This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).

注:所有本实验是按照昆士兰大学的地方动物伦理规程（经昆士兰大学生物科学解剖伦理委员会）执行。 1.胰岛分离<ol><li>试剂的准备<ol><li>酶的混合物<ol><li>对于胰腺消化，使用LIBERASE和IV型胶原酶的混合物。稀释LIBERASE TL（嗜热低）1瓶5毫克用26毫升的DMEM（贝科改良Eagle培养基）。 </li><li>准备在Hank氏缓冲IV型胶原酶在浓度为0.5mg / ml的，补充有5mM的HEPES，0.5mM的氯化钙 ，为0.1mg / ml的DNA酶和1mg / ml的牛血清白蛋白。 </li><li>混合LIBERASE TL和胶原酶溶液以4:1的比例（按体积计）， 即加入4ml LIBERASE TL +1毫升胶原酶。等分的酶的混合物在-20℃至2.5毫升每个和存储。 注:该组织孵化时间vARY酶批次之间的位（30秒到1分钟差）。因此，一批测试可能需要。 </li></ol></li><li> RPMI媒体隔离<ol><li>溶解的RPMI 1640中粉一个小瓶在1升水中在RT和补充与2g 碳酸氢钠 ，4.02克HEPES。 </li><li>结合的RPMI隔离介质在一个大锥形瓶中，用磁力搅拌器混合，调节至pH7.4，用NaOH，过滤灭菌，并在4℃下存储。 </li></ol></li><li> RPMI文化传媒</li><li>使用的RPMI培养基，含10％胎牛血清和1％抗生素（ 即 ，青霉素100U /毫升，链霉素100微克/毫升）。 </li></ol></li><li>胰岛分离和培养<ol><li>牲<ol><li>牺牲小鼠根据当地机构的伦理程序颈椎脱位或CO 2窒息。用70％乙醇彻底喷鼠标主体。 </li></ol></li><li>胰腺灌注<ol><li> ü本身2长切口横向穿过腹部皮肤和腹膜，使V形翼板。拉扯皮肤向上的襟翼揭开整个腹腔，抛开肠和保持就位肝经折叠薄纸以显示胆总管（ 图1和2）。把动物使头部朝向外科医生和尾部远离外科医生。 </li><li>应用血管钳在壶腹在十二指肠壁进入十二指肠（ 图1）阻断注射酶。 注意:这是一个重要的步骤，因为它决定了注入酶是否将进入胰管和灌注胰脏或回流到注射部位（超过钳位）或进入十二指肠（下夹子）。 </li><li>导管插入胆总管有一个31 G针肝总管和胆囊管（ 图1）的交界处附近，留有余地，第二次注射，如果日Ë胰腺不能灌注的第一次。慢（超过约10秒）注​​资约冷酶混合物2毫升到小鼠胆总管。 注:胰腺将很快灌注如果夹具是在正确的位置。 </li><li>调整夹子的位置，如果所述针是胆总管内但不能灌注胰脏（ 即 ，从肝脏如果超过夹紧向上移动夹具有点远或移动下来一点到肝脏如果下夹紧）。 注:针有时进入周围胶囊而非导管的内腔中。一种有用的标志来检查针是否是内部胆总管是导管注射时的膨胀。如果胰脏不能灌注，另一种是酶混合物可以注入到具有较低产量预期胰腺的多个站点。胰腺（脾叶）的左侧部位的灌注是胰岛的收获量的最大化很重要的。 </li><li>后胰腺为perfused，迅速取出胰腺，放入小瓶（约20毫升，玻璃）和孵育〜19分钟，在37℃水浴。 