我们感谢 Ninov 实验室的成员对手稿的评论, 再生疗法中心的成员德累斯顿 (CRTD) 鱼和显微镜设施的技术援助。N.N. 的资助来自于 DFG-CRTD, 在土德累斯顿, 德国研究基金会 (DFG) 和德国糖尿病研究中心 (DZD) 的杰出群体。

所有的程序, 包括动物被批准的动物福利法案, 并征得 Landesdirektion Sachsen, 德国 (亚利桑那州 24–9168.11-1/2013-14, T12/2016) 的许可。
1. 准备
注: 本协议是对斑马鱼原胰岛从双转基因 Tg (Renilla-mKO2; cryaa: CFP) 的体外成像;Tg (ins: GCaMP6s; cryaa: mCherry) 19 斑马鱼。在这一转基因线, 胰岛素启动子 (ins) 驱动β细胞特异表达两个转基因: nls-Renilla-mKO2, 这标志着β细胞细胞核与单体 Kusabira 橙 2 (mKO2) 荧光;和 GCaMP6s15, 它发出绿色荧光反应增加细胞内钙水平。GCaMP6s 的β细胞特异表达能够研究β细胞的葡萄糖反应性, 而不受周围细胞类型钙变化的干扰。
<ol><li>在2毫升离心管中, 将10毫克的牛纤维蛋白原溶于1毫升的钙2 +/毫克1.5 +含汉克斯的平衡盐溶液 (HBSS), 制备新鲜纤维蛋白原 (10 毫克/毫升)。漩涡大力, 直到纤维蛋白原粉完全溶解。使溶液在室温下保持至少15分钟。 注: 如果溶液开始聚合并变得粘稠, 可以在室温下保存2–3。</li>
	<li>通过稀释 HBSS 中的纤维蛋白原三倍, 制备纤维蛋白原工作溶液 (3.3 毫克/毫升)。例如, 将300µL 的纤维蛋白原储存在600µL 的 HBSS 中, 准备900µL 的纤维蛋白原工作溶液。</li>
	<li>在1毫升的 HBSS 或磷酸盐缓冲盐水 (PBS) 中溶解10单位凝血酶, 制备凝血酶溶液 (10 U/毫升)。 注: 本解决方案可提前准备, aliquoted 50 µL 部分, 冷冻-20 摄氏度。</li>
	<li>在50毫升水中溶解1.8 克 d-葡萄糖, 制备200毫米 d-葡萄糖溶液。保持4摄氏度, 长期贮存。</li>
	<li>在50毫升水中溶解1.1 克氯化钾, 制备300毫米氯化钾溶液。保持4摄氏度, 长期贮存。</li>
	<li>采购35毫米直径的玻璃底部的菜肴, 以安装胰岛。</li>
	<li>对于胰岛的解剖和安装, 使用装有荧光灯和红色过滤器立方体的立体声显微镜 (TRITC: 励磁: 532–554 nm, 发射: 570–613 nm; 或得克萨斯红色: 励磁: 540–580 nm, 发射: 592–667 nm)。</li>
	<li>为了成像β细胞中的钙流入, 使用一个倒置共焦显微镜 (蔡司 LSM 780 或类似) 与 20X (0.8 NA) 的空气目标, 并配备一个板持有人为35毫米直径的玻璃底菜。</li>
	<li>为 euthanizing 斑马鱼 tricaine 甲烷磺酸 (MS222) 制备200毫克/升溶液。</li>
	<li>为斑马鱼解剖采购了 90 mm 培养皿。</li>
</ol>2. 斑马鱼主要胰岛解剖和安装
<ol><li>弄死在 MS222 中使用长时间孵化的鱼。为此, 用渔网轻轻地将鱼从鱼缸中取出, 在含有 MS222 溶液的培养皿中孵化, 直到动物不显示鳃盖 (鳃) 运动;通常, 这需要5分钟. 将鱼转移到含有 HBSS 溶液的培养皿中, Ca2 +/毫克2 +。</li>
	<li>
在装有荧光灯和红色过滤器立方体的立体显微镜下, 解剖动物腹部右侧的皮肤, 以隔离胰腺。
	<ol>
