This work was supported by NIDDK grants DK098143 and DK101530 from the NIH (JPA), Stanford's Spectrum Child Health Research Institute (CHRI) and SPARK (UL1 TR001085, JPA).

该协议是根据医学的斯坦福大学医学院的机构动物护理和使用委员会（IACUC）进行。 300克（8 - - 9周龄）的雄性Sprague Dawley大鼠，这足以产生228井胰岛细胞复制评估384孔板的所描述的协议从六个250缩放为胰岛分离。 1.材料准备<ol><li>之前通过在15厘米的组织培养皿28收集维持在合流3天804G大鼠膀胱癌细胞的条件培养基（20毫升RPMI1640的）发起胰岛分离制备涂覆介质 。无菌过滤并储存（-20℃）以备后用的条件培养基。 </li><li>消毒手术工具（一个12厘米齿组织钳，一是11.5厘米精剪，一是14.5厘米手术剪，二16厘米弯钳，一是12厘米弯止血钳，一是12厘米手术刀手柄）和初始化之前一30目组织筛iating胰岛分离。 </li><li>在60ml 1×的Hanks&#39;平衡盐溶液（HBSS）中的补充有钙和镁- Ⅱ类胶原酶（40万台300,000总胶原酶活性）:通过溶解60％制备胰消化溶液 :纯化的I类的40％混合物中。负载10毫升胰消化缓冲液到10ml注射器（六）具有一个1"22克针和放置在冰上。 注10毫升消解液每鼠注射。相应的规模。 </li><li>通过混合1.3毫升氯胺酮（100毫克/毫升），0.2赛拉嗪的混合物（100毫克/毫升）和3毫升的PBS制备4.5毫升麻醉剂鸡尾酒在通风橱。启动胰岛分离的1小时内准备麻醉的鸡尾酒。 </li><li>通过加入25ml的新生小牛血清的至500ml的HBSS制备洗涤缓冲液 。放置洗涤缓冲液在冰上待用。 </li><li>制备1瓶胰岛介质，其为500 mL的低葡萄糖Dulbecco氏改良的Eagle培养基（DMEM），5的0毫升胎牛血清（FBS），青霉素5000单位/ ml和5000微克/毫升链霉素。 </li><li>准备从功能/存活率溶液（500毫升），用2％FBS，2mM谷氨酰胺，5mM葡萄糖，青霉素5000单位/ ml和5000微克/ ml链霉素的胰岛功能介质 。存储供以后使用胰岛功能的媒体 （4℃）。 </li><li>通过加入2.5毫升驴血清和0.12毫升三硝基甲苯X-100至37.38毫升1×磷酸盐缓冲盐水（PBS）中制备40毫升封闭缓冲液。供以后使用商店封闭缓冲液（4℃）。 </li><li>事先得到启动协议所需的抗体。 注意:PDX-1（TRITC）和Ki-67（FITC）共染色用于主β细胞复制的筛选。对于具体的谱系复制分析，进行免疫荧光染色额外的细胞特性标记（胰岛素，胰高血糖素，波形或生长抑素; TRITC）核（DAPI），PDX（Cy5的）和Ki-67（FITC）染色一起。另类代表lication标记， 例如 ，PCNA，也可以使用。 </li><li>准备在一式四份执行的每个处理条件的228井的治疗方案。包括正性（5-Iodotubercidin [1μM]或双嘧达莫[15μM]）和车辆控制（DMSO 1:666稀释）井。 </li></ol> 2.胰腺灌注<ol><li>由麻醉剂稀释液的鸡尾酒（0.30毫升/ 100克体重）腹腔注射麻醉大鼠。一旦老鼠是完全麻醉，反应迟钝有害刺激，进行颈椎脱位。 </li><li>放置在仰卧位（头脚方向）纸巾的叠层上安乐死大鼠和喷雾用70％乙醇腹部区域。 </li><li>具有U切口（基于耻骨区域）打开腹腔，并在延髓方向上胸部回缩皮肤以暴露腹腔。 </li><li>仔细抢使用两对弯钳十二指肠，找到共同的b的进入十二指肠ILE管（奥狄括约肌）和管夹在乳头用弯止血钳十二指肠开放。 </li><li>解除用于夹紧胆总管乳头和抢用弯钳胆管接近肝脏的弯止血。使用钝性分离与弯钳分离和暴露胆管。 </li><li>重新定位与头部近端和脚远端的老鼠。 </li><li>导管插入在胆总管（CBD），或只是远端与胰消化溶液胆囊和常见肝管的交界-loaded 10毫升注射器慢慢注入10 胰腺消化溶液 。同时注入，支持CBD用手术刀手柄。