注:孵育时间可以改变一点取决于小鼠品系和年龄。 </li><li>后的培养时间，加入约20毫升冷的隔离介质的停止消化过程。摇动小瓶轻轻地破坏胰腺，并通过正常的茶筛（1毫米孔径）倒入一个无菌的50ml管中。顶部与隔离媒体和离心管400 XG在4℃下1分钟，摘下。 </li><li>再次溶解在隔离介质颗粒与传输两个15ml试管，然后离心，在400×g离心1分钟，在4℃下与制动切断。吸出上清液后，通过涡旋或上下抽吸混合两种粒料以及用6ml密度梯度细胞分离介质（的Histopaque 1077溶液）每管中。然后轻轻加〜6毫升隔离介质在管的侧面上的顶密度梯度分离介质和离心机在100×g离心15分钟，在4℃下与制动切断。 </li><li>收集在上部（分离介质）上清，中部和下部（密度梯度细胞分离介质）层在3个不同的菜肴。精选在这些三个培养皿胰岛，用解剖显微镜下的吸移管。 注:中间层包含了大多数的胰岛。胰岛被认为是约50至500微米的尺寸紧凑的结构（ 图3）。我们获得〜200胰岛在WT小鼠和db / db小鼠300- &lt;100小岛。 </li></ol></li><li>胰岛培养<ol><li>为了帮助胰岛从酶消化回收，40胰岛中的25cm培养皿6毫升的RPMI培养基中（10mM葡萄糖）培养基，在37℃，5％/ 95％的CO 2 / O 2]为前活2天-cell双光子成像。每天更换培养基。 </li></ol></li></ol></li></ol> 2。活细胞-2-光子成像<ol><li>试剂准备<ol><li>外缓冲区<ol><li>制备的钠丰富细胞外缓冲器用于与140 mM氯化钠，5mM的氯化钾，2.5mM的氯化钙 ，1mM的MgCl 2的，5mM的碳酸氢钠和10mM HEPES的2光子检测。 </li></ol></li><li>外染料<ol><li>对于双光子测定中，准备股票SRB 8mM的胞外缓冲液，分装成小管并储存在-20℃。稀释股票SRB 1:10新鲜外缓冲器得到的0.8毫米的工作浓度。 </li></ol></li></ol></li><li>光子成像<ol><li>首先，预孵育2天的培养的胰岛在3毫米的葡萄糖胞缓冲液30分钟。 注:我们在培养2至5天使用的小岛。 </li><li>然后，将载玻片上的热控制腔装在显微镜载物台上。使用润滑脂以防止溶液泄漏。 </li><li>覆盖用500μl细胞外的腔室ular缓冲器含有0.8毫SRB具有不同葡萄糖浓度（3mM的，6毫15 20mM和20mM葡萄糖）。 </li><li>下一步，将预孵育的胰岛到室的中部和聚焦在3-4细胞层进入胰岛，其中，在啮齿动物胰岛，免疫染色胰岛素表明大部分细胞是β细胞。 注:SRB将概述胰岛所有细胞膜迅速，胰岛准备图像。 </li><li>使用双光子显微镜用60X油浸物镜（NA 1.42）和图像处理软件（ 例如 ，ScanImage 11）。 图4示出了一个典型的显微结构。在550-650纳米的检测采用SRB作为膜透性荧光标记物外，在880 nm光激发飞秒激光脉冲图片胞吐事件，有荧光。记录胰岛反应的刺激。 </li><li>由小荧光点的突然出现识别单胞吐事件（分辨率10像素/1;米），并通过荧光信号的快速上升阶段确认在感兴趣的区域）（〜0.78μm2以下。 </li></ol></li></ol>

