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

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

<ol>
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作者宣布, 他们没有竞争的金融利益。

这份手稿描述了一种技术, 可以用来可视化胞胰岛素颗粒的β细胞在完整的胰岛的共聚焦显微镜。它使用 NPY-pHluorin 作为荧光记者克隆成腺病毒, 以确保高转染效率。
虽然该方法在我们手中是高效的, 但它可能需要一些修改, 主要取决于两个参数: 1) 胰岛的质量和 2) 的病毒库存的效价优化感染条件。在胰岛病毒感染之前, 允许胰岛准备在37° c 过夜时从隔离和运输压力中恢复。检查在光显微镜下的胰岛形态在整个感染/表达期间, 并检查是否有降低活力的迹象, 如细胞凸出从胰岛表面或存在缺氧细胞 (较暗的细胞) 在胰岛中心18.每种病毒的股票在效价和感染效率上可能有所不同。在显微镜检测之前, 一定要根据病毒的滴度和培养时间来调节病毒的体积。最关键的一步是优化感染条件 (例如, 病毒微粒的数量, 检测前的培养时间), 以便足够的记者表达和胰岛的完整性和响应性保持。在优化感染协议和每一个新的病毒批次, 收集几个小岛在不同的时间点, 并检查是否胞可以诱发氯化钾 (30 毫米) 和/或高糖 (16 毫米)。我们发现, 该检测工作更好, 胰岛细胞保持健康和葡萄糖反应时, 莫伊是低 (约 2) 和文化时间延长 (约5天), 以提高表达水平的融合结构。我们的结果与先前的研究一致, 表明基因产品的有效表达在腺病毒载体的低染浓度下可以更好地实现, 同时保留胰岛函数19。
然而, 这种技术有一些局限性。一是, 使用 NPY-pHluorin 作为记者, 颗粒不能被形象化之前, 他们与等离子体膜和融合毛孔打开。为了研究胰岛素颗粒的贩运而不是胞, 可以使用腺病毒编码 NPY-eGFP。另一个限制是, 在较低的程度, NPY-pHluorin 可以针对胰岛素含有颗粒。然而, 大多数的细胞, 被感染的 NPY-pHluorin 也表达胰岛素和少于10% 的细胞表达胰高血糖素或生长抑肽。事实上, 颗粒分泌物的葡萄糖依赖性, 其搏动模式和胰岛素共存, 表明大多数分泌事件代表了从胰岛素颗粒释放的货物。为了保证对胰岛素胞的监测, 应在刺激胰岛素分泌的条件下进行检测 (如, 高糖)。然而, 为了提高β细胞表达的特异性, NPY-pHluorin 可被置于更具选择性的启动子 (如大鼠胰岛素启动子) 的控制之下。该技术的另一个限制是, 它需要外源蛋白在胰岛素颗粒中的表达, 这可以扰乱他们的生物和/或行为20。在解释实验结果时应考虑到这一点, 如果可能的话, 应使用其他的胰岛素颗粒货物蛋白 (例如, 如, C 肽, 胰岛淀粉样多肽) 作为记者来验证结论。
本议定书还建议包括提高营剂 (IBMX 和佛司可林) 增强高糖对胰岛素颗粒胞的影响。这种刺激的协议模仿的激活生理相关的放大通路 (由 incretins 或分泌和神经信号分子触发), 增强胰岛素分泌通过增加阵营在胰腺β细胞。然而, 在缺乏 cAMP 培养剂的情况下, 高糖 (16 毫米) 也会诱发颗粒胞, 但观察到的分泌事件的数量却较低。颗粒仍然出现在离散爆发, 与平均细胞反应同步, 并显示类似的爆裂期: 1.4-6.6 分钟的高葡萄糖加 IBMX/佛司可林 vs. 1.5-10 分钟的高葡萄糖单独。
本文所描述的分析对于现有的方法有很大的意义, 因为它有足够的空间和时间分辨率来可视化完整的胰岛内的β细胞中的单个融合事件的实时性。举例来说, 它揭示了高同步胰岛素颗粒释放在人类胰岛后葡萄糖刺激。此外, 如果胰岛很好地附着在片上, 它们可以长时间地成像 (至少 15-20 分钟)。这是用来表明, 在人类胰岛胞发生在不同的爆发, 出现在类似的周期搏动性的在体内胰岛素分泌。该分析还允许测试几个刺激对胰岛素胞使用小鼠或人类胰岛的影响。此外, 因为它不需要像其他方法的胰岛细胞离解, 它可以用于研究β细胞的行为在一个小岛。事实上, 我们观察到相邻的β细胞形成了 5-10 细胞的簇群, 其中的活动是同步的。同一簇中的β细胞可能是由连接蛋白 3621的缝隙接合通道耦合而成。他们的同步反应说明了β细胞的连接性, 这对于正确的胰岛素分泌是必不可少的22。
在未来, 这项技术可以与其他荧光染料的功能成像, 以实时可视化的变化, 胞浆钙或膜电位, 例如, 与颗粒胞的β细胞。特别是, 由于新的 pH 敏感的红色荧光蛋白是可用的14,23 , 可以融合到 NPY 或其他胰岛素颗粒货物蛋白质, 绿色染料的功能成像可以使用。此外, 这项技术可以与鼠标模型相结合的活体成像的血管化胰岛移植到前室的鼠标眼睛24。胰岛可感染腺 48-72 小时前移植到眼睛。如果移植人类胰岛, 应使用免疫缺陷小鼠。利用该模型, 胰岛素颗粒分泌的亚细胞空间模式可与血管排列25相关。