重新定位针如果管道和胰腺不能夸大。 </li><li>删除使用弯钳和细剪刀将其从降结肠，肠，胃，脾分离充气胰腺。置于冰上膨胀胰腺中含有5毫升50毫升管<eM&gt;胰腺消化液。 注意:为了获得最大的胰岛收获，收集每次50ml消化管30分钟和地点只有1或2胰腺内的所有胰腺。 </li></ol> 3.胰腺的解离<ol><li>一旦所有胰腺被收集，该消化管放入37℃水浴15分钟。轻轻旋转每3分钟的管子。 </li><li> 15分钟后，大力摇晃（垂直）管的10倍，并添加10毫升冷洗涤缓冲液 。大力摇晃管额外的5倍，并置于冰上。 </li><li>颠倒试管5次填补消化管到50毫升冷洗涤缓冲液和组合。 </li><li>离心在97 XG管在4℃下1分钟，倒出上清液。要小心，不要打跑沉淀组织。 </li><li>加入25 1ml 洗涤缓冲液并通过温和涡旋重新悬浮组织。重复步骤3.4和3.5倍以上。 </li><li>将30目筛组织通过STerile容量250ml的烧杯，并倒入组织悬液到筛。冲洗消化管与另外20 1ml 洗涤缓冲液倒到网格。未消化的胰腺和脂肪组织是在此步骤中去除。 </li><li>倒入过滤材料成两个新鲜的50毫升管，并通过在4℃以97 XG纺丝1分钟沉淀组织。倒出上清，逆变管排出多余的缓冲区。 </li></ol> 4.胰岛分离纯化<ol><li> 20ml冷多聚蔗糖/钠泛影葡胺溶液（1.119克/ ml的密度）添加到沉淀胰腺组织。轻轻吹打上下五次均匀重新悬浮每个管中的内容。 </li><li>轻轻覆盖10毫升HBSS的多聚蔗糖/泛影葡胺钠溶液。通过沿管壁缓慢加入HBSS保持锋利液体界面。 </li><li>离心在560 xg离心消化的胰腺15分钟（4℃）以缓慢的加速和不制动。 </li><li>有限公司在新鲜的50毫升管使用10毫升吸管和地点胰岛llect从界面胰岛层。不要在这个阶段集中小岛。 </li><li>在140 xg离心加40 1ml 洗涤缓冲液中，以每50ml管中并旋转1分钟，在4℃。 </li><li>倒掉上清液，并通过反相管几次重新悬浮在50ml 洗涤缓冲液汇集小岛。离心1分钟的管子在97 XG在4℃收集的胰岛中的颗粒。 </li><li>倒掉洗涤液 ，重复清洗。把胰岛进入组织培养罩和吸出上清液。 </li><li>重悬胰岛每管6毫升胰岛介质 。 </li><li>传送从每次50ml管中的胰岛成六个孔组织培养皿的孔中。摇动6孔皿收集胰岛入井的中部，并在显微镜下观察胰岛质量和纯度。 </li><li>手动收集使用1毫升吸管小岛和拾取胰岛转移到邻近的以及含6毫升胰岛媒体。重复胰岛旋流和采摘直至&gt; 90％纯度的实现。 </li><li>转移纯化胰岛含20ml 胰岛培养基在10cm组织培养皿中。 </li><li>在组织培养箱地方胰岛（37℃，5％CO 2）过夜（ 图1A）。 </li><li>在制备镀胰岛细胞，外套与804G条件培养基的40微升每孔384孔平板的孔中。在组织培养箱孵育过夜。 </li></ol> 5.分散和胰岛细胞的电镀<ol><li>翌日，轻轻分离从10cm皿用细胞刮刀胰岛和胰岛转移到50毫升管中。 </li><li>沉淀通过离心胰岛1分钟，在室温下22 XG。吸出上清液。 </li><li>用20毫升温的PBS洗涤小岛和如上倒出。 </li><li>重悬在0.25％胰蛋白酶的胰岛（每只大鼠胰腺150微升），在37℃下孵育10分钟。吸管胰岛并用1ml移液器后5和10分钟温育的上下10次。 </li><li> 10分钟后，评价在显微镜下胰蛋白酶化的胰岛的20微升样品，以确保完全消化并使用血细胞计数器计数的胰岛细胞。如果未消化的胰岛仍然存在，继续培养额外的3 - 5分钟，吸管上下10次以上。根据需要重复。 注:典型的产量〜125000 - 每个大鼠胰岛150000细胞。 </li><li>从培养箱除去804G空调涂覆介质384孔板并用12孔歧管抽吸除去介质。 </li><li>暂停胰岛细胞以每毫升45000细胞胰岛功能介质的密度和板每孔70微升（3150细胞）与多道移液器（图1B）。 注意:由于位于外井β细胞倾向于朝向板使用的行C的周长为N和列4迁移到21至板228的孔与来自6只大鼠中分离的胰岛细胞。 </li><li>放置在组织培养孵化该板48小时，以允许细胞粘附。 </li></ol> 6.胰岛细胞治疗文化，固定和染色<ol><li>制备200μl的车辆和化合物（处理条件以一式四份进行）在胰岛功能介质 2倍的浓度。安排在无菌96孔板化合物，以促进多井移液。 </li><li>小心地取出胰岛培养基12孔歧管吸引和用12孔吸管胰​​岛功能介质（35微升/孔）更换。取出和更换时间从一行媒体限制干燥（图1C）的风险。 </li><li>通过转移35微升/孔的2X化合物溶液开始治疗。返回板的孵化器为48小时的治疗持续时间。 </li><li>处理48小时后，通过倒转板倒出处理介质。&lt;/ LI&gt; <li>轻轻地洗胰岛细胞每1×PBS（1分钟）以及50微升。使用多通道移液器添加PBS中。 </li><li>倒出PBS中并通过加入每冷的4％多聚甲醛（PFA）的孔50μl在1×PBS中稀释固定细胞。孵育细胞在4℃下15分钟。 </li><li>通过倒转板以除去PFA和轻轻加入60微升的PBS每孔如上洗涤细胞三次（每次5分钟）。 </li><li>通过混合14.25毫升甲酰胺加入0.75ml 0.15M的柠檬酸钠（pH值为6）制备15毫升抗原修复溶液。使用前的抗原修复立即解决方案。 </li><li>通过倒转板，从孔中除去PBS中，添加60微升抗原修复溶液每个孔中。加热在70℃的水浴的板45分钟。将此温育过程中在板的顶部的加热块，以防止该多孔板的翘曲。 注:水应该是半山腰板裙子;该板不应该被淹没。 </li><l我&gt;移除从水浴板并让其冷却，在室温下20分钟。确保该板不通过视觉检查翘曲。 </li><li>清洗细胞，每孔60微升的1×PBS清洗3次（每次5分钟）。添加PBS中的一个多通道移液器和通过反转所述板倒出PBS中。 </li><li>使用多通道移液器以每孔加入封闭缓冲液的40微升孵育在室温下30分钟。通过倒转板和倾析除去阻断缓冲液。 </li><li>添加小鼠抗人Ki-67的（1:200）的40微升和山羊抗人PDX-1（1:100），在封闭缓冲液到每个孔中稀释的抗体并在4℃下孵育过夜。 </li><li>第二天，弃去抗体溶液并用1×PBS洗涤细胞三次（每次5分钟），如上所述。倒出PBS。 </li><li>添加40微升每稀释的井（1:200）生物素化的驴抗小鼠IgG和罗丹明缀合的驴抗山羊IgG在封闭缓冲液。孵育1小时在室温下。 </li><li>清洗细胞用1×PBS清洗3次，每次5分钟。添加40微升稀释（1:400在封闭缓冲液）的荧光素缀合的链霉抗到每个孔中。在室温下孵育30分钟。 </li><li>清洗细胞用1×PBS清洗3次，每次5分钟。添加40微升每DAPI的井（300纳米在封闭缓冲液中），并孵育在室温下15分钟。 </li><li>洗涤细胞用1×PBS两次并离开细胞在40微升1×PBS中。该板现在准备进行分析。 </li></ol> 7.β细胞复制分析注:测试方案必须用来测量β细胞复制的高内涵筛选显微镜成立。在其最简单的组合物，该协议是一两色测定法，其中的标识标记是用来定义β细胞（PDX-1 + -细胞）和一个复制标记（Ki-67的）被用来定义细胞分裂事件。 <ol><li>标识基于所述averag对象（β细胞）β型细胞核（图2A）的预期像素区域内:（宽度&lt;1.6的长度）的大致圆内PDX-1染色（549纳米过滤器）电子荧光强度。 </li><li>其次，建立设置来识别PDX-1 +区域（图2B）内根据平均荧光强度（485纳米过滤器）的复制事件（KI-67阳性）。 注意:曝光时间必须固定到允许跨图像的像素强度的比较。测定协议参数被调整，以使得机器呼叫始终排除非特异性染色，碎片和细胞聚集体，可能的数据质量造成不利影响。 </li><li>分析的最小每孔500β细胞以最大化精度和限制可变性。 注意:在大鼠胰岛培养物的基底β细胞复制索引预期为0.5 - 3％。通常情况下，每孔40幅影像是绰绰有余，以确定500β细胞。 </li><li>此前分析整个板，分析所选的负（DMSO处理）和阳性对照（5- iodotubercidin [1μM]或双嘧达莫[15μM]）处理的孔中，以评估该测定方案的有效性。当被正确建立的测定中，阳性对照化合物诱导至少在复制β细胞的百分比增加两倍。 </li><li>阅读整个盘中一度测试方案进行了验证。 </li></ol>