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The authors declare they have no competing financial interests.

在胰岛分离中最关键的因素是胰腺的初始灌注;一个下灌注胰脏结果在相当低的胰岛的收获量。其它因素也影响了隔离质量如消化时间和摇晃可能部分弥补灌注的水平的水平。例如，一个完全灌注胰脏将需要在37℃下被孵育〜18分30秒，轻轻振动，与此相反的下消化胰，可能需要约20分钟的消化与较硬晃动。两种酶消化我们手中的混合物提供更好的胰岛分离比任何一个单独的。对于胶原酶唯一方法，消化时间主要影响胰岛产量只有30秒的时间差，可以显着地影响质量。对于LIBERASE只，产量是可以接受的，但在消化有时会发生，我们认为，在加入胶原酶有助于消化结缔组织更好。 有若干可在胰岛已经分离之后进行可能的实验。胰岛可以固定在分离之后立即多聚甲醛或甲醇与用于免疫荧光染色，以确定感兴趣的10种蛋白的位置。胰岛可以分散在单细胞。这可以在胰岛分离的同一天或之后胰岛培养几天来完成。然后将单细胞铺板出来，这是该制剂已经作为支柱如膜片钳和全内反射显微技术。在该实验中测量胰岛素的分泌，以及用于活细胞2光子成像，我们已发现，胰岛需要在培养2天以得到再生的数据。什么是发生在文化不是很清楚，但被认为是一个周期从隔离和消化过程的恢复。 这里介绍的双光子技术使研究人员能够图像的胰岛素分泌过程在单个颗粒融合从完整的胰岛内的许多细胞中的水平。我们已发现，颗粒融合事件和它们的定时的数量是与量和胰岛素分泌8的时间曲线相一致。这表明该方法的检测的大多数胰岛素颗粒融合事件。鉴于在到定影颗粒染料项不是正在研究7,8检测，我们已经竭尽全力胰岛素颗粒融合事件进行对照实验证明β细胞胰岛素颗粒融合的具体方法</sup&gt;。然而，这种证据的一个组成部分是依赖于啮齿动物胰岛，其中β细胞富集的胰岛的芯的结构。人胰岛，其中不同的内分泌细胞分散在整个胰岛体积，所述的不同的结构将需要额外的方法来识别β细胞。 有许多的技术问题与双光子显微镜是通用于所有的成像方法。例如，我们仔细平衡试图获得足够的光线中的胰岛，使我们能够降低探测器的增益的对立因素，因此降低噪音，与把这么多的光中，我们造成细胞损伤胰岛。在我们的系统中，Pockels盒用来衰减脉冲激光（ 图3）;所有商业系统有某种机制来调节光的落在到试样的强度。 总之，仔细隔离分贝的组合/ Db胰岛和全胰岛2光子显微镜本新的机会来研究胰岛素分泌的方法，并确定在2型糖尿病的主要故障元件。

在疾病的β细胞功能障碍中的作用已被广泛认可3,4。细胞系如MIN6和INS-1是有用的工具来理解的β细胞行为的生物学。然而，在生理控制胰岛素分泌的需要胰岛内进行。这些胰岛包含数千紧密包装的β细胞，以及血管和其它内分泌细胞类型。胰岛内这样的环境影响胰岛素分泌和很可能是在糖尿病重要。因此，要理解的生理控制胰岛素分泌，以及疾病的病理生理学，有必要研究完整的胰岛。 胰岛分离从2型糖尿病患者活人胰岛和，特别是人胰岛是很难获得的。此外，人类的胰岛有实验分子操作的可能性有限。因此，研究人员使用的是允许从动物和2型糖尿病的动物模型。一个这样的疾病模型是的db / db小鼠。这是模型2型糖尿病的表型发展的非常类似于人类疾病5,6自发突变。从糖尿病db / db小鼠呈现此处胰岛分离的协议具有许多共同的步骤与其它基团的一​​些精炼为更好产量，纯化和增强胰岛存活。 2光子成像此处描述的活细胞2光子检测使研究人员能够量化数和从糖尿病7和野生型胰岛8,9-许多细胞评估单含胰岛素的颗粒的特征。

