我们感谢中央研究院纳米科技项目和台湾科技部的资助 (101-2113 米-001-001-MY2; 103-2113 米-001-028-MY2)。

注意: 该协议描述了对转换辅助活化的一个详细的仪表修改, 这是一种生成笼式 PKA-UCNP 的合成过程, 它是由二氧化硅涂层的 UCNP 的透射电子显微镜 (TEM)和笼 PKA-UCNP 样品, 紫外线和近红外光解设置, 细胞制备, PKA UCNP 显微注射, 活化研究, 和 REF52 细胞的应力纤维染色. 
 1. 转换频谱测量的 Fluorimeter 设置 <ol> <li> 在荧光光谱仪的试管持有者旁边安装4X 物镜, 以便激光聚焦在试管的中间. </li>
	<li> 将可调谐的 0.5-至 8 W 980 nm 半导体激光二极管90和 #176; 与物镜匹配. 
	注意: 操作人员需要具备激光安全知识, 并佩戴近红外激光护目镜. </li>
	<li> 在物镜和激光器的光路相交处安装全反射镜。放置一个黑色阳极氧化金属板, 以阻止光谱的励磁窗口和保护内部部分. </li>
	<li> 将试管的二氧化硅涂层 UCNP 溶液 (0.2-0.6 毫克/毫升) 放在试管支架上, 使明亮的转换光束可以可视化, 并可用于调整镜像安装的最佳光对齐方式. </li>
	<li> 将光谱学转换为最小 PMT 电压设置的发光模式 (如果信号太弱则调整到更高的设置)。将镜像调整到最佳对准, 其中光束位于试管的中间, PMT 拾取最强的信号. </li>
	<li> 对齐后测量转换频谱, 并使用所需的电源设置。使用热堆功率计, 在激光电源上的电流读数上建立激光功率的校准曲线。不断检查激光性能, 确保性能在校准范围内. </li>
</ol> 2。显微镜设置 注意: 以下设置适用于配备双层光路的显微镜。如果用户打算在后端安装一个励磁光源开关, 使近红外激光器和卤化物灯共享同一端口, 则在后端端口的准直器将显著阻挡近红外光谱, 因为它是为减少样品加热而设计的。用户必须作出困难的选择: 保持准直器在和遭受非常低的转换效率, 或删除的准直和牺牲强度的励磁光的荧光. 
 注意: 显示修改后的光的光路方案的剖面图、转换发光和近红外解离模式的显微镜、荧光和 UV 光解模式, 以及实验设置用于此实验的说明如 图 2 所示. 
<ol> <li> 将荧光显微镜与金属卤化物灯导轨一起配置到后端. 
	注意: 在这个上层的光路中, 有一个立方体炮塔, 一个立方体的位置必须是空的, 允许从较低层光路径来的近红外光谱. </li>
	<li> 在右侧端口上安装可调谐的0.5 到 8.0 W 980 nm 激光二极管, 并打开侧面端口准直器. 
	注: 这将激光光斑放大到大约500和 #181; 当使用10X 物镜时, 直径为 m。除去这个准直器会产生微米范围的光点, 使近红外细胞照射变得不切实际。这种准直器是近红外透明的. </li>
	<li> 在下层光路中安装一个分色镜 (850 nm 短路) 和一个励磁滤镜 (950 nm long-pass). </li>
	<li> 使用 UCNP 的解决方案来可视化近红外光点。调整激光的位置, 使近红外光束在视野中居中, 以完成对准. </li>
</ol> 3。笼型 PKA UCNP 的制备与表征 <ol> <li> 孵化重组 PKA (9 和 #181; M) 与0.5 毫米 2-nitrobenzylbromide (NBB) 在锁定缓冲 (20 毫米三盐酸, 100 毫米氯化钠和10毫米氯化镁 2 ; 8.5-1 h 在2和 #176; c. 淬火过剩的 NBB 与10毫米 dthiothreitol (数码), 然后透析反对 4-(2 羟乙基) 哌嗪-1-磺酸 (HEPES) 缓冲 (10 毫米, pH 7.5) 去除多余的试剂和盐. 