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The authors declare that they have no competing financial interests.

为研究控制β细胞的生长和再生的分子通路的实验方法对于糖尿病研究的重要工具。在此，基于大鼠胰岛筛选平台，以确定和描述β细胞复制的小分子刺激呈现。 虽然该协议的大部分内容很容易被经验丰富的研究人员进行了几步需要特别的技术。首先，胰岛分离过程中，而不破坏其完整性胆管插管需要实践。一个有用的策略是最低限度地膨胀胆管，以确保充分配药胰腺消化液?...

糖尿病包括病症共享打乱葡萄糖稳态的共同终点的集合。虽然糖尿病亚型的致病机理是不同的，它们共享降低β细胞量， 即 ，胰岛素生产能力1,2-损失的后果。目前，糖尿病的治疗策略依赖外源性胰岛素，胰岛素的产生或增强胰岛素敏感性的药理学刺激的长期管理，也很少，胰岛或全胰腺3,4移植。令人遗憾的是，这些策略的成功是短命的和/或不能充分概括内源性胰岛素产生的作用。尽管显影刺激β细胞再生的方法的实用性，没有这样的方法存在。因此，一个重要的糖尿病研究的目标是开发的方法来产生新的β细胞或展开内源性β细胞量5 </sup&gt;。虽然来自可再生资源，如胚胎干细胞β细胞再生正在推进，安全性和效率的关注使得替代策略，包括膨胀成熟β细胞，优先级6,7的追求。重要的是，新的β细胞在体内的主要来源是预先存在的β细胞，而不是专门祖细胞8,9。虽然β细胞看起来具有有限的复制能力，在β细胞量略有增加（〜30％）可足以在许多糖尿病患者恢复葡萄糖动态平衡。另外， 在 β细胞量的原位药物刺激是一种潜在的廉价的和可扩展的治疗策略。这里提出了识别和表征刺激β细胞生长的小分子的高含量筛选方法。 各种体外实验方法可以用于鉴定基因产物和/或分子塔ŧ促进初级β细胞复制。用于测量β细胞复制诱导早期的努力用于胎儿啮齿动物的胰腺文化或完整隔离胰岛文化来衡量响应特定的处理条件10 [3 H]胸苷掺入，BrdU的醛，硫堇或胰岛素染色人群中掺入或有丝分裂体， 11。 这些体外的方法和密切变体具有一些局限性。突出的缺陷包括:（1）使用胎儿细胞，不同于成熟β细胞，显示高基底β细胞复制速率和在明显的方式12调节生长; （2）β细胞复制事件实验者相关裁决的主观性; （3）β细胞复制事件实验者依赖计数的劳动和时间密集性质阻碍了实验的吞吐量; （4）利用核掺入/染色/外观识别复制甚至TS和一个非重叠的胞质染色，以确定β细胞导致邻近非β细胞复制事件对β细胞的错误认定。 最近成熟原β细胞已被用于评估转基因过度表达对β细胞复制13-16的影响，以及基因产物或化合物处理。然而，这些研究还依赖于复制事件，对β细胞鉴定和/或限制通过劳动密集型的步骤， 例如 ，细胞或完整胰岛个体滑动孔电镀细胞质staining-或非特异性方法的主观计数石蜡包埋和处理17。值得注意的是，基于图像的人β细胞复制的筛选方法，类似于本文中所呈现的人，已发表​​18;然而，成功地利用该测定的尚未得到证实和利用对于初筛人胰岛的可能不是大致FEAsible。 识别复制促进物质的另一种策略是评估的β细胞系的生长诱导。最初的努力用于转化的β细胞，系如MIN6细胞或INS一十三分之八百三十二细胞14,19-21。然而，这些细胞系中表现出奔放增长，没有什么相似之处分化良好的β细胞22。因此，增长的感应能力是最小的，目前还不清楚相关的，有时难以概括。基于细胞系的筛选改进策略采用"可逆转变"是生长在没有四环素（强力霉素）的逮捕依赖SV40 T抗原表达23,24细胞。然而，目前还不清楚这些细胞是否恢复到在强力霉素除去一个"正常"β细胞样状态。不幸的是，使用这些细胞已经产生，不会出现有直接效用广义生长促进化合物24。总体而言，使用的细胞系来研究细胞类型中显示最小的自发复制活动的生长调控可能具有有限的适用性。 本文中所呈现的β细胞复制筛选平台利用成熟原代大鼠β细胞在体内的生长调节以保持在可能的范围内，混合细胞型组合物，以便能够谱系限制性生长促进性活动的胰岛细胞培养物，多-Well格式以最大化吞吐量和自动化的分析，以消除偏见和促进吞吐量。成功地利用这个平台，使促进胰岛β细胞的复制25,26几种化合物的鉴定。此外，该测定已被用于结构 - 活性关系的研究和化学上位实验以提供机械见解β细胞复制的分子调控。所提出的平台成功地适应FOβ细胞复制的RNA干扰基于-R的慢病毒调查通路25。该测定的局限性包括限制可扩展性（使用原代细胞），利用啮齿动物，而不是基于抗体的成像和初级胰岛使用，使用相关联的人胰岛细胞（尽管该测定可适于人胰岛研究），费用的的分散的胰岛（胰岛破坏架构），以方便自动图像采集和依赖性在与图像采集和分析能力的自动显微镜的可用性。虽然用于鉴定基因产物或刺激原位 β细胞再生化合物的简便体内筛选方法将是理想的，这样的平台尚不可用27。因此，所描述的平台是适合研究者感兴趣的研究β细胞复制的大多数方面。