<table><tbody><tr><td>Liberase TL 5 mg</td><td>Roche</td><td>-5401020001</td><td></td></tr><tr><td>Collagenase type IV-1g</td><td>Gibco-Life Technologies</td><td>17104-019</td><td></td></tr><tr><td>Histopaque 1077 500 ml</td><td>Sigma Aldrich</td><td>10771</td><td></td></tr><tr><td>RPMI 1640 Medium (10x) 1L</td><td>Sigma Aldrich</td><td>R1383</td><td>to prepare isolation media</td></tr><tr><td>RPMI 1640 (1x) 500 ml</td><td>Gibco-Life Technologies</td><td>21870-076</td><td>to prepare cultured media</td></tr><tr><td>Penicillin-Streptomycin 100 ml</td><td>Gibco-Life Technologies</td><td>15140-122</td><td>to prepare cultured media&amp;nbsp;</td></tr><tr><td>Fetal bovine serum 500 ml</td><td>Gibco-Life Technologies</td><td>10099-141</td><td>to prepare cultured media</td></tr><tr><td>DMEM (Dulbecco&#039;s Modified Eagle Medium)</td><td>Gibco-Life Technologies</td><td>11966-025</td><td>to dilute the liberase</td></tr><tr><td>Metamorph program</td><td>Molecular Devices, USA</td><td></td><td>to analyze the 2-photon images</td></tr></tbody></table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

2型糖尿病是一种慢性疾病，影响到3.82亿人，2013年，预计将上升到5.92亿，到2035年1。在过去的二十年中，β细胞功能障碍的2型糖尿病患者已明确规定2的作用。研究进展所需的胰岛的分离方法。胰岛分离的协议这里提出股与来自其它组协议许多共同的步骤，具有一些修改，以提高来自野生型和糖尿病瘦素受体​​分贝 （的db / db）小鼠分离的胰岛的产量和质量。的实时小区2光子成像方法被提出可用于研究胰岛素分泌胰岛内的控制。

胰岛产量和纯化对于一个正常的野生型小鼠，大约200胰岛预期。健康的胰岛看起来明亮，圆形并有光滑的边框。过消化隔离批次通常具有小和模糊胰岛而一个下消化批次具有较少的胰岛和腺泡细胞附着胰岛（ 图3）。糖尿病db / db小鼠，胰岛的收获量和外观取决于疾病进展具有更好的血糖小鼠具有更大，更明亮，更胰岛（高达300胰岛）相比，更坏的血糖小鼠具有较小的胰岛具有半透明的外观（在100胰岛） 2光子检测 2光子成像是一种间接测定法来测量胰岛素分泌以单一胰岛素颗粒融合的水平。响应于刺激，例如葡萄糖或高钾，胰岛素颗粒熔合质膜和细胞外染料SRB进入第Ë颗粒，这导致突然荧光斑点〜400nm的直径的外观（ 图5）。各种实验已经进行了验证该测定作为融合含有胰岛素的颗粒，如葡萄糖剂量依赖性增加的颗粒融合事件数目与胰岛素分泌的期望量，所述响应单元中的2的大小一致的的测量光子与β细胞的，由2光子和电子显微镜测得的类似颗粒直径，染料摄取和胰岛素的共定位染色8。 内记录时，响应于刺激识别和事件的像吻 - 运行或全融合的融合事件的位置，外观的时间，持续时间和类型的所有胞吐事件的特征（ 图5，6）。在融合过程中，这些特性可用于评估有价值<html>在2型糖尿病模型的颗粒融合的任何缺陷。我们使用这种方法以前的报告显示，在糖尿病的db / db胰岛缺陷为满颗粒融合的损失，而不是在单个颗粒融合7动力学的特性的变化。此外，我们表明，在降低胰岛素分泌的疾病的最大因素是在应答细胞7的数量的减少。 <img alt="图1" src="/files/ftp_upload/52632/52632fig1.jpg" /> 图1:夹紧和喷射的部位的图注入的酶的肝总管和胆囊管的交界处附近的胆总管。夹紧瓦尔特壶腹通过血管钳，以方便注射酶到胰管。 2632fig2.jpg"/&gt; 图2:在小鼠中的注入过程 。顶部图说明胆总管施加在瓦尔特壶腹血管钳内的针。下图显示了注射后LIBERASE灌注胰腺。 <img alt="图3" src="/files/ftp_upload/52632/52632fig3.jpg" /> 图3:野生型和的db / db胰岛典型图像（A）野生型胰岛。 （B）的db / db孤立小岛。 （C）在消化胰岛附带腺泡细胞（箭头）。 （ 四）在消化胰岛小和模糊的小岛（箭头）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" sRC ="/文件/ ftp_upload / 52632 / 52632fig4.jpg"/&gt; 图4:订造2光子显微镜的主要组成部分 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52632/52632fig5.jpg" /> 图5:为记录有2光子显微镜单一胰岛素颗粒融合事件的实施例左数据代表前（A）和后（B）一种胞吐事件（箭头）响应于15mM的外观葡萄糖刺激的细胞。 （C）表示与细胞外染料SRB的进入颗粒的SRB标记，当它与质膜融合。 （D）示出以上的胞吐事件的感兴趣区域的荧光强度以及sequentia此事件胞吐（比例尺1微米）的L-图像。图片来源做等人 2014年7修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52632/52632fig6.jpg" /> 图6:双光子显微镜用SRB作为细胞外染料下的野生型和糖尿病的db / db胰岛的响应 。在这些实验中胰岛素颗粒融合事件响应于15mm的葡萄糖刺激被记录。黄色圆圈是胞吐事件的过程中记录的20分位点。图片来源做等 2014 7修改。