	注: 在透析过程中, 锁定溶液的 ph 值从8.5 降低到 7.5, HEPES ph 值7.5 用于随后的固定化反应. </li>
	<li> 混合稀土盐-Y (CH 3 CO 2 ) 3 水合物 (99.9%, 0.4527 克, 1.38 摩尔), Yb (ch 3 CO 2 ) 3 水合物 (99.9%, 0.2533 g, 0.60 摩尔) 和 Tm (ch 3 co 2 ) 3 水合物 (99.9%, 0.0168 g, 0.02 摩尔)-在十八 (30 毫升) 和油酸 (12 毫升), 加热混合物到115和 #176; c 为30分钟, 冷却到50和 #176; c。然后, 将反应混合物溶解在甲醇溶液 (20 毫升) 中, 用0.2 克 (5.0 摩尔) 的氢氧化钠颗粒和0.2964 克 (8 摩尔) 的氟化铵超声为15分钟. 将反应温度提高到300和 #176; C 获取 UCNP 核心 (#946;-NaYF 4 : 1.0% Tm 3 + /30% Yb 3 + ) 纳米颗粒。
	<li> 溶解在环己烷 (10 毫升) CO-520 (0.5 毫升) 的核心纳米颗粒 (25 毫克)。添加氨水 (重量30% 伏/v, 0.08 毫升) 和 tetraethylorthosilicate (乙, 0.04 毫升), 并在室温下应用恒定搅拌12小时, 以获得二氧化硅涂层和 #946;-NaYF 4 : 1.0%Tm 3 + /30%Yb 3 + 核心纳米粒子. </li>
	<li> 孵育 PKA (6 nmol) 或笼 PKA (6 nmol) 与 UCNP (1 毫克) 15 在 HEPES 缓冲区 (10 毫米, pH 7.5) 在4和 #176; C 为 3 h 固定 PKA 在硅涂层的 UCNP 通过 静电相互作用. </li>
	<li> 使用 PVDF 过滤器过滤混合物 (0.45 和 #181; m), 离心机在 1.7万 x g 为10分钟在4和 #176; C, 和 redisperse HEPES (50 和 #181; L, 10 毫米, pH 7.5) 含有0.1% 蛋白稳定剂, 以获得纯化的 PKA-UCNP 复合物的颗粒。离心机在 1.7万 x g 为10分钟在4和 #176; C 和收集上清为布拉德福德化验在1.5 毫升管. </li>
	<li> 分析从步骤3.5 中保存的上清中的无界 PKA, 用布拉德福德法对 UCNP 粒子的 PKA 置换水平进行反计算, 后者通常具有 4.5 nmol 每毫克粒子的 PKA 的替代水平. 
	注意: 布拉德福德化验显示了一个强烈的蓝颜色的颗粒, 这意味着高水平和稳定的蛋白质固定不脱附, 而笼 PKA-UCNP 溶液的上清分数显示一种颜色类似 HEPES 缓冲区 ( 图 4 c-4e ). </li>
</ol> 4。特性 注意: 激酶测定用于量化丙酮酸激酶的特定活性。简言之, 与丙酮酸激酶和乳酸脱氢酶结合的肽的磷酸化, 导致 NADH 氧化。后者的形成是通过测量 NADH 在 340 nm 的吸光度下降来监测的. 