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

<table width="845">
	<tbody>
		<tr height="63">
			<td height="63" style="width:280px;height:63px;">Upright laser-scanning confocal microscope</td>
			<td style="width:232px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:167px;">TCS-SP5</td>
			<td style="width:167px;">includes LAS AF, the image acquisition software</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Imaging chamber platform</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">PH-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">22x40 glass coverslips</td>
			<td style="width:232px;">Daiggerbrand</td>
			<td style="width:167px;">G15972H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum silicone grease</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Multichannel perfusion system</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">VC-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Single inline solution heater</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">SH-27B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Temperature controller</td>
			<td style="width:232px;">Warner instruments</td>
			<td style="width:167px;">TC-324C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Peristaltic Suction pump</td>
			<td style="width:232px;">Pharmacia</td>
			<td style="width:167px;">P-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:280px;height:42px;">35 mm Petri dish, non-tissue culture treated</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">10861-586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">CMRL Medium, no glutamine</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">11530037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">FBS, heat inactivated</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">16140071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">L-Glutamine 200mM</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">25030081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">5M NaCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">S5150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3M KCl solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">60135</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M CaCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">21115</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="width:280px;height:25px;">1M MgCl2 solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">M1028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Bovine Serum Albumin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">1M HEPES solution</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Vacuum filter</td>
			<td style="width:232px;">VWR</td>
			<td style="width:167px;">431098</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">D-Glucose</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Poly-D-lysine hydrobromide</td>
			<td style="width:232px;">Sigma-aldrich</td>
			<td style="width:167px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Di-8-ANNEP</td>
			<td style="width:232px;">ThermoFisher</td>
			<td style="width:167px;">D3167</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">3-isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">I5879</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:280px;height:21px;">Forskolin</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:167px;">F3917</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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