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

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, ...

leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Nanyang Technological University

Research

JoVE Journal

Medicine

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

9.4K 次观看.  The University of Queensland. 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.

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

Near Infrared Optical Projection Tomography for Assessments of &beta;-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen's (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)2; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction2 and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations3. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.

Near Infrared Optical Projection Tomography for Assessments of &#946;-cell Mass Distribution in Diabetes Research

We describe the adaptation of optical projection tomography (OPT)1 to imaging in the near infrared spectrum, and the implementation of a number of computational tools. These protocols enable assessments of pancreatic &beta;-cell mass (BCM) in larger specimens, increase the multichannel capacity of the technique and increase the quality of OPT data.

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of ...

科研

医学

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

生物学

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

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

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

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

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 ...

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 ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

急性小鼠胰腺组织切片钙动力学的共聚焦激光扫描显微镜

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

移植胰岛:一种将胰岛植入小鼠眼前房进行体内成像的方法

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...

JoVE Encyclopedia of Experiments

癌症研究

胰腺癌

Assays & techniques

Intravital Imaging: A Method for Cellular Level Observation of the Mouse Pancreas Through a Stabilized Imaging Window

Source: Park, I. and Kim, P. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62538">Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window.</a> J. Vis. Exp. (2021).
In this video, we describe the intravital imaging of a mouse pancreas through a stabilized window for long-term visualization of cellular dynamics.

活体成像:一种通过稳定成像窗口对小鼠胰腺进行细胞水平观察的方法

Source: Park, I. and Kim, P. Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital ...

瘦素受体

摘要

探索更多视频

此视频中的章节

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

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

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

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

小鼠胰腺胰岛细胞亚群中的钙动力学成像

急性小鼠胰腺组织切片钙动力学的共聚焦激光扫描显微镜

移植胰岛:一种将胰岛植入小鼠眼前房进行体内成像的方法

活体成像:一种通过稳定成像窗口对小鼠胰腺进行细胞水平观察的方法

瘦素受体

摘要

探索更多视频

此视频中的章节

近红外光学投影层析成像技术在糖尿病研究β细胞质量分布的评估

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

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

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

稳定纵向 在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口

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

小鼠胰腺胰岛细胞亚群中的钙动力学成像

急性小鼠胰腺组织切片钙动力学的共聚焦激光扫描显微镜

移植胰岛:一种将胰岛植入小鼠眼前房进行体内成像的方法

活体成像:一种通过稳定成像窗口对小鼠胰腺进行细胞水平观察的方法

稳定纵向在Vivo 细胞级可视化胰腺在穆林模型与胰腺内活性成像窗口