<ol> <li> 确定60和 #181 中的 PKA 和 PKA UCNP 的活动; 含 4-morpholinepropanesulfonic 酸 (拖把, 100 毫米, pH 7.5), 氯化钾 (100 毫米), 丙酮 (PEP, 1 毫米), 三磷酸腺苷 (ATP, 1 毫米), 和 #946 的测定混合物的总容积。烟酰胺腺嘌呤核苷酸, 减少的形式 (NADH, 0.8 毫米), 氯化镁 (氯化镁 2 , 1 毫米), kemptide (0.4 毫米) 和丙酮酸激酶/乳酸脱氢酶混合物 (PK/LDH 的30/40 单位). </li>
	<li> 测量 PKA 溶液 (2-#181; L) 加入测定混合物后的 NADH 吸收量的减少。计算直线的斜率, 以直接反映被测激酶的活动. </li>
	<li> 测量 PKA-UCNP 15 和整个活动的活动, 该行为应与本机 PKA 特定活动的乘法乘积以及计算的 PKA 表面涂层量匹配. </li>
</ol> 5。光解设置 <ol> <li> 多边环境协定使用热堆传感器和计算功率密度 (PD = 功率 (W)/面积 (cm 2 )) 的所有光源功率, 基于光点区域的 18 . </li>
	<li> 执行 体外 紫外线光解实验在一个商业系统与 365 nm LED (200 兆瓦/cm 2 ) 15 。
	<li> 对于显微镜阶段的 UV 活化, 用商业立方体过滤的激发光照亮 PKA UCNP 溶液 15 . 
	注: 功率密度为 ~ 15 兆瓦/cm 2 , 无物镜聚焦. </li>
	<li> For 体外 近红外活化在显微镜阶段, 用 980 nm 激光照射 PKA-UCNP 溶液, 在下层光路上安装有定制的转换滤镜/镜像设置, 分色镜 (850 nm 短路),励磁过滤器 (950 nm long-pass). 
	注: 在4X 物镜聚焦前, 输出功率密度为5瓦特/厘米 2 。在4X 物镜聚焦后, 功率密度估计为68瓦特/cm 2 . </li>
</ol> 6。PKA-UCNP 配合物的细胞样品制备及显微注射 <ol> <li> 通过0.45 和 #181 对样本进行预筛选; m PKA 固定后的过滤器 (步骤 3.5). </li>
	<li> 在注射期内孵育含有 L-15 培养基的10% 胎牛血清 (FBS) 中的细胞, 以便在恒温箱外进行稳定的 pH 控制. 
	注: 要确认微注射完成, co-inject 原 PKA-UCNP, 笼 PKA UCNP, 或乙二醇 UCNP (7.5 毫克/毫升) 15 具 5 (6)-四 (TAMRA) (10 和 #181; M) 到单元格中. </li>
	<li> 调整 PKA UCNP 颗粒的浓度, 使其在微注射后, 胞浆浓度为 1-3 和 #956; M PKA-一个细胞内 PKA 的生理浓度范围, 假设注射量是1/10 的细胞体积.</li>
	<li> 使用带有单轴液压微 ( 图 3 ) 的微量设备对单元 (步骤 6.2) 执行微注射. </li>
	<li> 使用数字压力表监测微量所施加的压力在操作范围内 (50-200 hPa), 并在定制加压室中密封分配器阀, 以提供微注射的补偿压力。使用拉拔器将针向后拉出. </li>
	<li> 保持50和 75 hpa 之间的注射压力, 注入时间为200-300 毫秒, 15 hpa 的补偿压力, 以防止培养基通过毛细管作用回流到针头中. </li>
	<li> 检查针头的尖端开口, 以确保它们的外径介于1.3 和1.5 和 #181 之间; m, 可以通过使用40x 物镜拍摄图像来确认. </li>
	<li> 用2毫升的 L-15 培养基在注射后用 10% FBS 冲洗细胞。观察 5 (6)-carboxytetramethyl-罗丹明 (TAMRA) 的细胞荧光成像, 以确认微注射完成. </li>
</ol> 7。在活细胞中使用紫外或近红外光的活化笼 PKA UCNP <ol> <li> 在 PKA-UCNP 微注射后, 在3.5 和 #181 上生长 REF52 细胞; m 盘, 将它们放在显微镜舞台上, 然后用 uv 光照射它们立方体为3分钟, 没有物镜聚焦. 
	注: REF52 细胞保持在 DMEM 10% FBS 37 和 #176; C 在 5% CO 2 孵化器中, 当70-100 每90% 天达到2-3 时, 细胞被继. </li>
	<li> 对于 PKA-UCNP 微注射后的近红外活化, 请按照步骤5.4 中的说明, 从较低层的滤镜/镜像设置中照射 980 nm 的激光器上的单元格. 