<table><tbody><tr><td>250 g male Male Sprague Dawly Rat</td><td>Charles River</td><td>Stain # 400</td><td></td></tr><tr><td>12 cm teeth tisuue forceps</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>11.5 cm fine scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>14.5 cm surgical scissors</td><td>Fine Science Tools</td><td>14001-14</td><td></td></tr><tr><td>16 cm curved forceps</td><td>Fine Science Tools</td><td>11003-16</td><td></td></tr><tr><td>12 cm curved hepostat</td><td>Fine Science Tools</td><td>13011-12</td><td></td></tr><tr><td>12 cm scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Tissue sieve-30 mesh</td><td>Bellco Glass</td><td>1985-85000</td><td></td></tr><tr><td>Cizyme RI, 375,000 CDA units</td><td>VitaCyte</td><td>005-1030</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution (Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;++&lt;/sup&gt;)</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>Ketamine HCl (200 mg/20 ml)</td><td>JHP Pharmaceuticals</td><td>NDC# 42023-113-10</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Xylazine (5 g/50 ml)</td><td>LLOYD</td><td>NADA# 139-236</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>H-1077</td><td>to make histopaque 1100</td></tr><tr><td>Histopaque 1119</td><td>Sigma</td><td>H-1119</td><td>to make histopaque 1100</td></tr><tr><td>Newborn Calf Serum 500 ml</td><td>Hyclone</td><td>SH30118.03</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution</td><td>Hyclone</td><td>SH30268.01</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Low Glucose&amp;nbsp;</td><td>Hyclone</td><td>SH30021.01</td><td></td></tr><tr><td>Functionality/Viability Solution&amp;nbsp;</td><td>Mediatech</td><td>99-768-CV</td><td></td></tr><tr><td>RPMI1640 media&amp;nbsp;</td><td>Hyclone</td><td>SH30096.01</td><td>to make conditioned medium</td></tr><tr><td>804G rat bladder carcinoma cell-line</td><td>Available upon request</td><td></td><td>to make conditioned medium</td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>26160</td><td></td></tr><tr><td>GlutaMax-I</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin (5,000 IU/ml/Strptomycin (5 mg/ml)&amp;nbsp;</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Formamide 500 ml</td><td>Fisher BioReagents</td><td>BP227-500</td><td></td></tr><tr><td>Antigen Unmasking Solution 250 ml (pH 6.0)</td><td>Vector Laboratories</td><td>H-3300</td><td>to make 0.15 M Sodium Sitrate solution</td></tr><tr><td>Dextrose, Anhydrous</td><td>EMD Chemicals</td><td>DX0145-1</td><td>to make 1 M glucose solution</td></tr><tr><td>Nomal Donkey Serum (Powder)</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-100ML</td><td></td></tr><tr><td>Mouse anti-human Ki67 antibody</td><td>BD Biosciences</td><td>556003</td><td></td></tr><tr><td>Goat anti-human PDX-1 antibody</td><td>R&amp;amp;D Systems</td><td>AF2419</td><td></td></tr><tr><td>Polyclonal Guinea Pig anti-insulin antibody</td><td>Dako</td><td>2016-08</td><td></td></tr><tr><td>Polyclonal Rabbit anti-glucagon antibody</td><td>Dako</td><td>2014-06</td><td></td></tr><tr><td>Polyclonal Rabbit anti-somatostatin antibody</td><td>Dako</td><td>2011-08</td><td></td></tr><tr><td>Polyclonal chicken anti-vimentin antibody</td><td>abcam</td><td>ab24525</td><td></td></tr><tr><td>Biotin-SP-conjugated, Donkey Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>715-065-150</td><td></td></tr><tr><td>StreptAvidin, Alex Flour 488 conjugated&amp;nbsp;</td><td>Invitrogen</td><td>S32354</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Goat IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>705-025-147</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-025-148</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-025-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Goat IgG</td><td>Jackson ImmunoResearch</td><td>705-175-147</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-175-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Chicken IgG</td><td>Jackson ImmunoResearch</td><td>703-175-155</td><td></td></tr><tr><td>DAPI</td><td>Millipore</td><td>S7113</td><td></td></tr><tr><td>Disposable Reagent Reservoir 25 ml</td><td>Sorenson BioScience</td><td>39900</td><td></td></tr><tr><td>384 well, black/clear, tissue culture treated plate</td><td>BD Falcon</td><td>353962</td><td></td></tr><tr><td>96 well, black/clear, tissue culture treated plate</td><td>Costar</td><td>3603</td><td></td></tr><tr><td>Multi-channel pipettor</td><td>Costar</td><td>4880</td><td></td></tr><tr><td>12-channel vaccume aspirator</td><td>Drummond</td><td>3-000-096</td><td></td></tr><tr><td>Cell Scraper</td><td>Falcon</td><td>353085</td><td></td></tr><tr><td>Isotemp Water Bath Model 2223&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>High-content screening instrument: ArrayScan VTI</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