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

该技术的整个工作流显示在图 1中。简单地, 老鼠或人的小岛可以感染腺病毒编码 NPY pHluorin 和影像, 在几天的文化, 在共聚焦显微镜下。当颗粒与等离子体膜和开放的保险丝, 荧光的增加观察和可以量化 (图 1)。为了确定 NPY-pHluorin 是否确实是一个合适的工具, 以监测胰岛素颗粒动力学, 感染的胰岛 immunostained 与抗体抗 GFP 和不同胰岛激素胰岛素, 生长抑素或胰高血糖肽。大多数细胞表达 NPY-pHluorin (GFP 阳性) 是β细胞, 因为他们也表达胰岛素 (~ 90%;图 2A, 2B)。只有少数胰高血糖素阳性的α细胞或生长抑肽阳性的三角洲细胞是 GFP 标记的 (图 2B)。重要的是, 在受感染的细胞, NPY 融合 colocalized 与胰岛素 (皮尔逊的相关系数为0.61 ±0.04 的 gfp 和胰岛素 vs. 0.21 ±0.05 的 gfp 和胰高血糖素和0.07 ±0.01 的 gfp 和生长抑肽)。
我们证明, pHluorin 是一种有效的 ph 传感器, 增加细胞内 ph 值与50毫米 NH4Cl 导致 #62; 500% 增加细胞内荧光 (图 3A, 3B,视频 1)。由于颗粒内部的 pH 值随着等离子体膜的融合和融合孔的开启而增加, 含有 NPY-pHluorin 的颗粒在刺激胰岛素胞的条件下变得可见, 如氯化钾的膜除极化 (图4,视频 2)。
单个分泌事件的β细胞内完整的胰岛可以可视化的共聚焦延时成像 NPY-pHluorin 感染岛。这些发生的反应直接膜去极化与氯化钾或其他一些生理刺激 (图 4和图 7, 见下文)。在基础胞外葡萄糖浓度 (3 毫米), 很少分泌活动观察。然而, 与高糖 (16 mM) (图 5A,视频 3) 的刺激反应触发了与等离子膜的颗粒融合。通过放置不同大小和不同区域的 roi, 可以将分泌颗粒的反应动力学与近距离 (簇) (图 5B) 中的单个细胞或细胞组进行比较。这种类型的分析能够确定: 1) 个体分泌事件是非常同步和分泌在几秒钟内从平均细胞反应, 和 2) 的β细胞的密切接近形成功能集群的同步活动。
胰岛素在体内的分泌以搏动的方式发生。刺激 NPY-pHluorin 感染的小岛与高血糖浓度长期 (15-20 分钟) 引起了多个脉冲 (或爆发) 的分泌 (图 6,视频 4)。在每一个脉冲, 个别颗粒与等离子膜融合在一个同步的方式, 如前所示 (图 5)。每 3-4 分钟就能看到分泌活动的爆发。
在反应嘌呤激动剂 ATP (10 µM) 或毒蕈激动乙酰胆碱 (10 µM) (图 7) 中, 也可以观察到含颗粒的 NPY-pHluorin 中荧光的瞬态增加, 这两者都是已知的刺激人类胰岛的胰岛素分泌。如预期, 膜除极化与氯化钾 (30 mM) 也触发胰岛素颗粒胞 (图 5)。只要小岛在刺激之间被洗好, 几刺激可以适用于同一个胰岛准备。在重复实验时, 一定要改变应用程序的顺序。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56089/56089fig1.jpg"> 
图1。一个说明该检测方法的方案.对 pH 敏感的 GFP (pHluorin) 与 NPY 结合的腺病毒产生并用于感染小鼠或人胰岛。感染后四至六天, 胰岛被放置在一个片, 并在一个直立激光扫描共聚焦显微镜下成像。NPY-pHluorin 结构是用胰岛素颗粒在β细胞中表达的。它的荧光在成熟的胰岛素颗粒的酸性 ph 值上被淬火, 但在颗粒与细胞膜的融合和暴露于中性细胞外 ph 值7.4。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56089/56089fig2.jpg"> 
图2。在胰岛素颗粒中表达 NPY 融合结构.(A)完整的人类胰岛感染了 NPY-pHluorin, immunostained (绿色) 和胰岛素 (左面板, 红色), 生长抑素 (中, 红) 或胰高血糖肽 (右, 红色) 的共焦图像。细胞核以蓝色显示 (DAPI 标记)。缩放条形图 = 10 µm. (B)定量的 GFP 阳性细胞的分数也含有胰岛素 (Ins), 生长抑素 (Soma) 或胰高血糖肽 (谷氨酸)。显示的是平均±中小企业, n = 5 小岛。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56089/56089fig3.jpg"> 
图3。NPY-pHluorin 是一种有效的 pH 传感器.(A)在包含 3 mm 葡萄糖的胞外溶液中, 在含有 50 mm NH4Cl (下面板) 的情况下, 对感染了 NPY-pHluorin 的小鼠胰岛共聚焦图像的最大投影。缩放条形图 = 100 µm. (B)示踪在整个胰岛引起的平均荧光强度 (任意单位) 的变化, 由 nh4Cl. 虚线表示 nh4Cl 应用程序。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56089/56089fig4.jpg"> 
图4。含有 NPY pHluorin 的颗粒一旦与等离子体膜融合并打开, 就变得可见.人 isletsinfected 与 NPY-pHluorin 腺病毒 incubat用等离子膜染料 di-8-ANEPP (红色)。胰岛刺激与氯化钾 (30 毫米) 触发了荧光颗粒的突然和瞬态出现在细胞表面 (以绿色显示)。在 exocytotic 反应 (t = 0), 在 (t = 3-9 s) 和以后 (t = 十二年代) 之前, 人胰岛内的细胞共聚焦图像显示。对比度被调整以去除背景绿色荧光。缩放栏 = 20 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56089/56089fig5.jpg"> 
图5。高糖触发 NPY-pHluorin 融合与等离子体膜.(A)在基础葡萄糖浓度 (3G-3 mm 葡萄糖、左面板) 或高葡萄糖 (16G-16 mm 葡萄糖, 右面板) 中感染 NPY-pHluorin 的完整人胰岛共聚焦图像的最大投影。胞外溶液中包含了佛司可林和 IBMX 的营地饲养剂 (详见协议)。缩放栏 = 10 µm. (B)跟踪显示在 roi 绿色通道中的平均荧光强度 (任意单位) 的变化, 放置在单个分泌事件 (红色, 颗粒), 细胞 (绿色) 或细胞簇 (蓝色)。高糖用于5分钟后〜2.5 分钟, 在3G。荧光值被归入初始荧光值 (基线)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56089/56089fig6.jpg"> 