	注: 功率密度输出为5瓦特/厘米 2 前10X 物镜对焦和估计是300瓦特/厘米 2 后10x 物镜聚焦。转换是一个需要高光子密度的过程, 特别是当需要更大的反斯托克位移时。因此, 在活化中需要集中. </li>
	<li> 当需要更大的光点 (650 和 #181; m x 520 和 #181 m) 时, 用10x 物镜聚焦的近红外光谱照射这些细胞, 使准直器为 15 min. 允许在每5分钟照射后5分钟的暗间隔, 以避免发热. 
	注意: 当需要高空间分辨率 (亚细胞光点) 时, 使用40X 物镜, 并在导光指南的出口端安装针孔, 以生成亚细胞尺寸的光点 (5 和 #181; m x 4 和 #181; m)。在这种情况下, 1 s 的辐照是足够的近红外活化由于高通量的光子. </li>
	<li> 在 UV 或近红外光解之后, 允许单元格恢复在 5% CO 2 孵化器在37和 #176; C 为 1 h 在完全培养基产生细胞反应。随后, 将它们固定在1毫升的4% 甲醛/磷酸盐缓冲盐水 (PBS) 20 分钟前进一步染色. 
	警告: 甲醛是剧毒的;避免接触皮肤、眼睛或粘膜。在测量和准备过程中避免吸入粉末. </li>
</ol> 8。近红外活化引起的应力纤维崩解的可视化 <ol> <li> 在固定后, 使用 Alexa 594-笔 (添加5和 #181; l 6.6 和 #181; M 库存解决方案, 以200和 #181; 我的 PBS 染色; 在室温下孵育25分钟) 染色可视化应力纤维。使用滤镜集可视化 Alexa 594-笔 (前 581 nm/Em 609 nm) 的图像. </li>
	<li> 在室温下孵育4和 #39 中的细胞5分钟;, 6-diamidino-2-吲 (DAPI)。染色后, 用 PBS 清洗样品, 分别取应力纤维和细胞核的荧光图像. </li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-mediated+Construction+of+Hollow+Upconversion+Nanoparticles+for+Protein+Harvesting+and+Near-Infrared+Light+Triggered+Release.">DNA-mediated Construction of Hollow Upconversion Nanoparticles for Protein Harvesting and Near-Infrared Light Triggered Release.</a> Adv Mater. 26 (15), 2424-2430 (2014).</li><li>Gao, H. -. D., 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=Construction+of+a+Near-Infrared-Activatable+Enzyme+Platform+To+Remotely+Trigger+Intracellular+Signal+Transduction+Using+an+Upconversion+Nanoparticle.">Construction of a Near-Infrared-Activatable Enzyme Platform To Remotely Trigger Intracellular Signal Transduction Using an Upconversion Nanoparticle.</a> ACS Nano. 9 (7), 7041-7051 (2015).</li><li>Wehbi, V. L., Task&#233;n, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+for+cAMP-Mediated+Immunoregulation+in+T+cells+-+Role+of+Anchored+Protein+Kinase+A+Signaling+Units.">Molecular Mechanisms for cAMP-Mediated Immunoregulation in T cells - Role of Anchored Protein Kinase A Signaling Units.</a> Front Immunol. 7, (2016).</li><li>Pitchiaya, S., Heinicke, L. A., Custer, T. C., Walter, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Fluorescence+Approaches+Shed+Light+on+Intracellular+RNAs.">Single Molecule Fluorescence Approaches Shed Light on Intracellular RNAs.</a> Chem Rev. 114 (6), 3224-3265 (2014).</li><li>Gao, D., Tian, D., Zhang, X., Gao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quasi-one+dimensional+Propagationand+Tuning+of+Upconversion+Luminescence+Through+Waveguide+Effect.">Simultaneous Quasi-one dimensional Propagationand Tuning of Upconversion Luminescence Through Waveguide Effect.</a> Scientific Rep. 6, 22433-22442 (2016).</li><li>Roger, P. P., Rickaert, F., Huez, G., Authelet, M., Hofmann, F., Dumont, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+catalytic+subunit+of+cyclic+AMP-dependent+protein+kinases+triggers+acute+morphological+changes+in+thyroid+epithelial+cells.">Microinjection of catalytic subunit of cyclic AMP-dependent protein kinases triggers acute morphological changes in thyroid epithelial cells.</a> FEBS Lett. 232 (2), 409-413 (1988).</li><li>Curley, K., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+of+a+Signal+Transduction+Pathway+in+Living+Cells.">Photoactivation of a Signal Transduction Pathway in Living Cells.</a> J Am Chem Soc. 120 (33), 8573-8574 (1998).</li><li>Carling, C. J., Nourmohammadian, F., Boyer, J. C., Branda, N. 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作者声明他们没有竞争的金融利益。

此前, 霍夫曼和同事发现, 在自由 PKA19的微注射后, 在 REF52 细胞中观察到戏剧性的形态学改变。在另一项研究中, 劳伦斯小组证明, 笼 PKA 可以激活在体内, 导致形态学变化和应力纤维的解体时, 受到紫外线光解20。早先关于开发 upconverted 紫外光的报告活化显示了激活的几个 UCNP 协助, 笼, 小分子 bioeffectors21,22,</...