<li>为此, 用锋利的镊子将斑马鱼的皮肤从嘴里切开到肛门鳍上。剥去切开的皮肤露出腹部;这个截图暴露了内脏。采用 mKO2 在β细胞中的荧光表达, 通过显微镜下的目视检查确定胰岛的位置。如果需要, 取出肝脏的裂片, 因为它们可以覆盖胰岛, 使它很难找到。 注意: 主胰岛位于腹部前部, 通常在右侧。</li>
	</ol>
</li>
	<li>通过仔细去除周围组织如肝脏和脂肪细胞来清洁主胰岛。采取预防措施, 不要伤害或戳胰岛;在清除周围的组织后, 在胰岛表面的单个细胞变得明显。</li>
	<li>将30µL 滴 HBSS 到玻璃底菜的中心。将解剖的胰岛转移到此下落。</li>
	<li>用 HBSS 和一次用30µL 的纤维蛋白原工作溶液 (3.3 毫克/毫升) 仔细清洗胰岛。确保避免干燥的胰岛在洗涤步骤, 因为没有这样做导致细胞死亡。</li>
	<li>慢慢地, 轻轻地加入10µL 的凝血酶溶液 (10 U/毫升)。离开胰岛和15–20分钟未触及的菜. 观察纤维蛋白原-凝血酶滴在此时会变得黏稠, 有点不透明。</li>
</ol>3. GCaMP 荧光强度在斑马鱼主要胰岛的体外活体成像研究
<ol><li>在模具顶部添加200µL HBSS, 并将盘子小心地放在共焦显微镜的板架上。使用 20X, 0.8 NA, 空气目标为共焦成像。使用 brightfield 选项定位胰岛。</li>
	<li>使用红色荧光过滤器查看β细胞中的核 mKO2 荧光, 重点放在胰岛上。单个原子核应该清晰可见。</li>
	<li>通过手动改变共焦显微镜的焦平面来定位清晰的成像平面, 以便沿着其 z 轴沿胰岛的厚度移动。确保成像平面包含足够数量 (50–100) 的β细胞用于成像, 核 mKO2 荧光的亮度是均匀的, 特别是在胰岛中心。</li>
	<li>使用 "智能设置" 菜单中的以下设置为 GCaMP6s 和 mKO2 荧光设置顺序获取: GCaMP6s, 激发: 488 毫微米, 发射: 500–555 nm, 假颜色: 绿色 (选择 "GFP");mKO2, 励磁: 561 毫微米 (mCherry), 发射: 570–630 nm, 假颜色: 红色 (选择 "mCherry")。 注: 使用此设置, 红色通道将记录β细胞核的位置, 而绿色通道将记录 GCaMP 荧光强度。</li>
	<li>在 "采集模式" 下, 将图像分辨率设置为 1024 x 1024 像素, 速度为 10, 平均为1。通过选择 "时间序列" 选项并设置500个周期的 "持续时间" 来启动连续录制, 每帧大约有2秒的捕获时间。 注: 时间序列的前50帧对应于β细胞在5毫米葡萄糖浓度下的活动。这是基线响应。反应β细胞将显示在绿色荧光强度与时间的打蜡和减弱。我们观察到, 一些 (1–5%) 的β细胞振荡5毫米葡萄糖。</li>
	<li>
注意成像周期。在头50帧之后, 将周围溶液的葡萄糖浓度提高到10毫米而不停止记录。
	<ol>
<li>在不扰动图像采集的情况下, 轻轻地将200毫米 d-葡萄糖溶液的5µL 放在保持胰岛的凝胶上。获得150帧在10毫米葡萄糖。 注: 葡萄糖浓度的增加将增加在绿色通道中 GCaMP 荧光振荡的β细胞数量。</li>
		<li>确保细胞细胞核在过程中保持稳定。如果胰岛在采集过程中有很广泛的震动, 原子核正从焦平面中移出, 则丢弃样本 (如果需要)。</li>
		<li>等待足够的时间, 使纤维蛋白原-凝血酶模聚合, 以确保后续样品的稳定性。</li>
	</ol>
</li>
	<li>在200帧, 进一步增加溶液浓度的20毫米, 轻轻吹打10µL 的200毫米 d-葡萄糖溶液。获得150帧的20毫米浓度。</li>