a high-content in vitro pancreatic islet &#946;-cell replication discovery platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

为了评估β细胞或α-细胞复制，需要一个四色测定方案。首先，对象由DAPI染色（通道1，386纳米）来识别。接下来，β细胞（事件1）计数:的对象共表达PDX-1 +（通道2，650纳米）和近郊核胰岛素（通道3，549纳米）。随后，复制β细胞（事件2）被计算:β-细胞（事件1），该共表达Ki-67的（信道4中，485纳米）（图3）。复制β细胞的百分比来计算:（事件2 /事件1...

Watch this Scientific Journal Video about A High-content In Vitro Pancreatic Islet Î²-cell Replication Discovery Platform at JoVE.com

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

对于糖尿病研究领域的关键挑战是要明白，调节胰岛β细胞的复制，并制定刺激β细胞再生方法的分子机制。在此高内涵筛选的方法来识别和评估小分子的β细胞复制促进活性表示。

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

a-high-content-vitro-pancreatic-islet-cell-replication-discovery

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

11.1K 次观看.  Stanford University School of Medicine. 对于糖尿病研究领域的关键挑战是要明白，调节胰岛β细胞的复制，并制定刺激β细胞再生方法的分子机制。在此高内涵筛选的方法来识别和评估小分子的β细胞复制促进活性表示。 

视频: A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

体外测定菌落为表征的三效祖细胞从成人胰腺小鼠隔离

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

科研

发育生物学

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

复制和β细胞功能评估Adenovirally转分离的啮齿动物胰岛

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

医学

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

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

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

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

β细胞的有丝分裂原持续管理局完整小鼠胰岛<em&gt;离体</em&gt;使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

生物工程

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

在2D培养系统中将人多能干细胞分化为胰腺β细胞前体

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors 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 ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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 ...

生物学

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 ...

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, ...

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