图6。分泌事件产生的离散脉冲分泌后, 高糖.在持续刺激16毫米葡萄糖的过程中, 在一个完整的人类胰岛周围的不同细胞内放置 roi 的平均荧光强度 (任意单位) 的变化。每种颜色代表单个单元格。爆发的分泌活动, 可以看到每 3-4 分钟.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56089/56089fig7.jpg"> 
图7。NPY-pHluorin 与细胞膜的融合是由已知的胰岛素分泌刺激引发的.在氯化钾 (30 mm)、ATP (10 µM)、高葡萄糖 (16G、16 mm) 和乙酰胆碱 (即10µM) 的完整人胰岛细胞内的单个分泌事件周围, roi 中的平均荧光强度 (任意单位) 的变化。氯化钾和 ATP 应用于基础葡萄糖浓度 (3 毫米)。黑线显示平均颗粒和灰色线反映 SEM 值。绿色跟踪显示相应的单元格响应。水平时间刻度 (2 分钟) 适用于所有图形。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/56089fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video 1" src="/files/ftp_upload/56089/56089video1.jpg"> 
视频1。NPY-pHluorin 腺病毒有效感染胰岛细胞和感觉 pH 变化。 小鼠胰岛被感染4天的腺病毒编码 NPY-pHluorin。感染的胰岛被放置在一个片上, 并安装在成像室。为了增加细胞内 pH, 胰岛被灌入50毫米 NH4Cl 添加到胞外溶液含有3毫米葡萄糖。在 NH4Cl 应用程序中可以看到快速而强烈的荧光增长。缩放条 = 100 µm. 电影速度 = 5 fps。电影的总持续时间是九十年代.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/Video_1_JoVe.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Video 2" src="/files/ftp_upload/56089/56089video2.jpg"> 
视频2。含有 NPY-pHluorin 的颗粒一旦与等离子体膜融合并打开, 就变得可见。 人胰岛感染 NPY-pHluorin 腺病毒, 孵化与等离子体膜染料 di-8-ANEPP。胰岛刺激与氯化钾 (30 毫米) 触发了突然和瞬态的外观荧光颗粒在细胞表面。给出了共焦平面的最大投影。对比度被调整以去除背景绿色荧光。缩放条 = 20 µm. 电影速度 = 5 fps。电影的总持续时间是六十年代.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/Video_2_JoVe.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Video 3" src="/files/ftp_upload/56089/56089video3.jpg"> 
视频3。高糖触发胞的颗粒含有 NPY-pHluorin。 在高糖 (16 mM) 刺激下, pHluorin 含有颗粒的融合反应触发。在基础胞外葡萄糖浓度 (3 毫米), 很少分泌活动观察。然而, 在高糖, 胰岛素颗粒瞬时出现在不同细胞的细胞膜上的同步方式。在 3G (16G 标签出现) 后2.5 分钟后应用高糖。给出了共焦平面的最大投影。缩放条 = 20 µm. 电影速度 = 10 fps。影片的总持续时间为7分钟.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/Video_3_JoVe.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<img alt="Video 4" src="/files/ftp_upload/56089/56089video4.jpg"> 
视频4。长时间的高糖刺激触发了几阵阵胞。 长时间的高糖刺激会产生多发的分泌胰岛, 其间颗粒瞬时出现。分泌活动的脉搏可以看到每〜 3-4 分钟的人类胰岛共焦平面显示。应用荧光强度的彩色方案。缩放条 = 20 µm. 电影速度 = 30 fps。影片的总持续时间为20分钟.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56089/Video_4_JoVe.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>

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

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

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

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

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

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

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

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

科研

生物学

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

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

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

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

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

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TIRF显微镜视网膜双极细胞的影像胞外分泌

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇

基于荧光测定活细胞中的钙进入商店经营:从培养的肿瘤细胞，骨骼肌纤维

单线粒体超氧闪烁在完整心脏或共聚焦成像

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

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

间隙连接通讯:天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

葡萄糖摄取测量和对胰岛素刺激的反应