	<li>350帧后, 去极化胰岛使用30毫米的氯化钾。为此, 添加20µL 的300毫米氯化钾库存解决方案。在这个阶段, 观察 GCaMP6s 荧光振荡将停止和固定在高强度;没有反应葡萄糖的β细胞也可能在氯化钾添加时显示绿色荧光强度的增加。</li>
</ol>4. 单个β细胞 GCaMP 荧光迹的量化
注: 为了追踪和量化单个β细胞对不同葡萄糖水平的反应, 量化整个成像周期的 GCaMP 荧光强度。量化是在细胞分辨率下进行的。为此, 使用斐济20从图像中提取 GCaMP 荧光的强度值 (步骤 4.1–4.6), 电子表格软件或 R21执行分析 (步骤 4.8–4.9)。
<ol><li>使用 "LSM 工具箱" 在斐济打开图像文件。为此, 请选择 "插件 |LSM 工具箱 |显示 LSM 工具箱 "。在 "LSM 工具箱" 中, 单击 "打开 lsm" 并选择图像文件。 注意: 对于 "LSM 工具箱" 不支持的格式, 首先将它们转换为 tiff 进行分析。</li>
	<li>使用斐济的感兴趣区域 (ROI) 工具提取单元格的响应。在 "工具" 下的 "分析" 菜单中打开 "ROI 管理器"。使用位于工具栏中的 "多边形选择工具" 手动绘制 ROI。</li>
	<li>在红色通道中绘制β细胞核可见的 ROI。选择 ROI, 使其覆盖大于细胞细胞核的区域, 以包括细胞的某些细胞质。确保在帧之间的 ROI 位置是一致的, 并在必要时调整位置。</li>
	<li>单击 "添加 [t]" 按钮, 将选定的 ROIs 添加到 "ROI 管理器" 中。选择并向 ROI 中添加多个 ROIs 以获取多个单元格上的数据。</li>
	<li>接下来, 从 "分析" 菜单中, 选择 "设置测量"。选择 "集成密度", 用于指定区域内总荧光强度的提取。</li>
	<li>转移到包含 GCaMP 荧光的绿色通道, 并在 "ROI 管理器" 中选择 "多指标"。 注意: 这将为整个时间序列中的细胞提供强度测量。</li>
	<li>如果由于胰岛的移动而需要调整 ROI 位置, 请手动编译不同帧中的强度测量。将这些值复制并粘贴到单独的电子表格中。</li>
	<li>从 "LSM 工具箱" 获取图像帧的时间戳。使用 "套用邮票 |应用 t 邮票 |文件名 |转储到文本文件 "以获取时间戳。使用 "另存为" 选项保存时间戳, 或将它们复制到电子表格中。</li>
	<li>在编译所有单元格的强度值时, 一次或自动执行一次单元格分析 (例如, 使用 Excel 公式或 R)。</li>
	<li>
在两个步骤中分析单个单元格。
	<ol>
<li>在第一步中, 计算基线以上的荧光强度。为此, 计算基线荧光强度 (F0) 作为前50帧 (5 毫米葡萄糖) 的荧光强度的平均值。然后从整个时间序列 (f-f0) 减去基线 (f0)。 注: 少数细胞可以在基底葡萄糖下显示清晰的 GCaMP 振荡, 通常在高浓度的刺激下继续。对于这样的细胞, 只有通过取细胞在荧光中的下降的初始帧的平均强度来估计 F0 。</li>
		<li>在分析的第二步, 通过对荧光强度的规范化, 获得最终的 GCaMP 荧光强度。 注意: 这是为了消除不同动物之间的小岛之间的差异。单个胰岛在葡萄糖刺激下显示不同水平的荧光发射。</li>
		<li>将荧光强度除以最高强度值来正常化。为此, 计算峰值强度 (f最大-f0), 并按峰值强度除以基线减去值, 以产生最终的 GCaMP 荧光强度 (f-f0)/(f最大-f0)。</li>
	</ol>
</li>
	<li>放弃在氯化钾刺激后没有表现出强度变化的细胞, 因为它们可能不健康或受损。</li>
	<li>要在 r 中执行分析 (步骤 4.9–4.10), 请使用 r 脚本 (plotcelltrace。R) 提供本手稿。</li>
</ol>

