞。在低血糖水平下很容易检测到α细胞中的钙尖峰，低血糖下的活动与肾上腺素和谷氨酸的反应之间具有很高的细胞相关性。Ghrelin 在低血糖下激活一些肾上腺素反应细胞，但对低血糖激活的大多数细胞中的钙动力学没有影响。当从部分频率分析，肾上腺素或格林刺激细胞显示显著增加的情况下，全或全无条件下，虽然基础尖峰和肾上腺素效应之间的整体变化是微妙的。相比之下，曲线下的部分区域对肾上腺素在所有细胞中引入的变化敏感，即使基底活性高。确保所有网格都与成像解决方案接触，并注意合理密集地定位组织以避免气泡。该方法可以扩展为包括人工智能，用于区分细胞类型。药理标记可以由模式识别技术取代，从而减少记录时间。

Research

JoVE Journal

Biology

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

近质膜和全球星形胶质细胞内钙动力学测量

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...