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P., 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=Different+developmental+histories+of+beta-cells+generate+functional+and+proliferative+heterogeneity+during+islet+growth.">Different developmental histories of beta-cells generate functional and proliferative heterogeneity during islet growth.</a> Nat Commun. 8 (1), 664 (2017).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676 (2012).</li><li>R Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A Language and Environment for Statistical Computing. , (2016).</li><li>Friedman, R. S., 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=An+evolving+autoimmune+microenvironment+regulates+the+quality+of+effector+T+cell+restimulation+and+function.">An evolving autoimmune microenvironment regulates the quality of effector T cell restimulation and function.</a> Proc Natl Acad Sci. 111 (25), 9223-9228 (2014).</li><li>White, R. M., 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Bader, E., 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=Identification+of+proliferative+and+mature+&#946;-cells+in+the+islets+of+Langerhans.">Identification of proliferative and mature &#946;-cells in the islets of Langerhans.</a> Nature. 535 (7612), 430-434 (2016).</li><li>Dorrell, C., 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=Human+islets+contain+four+distinct+subtypes+of+&#946;+cells.">Human islets contain four distinct subtypes of &#946; cells.</a> Nat Commun. 7, 11756 (2016).</li><li>Johnston, N. 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作者声明没有竞争的财政利益。

在这里, 我们演示了一种技术, 以定量的β细胞葡萄糖反应性的单细胞分辨率。这是可能的, 通过监测细胞内钙浓度使用基因编码钙指标, GCaMP6s。β细胞活性是通过在纤维蛋白原 -凝血酶模中安装胰岛来捕获的。该协议的一个关键步骤是模具的稳定性。需要足够的时间, 以使纤维蛋白原溶解在 HBSS 溶液中。如果没有这个, 模具在成像过程中不能聚合足够的稳定性。一个胰岛安装在纤维蛋白原-凝血酶模具和浸泡在细胞培养媒体可以保持至少一个星期的可行性 (数据没有显示)。可替代纤维蛋白原-凝血酶模, 如低熔琼脂糖, 可以用来登上胰岛22。另一个关键参数是胰岛的解剖。在这一步骤中, 胰岛周围的组织需要删除不伤害或戳胰岛。一个巧妙的解剖是与实践。
成像协议的限制是对一个胰岛的共焦平面的限制。这样做是为了捕捉在单个β细胞内钙流入的动态。一个 Z 栈通过整个胰岛的厚度导致低成像速度和损失的振荡信号从单个细胞。这种限制可以通过使用更快的共焦成像方法 (如旋转盘显微术) 来提高, 以便在3维度捕获钙动力学。另一个前沿将是体内的钙成像12。斑马鱼胚胎的透明性质或使用无色素菌株的斑马鱼成人23可以打开在体内成像未来的可能性。
在高空间和时间分辨率下的β细胞活性成像可以研究单个β细胞间的功能异质性。这种方法可以帮助揭示β细胞亚群的存在。最近, 多项研究表明, 在名义上均质β细胞24,25,26的亚群存在。体外成像可以与基因记者结合, 以描述亚群体对葡萄糖的反应。此外, 结合成像设置和药理刺激可以筛选的化合物, 可以改善β细胞功能。
总之, 这里提出的技术允许量化和比较的葡萄糖反应性的个别β细胞。它为β细胞功能提供了一个直接的窗口, 这是糖尿病发展的一个重要参数。

我们的血糖维持在一个狭窄的范围内, 在很大程度上是由于内分泌胰脏的功能。胰腺的内分泌作用是由胰岛进行的, 它含有分泌荷尔蒙的细胞。胰岛素分泌β细胞负责降低血糖水平后, 含有碳水化合物的膳食。β细胞胰岛素分泌不足可导致1的糖尿病, 其特点是持续高血糖。1型和2型糖尿病目前在全世界超过4亿人, 导致发病率和死亡率2。通过调查导致β细胞功能障碍的分子和环境因素, 我们将更好地了解2型糖尿病是如何开始和进展的。此外, 在体外将人类干细胞分化为功能性β细胞的能力可以为1型糖尿病的细胞置换治疗提供新的β细胞来源。为此, 研究基因模型生物体的β细胞功能和成熟度, 以获得在菜肴中制作功能β细胞所需的知识是非常重要的。
β细胞功能可以监测在整个胰岛水平, 通过量化的总数量的胰岛素分泌反应葡萄糖刺激。这种累积方法将胰岛作为一组单元进行研究, 而不区分单元格的单个属性。然而, 对单个β细胞的葡萄糖反应的分析揭示了β细胞功能特性的多样性和异质性3的存在。为了评估单个β细胞的功能, 有可能监测导致胰岛素分泌的细胞内变化4。胰岛素分泌之前, 葡萄糖进入β细胞。进入β细胞的葡萄糖迅速代谢成 ATP。高胞内 atp 浓度降低 atp 敏感性钾离子通道的开放概率, 导致β细胞退极化。退极化打开电压敏感的钙离子通道, 增加胞内钙。反过来, 钙触发胞吐作用胰岛素, 这是释放在循环和降低葡萄糖水平通过促进葡萄糖利用率5,6,7。
对β细胞的功能进行了一些研究, 包括对膜电位8的监测、胰岛素囊泡胞吐作用9的直接可视化和胞内钙2 +的量化;作为葡萄糖反应10的代表涌入。其中, 胞内 Ca2 +的成像提供了在同一个胰岛11,12的多个单独的细胞上扩大分析的优势, 允许直接比较葡萄糖反应之间单个β细胞。胞内钙2 +浓度可通过钙敏感荧光染料13或基因编码钙指标 (GECIs)14进行监测。而钙指示染料缺乏细胞型特异性, GECIs 可以通过特定的促进剂在特定细胞类型中表达。此外, 新一代的 GECIs, 如 GCaMP6, 提供了更好的信噪比和更快的时间动态15。在这里, 我们描述了新一代 GECIs 的效用, 特别是 GCaMP6s, 在单细胞分辨率下对β细胞中的钙进行可视化。我们将这种方法应用到斑马鱼的主胰岛作为我们的选择模型。在胚胎发育过程中, β细胞在主胰岛起源于背部和腹侧胰芽16。主胰岛位于斑马鱼胰腺内的一个定型解剖位置, 使其易于辨认和分离。β细胞在主胰岛是必要的葡萄糖调节, 因为他们的基因消融导致高血糖17,18。此外, 这些β细胞在早期斑马鱼的发育过程中成为葡萄糖反应的19。该协议也可以应用于成像的第二个胰岛, 形成在后胚胎阶段。该协议允许图像β细胞前体, 在后期的发展和定义的葡萄糖浓度。

<table border="0" cellpadding="0" cellspacing="0" width="1034">
	<tbody>
		<tr height="79">
			<td height="79" style="height:79px;width:276px;">Transgenic Zebrafish line: Tg(ins:nls-Renilla-mKO2; cryaa:CFP); Tg(ins:GCaMP6s; cryaa:mCherry)</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:291px;">By request from authors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Stereo microscope</td>
			<td style="width:123px;">ZEISS</td>
			<td style="width:344px;">495015-0001-000</td>
			<td style="width:291px;">SteREO Discovery.V8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Fluorescence lamp</td>
			<td style="width:123px;">ZEISS</td>
			<td style="width:344px;">423013-9010-000</td>
			<td style="width:291px;">Illuminator HXP 120 V</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Red Filter Cube</td>
			<td style="width:123px;">ZEISS</td>
			<td style="width:344px;">000000-1114-462&#160;</td>
			<td style="width:291px;">Filter set 45 HQ TexasRed</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Confocal Microscope</td>
			<td style="width:123px;">ZEISS</td>
			<td style="width:344px;">LSM 780</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine fibrinogen</td>
			<td style="width:123px;">Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HBSS (Hanks&#39; Balanced Salt solution)</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">35 mm diameter glass-bottom dishes</td>
			<td>ThermoFisher</td>
			<td>150680</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">tricaine methane sulfonate</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;">E10521&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Fine Forceps</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:344px;">11445-12</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">FIJI, using ImageJ Version: 2.0.0-rc-43/1.50e</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:291px;">https://fiji.sc/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">R, version 3.2.4</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:291px;">https://www.r-project.org/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">RStudio</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:291px;">https://www.rstudio.com/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">plotcelltrace.R</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:291px;">A custom script provided with the manuscript</td>
		</tr>
	</tbody>
</table>

analysis of beta-cell function using single-cell resolution calcium imaging in zebrafish islets

胰β细胞通过分泌荷尔蒙胰岛素来响应血糖浓度的增加。β细胞功能障碍导致高血糖和严重的危及生命的后果。了解β细胞在生理条件下如何运作, 以及什么基因和环境因素可能导致其功能障碍, 可能会导致更好的治疗方案, 为糖尿病患者。当钙离子的涌入触发胰岛素释放时, 测试β细胞中钙含量的能力是β细胞功能的重要指标。在这里, 我们描述了一个协议, 以监测葡萄糖刺激钙流入斑马鱼β细胞使用 GCaMP6s, 基因编码的钙传感器。该方法允许监测细胞内钙动力学的单细胞分辨率在体内安装的胰岛。在不同的葡萄糖浓度下, 同一胰岛内β细胞的葡萄糖反应能力可以同时捕获, 这表明斑马鱼β细胞间存在功能异质性。此外, 该技术提供了高的时间和空间分辨率, 揭示了钙流入的振荡性质葡萄糖刺激。我们的方法打开了门, 使用斑马鱼作为一个模型, 以调查基因和环境因素对β细胞功能和功能障碍的贡献。

利用上述协议, 分析了45天后受精 (dpf) 斑马鱼对胰岛β细胞的葡萄糖反应。为此, 主要胰岛是从安乐死的动物解剖, 并安装在纤维蛋白原-凝血酶模具中的一个玻璃底部的菜。胰岛浸泡在含有5毫米葡萄糖的 HBSS 中。葡萄糖浓度以步进的方式增加到10毫米和20毫米。记录了β细胞对葡萄糖浓度增加的反应。最后, β细胞偏振光使用30毫米氯化钾 (图 1)。使用氯化钾的退极化诱导健康β细胞中的钙进入。
利用斐济和数据分析软件, 提取和规范化了单个β细胞的 GCaMP6s 荧光强度 (图 2)。从荧光强度的痕迹来看, 单个β细胞在葡萄糖刺激时显示 GCaMP6s 荧光的振荡, 停止对氯化钾的刺激。该技术提供细胞分辨率的β细胞的葡萄糖反应性和一个窗口, 他们的功能。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57851/57851fig1.jpg"> 图 1: 用 GCaMP6s 在斑马鱼β细胞中进行钙流入的前体活体成像.从Tg (ins: Renilla-mKO2) 的主胰岛;Tg (ins: GCaMP6s)斑马鱼 (45 dpf) 被安装在纤维蛋白原-凝血酶模和孵化与5毫米 (基) 葡萄糖。β细胞用红色核标记标记, 而 GCaMP6s 荧光则存在于绿色通道中。胰岛被刺激与葡萄糖舷梯包括连续孵化与10毫米和20毫米 d-葡萄糖, 并且偏振光通过增加30毫米氯化钾. 箭头标记单个β细胞, 其活动被分析。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57851/57851fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57851/57851fig2.jpg"> 图 2: 规范化的 GCaMP6s 荧光强度-对单个β细胞的跟踪.规范化的 GCaMP6s 荧光强度-在图 1中用箭头标记的β细胞的跟踪。X 轴表示时间 (以秒为单位)。在顶部, 酒吧描绘了葡萄糖和氯化钾在 HBSS 培养基中的浓度。Y 轴表示时间序列中的规范化荧光强度。为此, 基线强度 (F0) 被计算为平均强度在孵化期间的5毫米葡萄糖。这从整个时间序列数据中减去 (f f0)。强度超过基线按单元格显示的最大强度 (f f0)/(f最大f0) 进行规范化。规范化的跟踪显示β细胞对葡萄糖的振荡反应, 当细胞偏振光30毫米氯化钾时稳定.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57851/57851fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets at JoVE.com

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

β细胞功能是重要的血糖稳态, 这是评估在单细胞分辨率使用基因编码的记者, 钙流入。

斑马鱼胰岛单细胞分辨率钙成像对β细胞功能的分析

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

analysis-beta-cell-function-using-single-cell-resolution-calcium

Nanyang Technological University

Research

JoVE Journal

Biology

14.6K 次观看.  TU Dresden. β细胞功能是重要的血糖稳态, 这是评估在单细胞分辨率使用基因编码的记者, 钙流入。

视频: Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

使用的视动反应研究斑马鱼的视觉功能

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

科研

生物学

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

斑马鱼肾功能评价采用荧光测定间隙

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

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 ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...