化学修饰的蛋白质, 可以光 (例如, PKA 笼的蛋白质) 已经发展成为一个新兴的领域, 在化工生物学性操作细胞间生化过程1,2 ,3。当激活这些笼中的蛋白质时, 利用光作为刺激提供了绝佳的时空分辨率。然而, 紫外线会导致不需要的形态学改变, 细胞凋亡和 DNA 损伤4,5。因此, photocaging 组的设计最近的发展重点是使 photocleavage 在较长波长或双光子激励下减少光, 以及增加深层穿透6,7。响应较长波长的锁定组允许我们选择合适的 uncaging 波长 (即、通道), 以便在两个或多个锁定组存在7时有选择性地激活 bioeffectors。鉴于这些有用的特征, 开发新的红光 photocaging 小组是非常重要的上游工作, 在光化学方法的生物研究范围内, 从探索的反应机制, 以控制细胞活动8。然而, 一个双光子锁定群通常过于疏水性由于融合芳香环结构, 和可见光锁定组通常是有机金属, 与芳香配体。当 bioeffector 是蛋白质或酵素时, 这种疏水/芳香物产不适合, 因为它使酵素或蛋白质的激活站点和导致作用损失, 即使共轭和光解仍然工作在化工水平上2 ,9。
UCNPs 是将近红外激发光转换为紫外线的有效传感器。这一独特而迷人的 UCNPs 的财产提供了现实的解决方案, 以应对与活化和触发控制释放小分子, 包括叶酸10, 顺铂衍生物11, DNA/siRNA12, 共聚物泡13, 空心粒子14。然而, 据我们所知, 目前为止, UCNP 辅助的酶或蛋白质活化还没有经过测试。因为没有成功的案例使用红光或近红外光谱来光酶, 我们被提示执行的近红外触发活化的蛋白质/酶结构组成的化学改性笼酶复合物与二氧化硅涂层,掺杂稀土的 UCNP15。在这项研究中, UCNP 被共轭的快速反应信号转导激酶的形式笼 PKA。PKA 控制糖原合成和骨架调节, 响应外部刺激通过循环腺苷磷酸 (阵营) 调节在胞16。本文研究了近红外辐照下细胞实验中酶激活的时间和空间方式的可行性。这种 UCNP 辅助的活化平台是一种新的方法, photoactivate 的酶使用近红外光谱和避免不受欢迎的信号转导反应由传统的紫外线照射2,4。
在细胞膜上转移大的 bioeffectors (例如,蛋白质) 很难控制细胞活动。虽然粒子固定化的蛋白质可能更容易通过吞转移到胞, 吞可能被破坏或退化通过内涵诱捕和随后的溶酶体退化2,4。即使笼状蛋白在膜移位后仍然功能正常, 移位量也不足以触发细胞响应2,17。与之形成鲜明对比的是, 微注射是一种直接和定量的方法, 可以将大的 bioeffectors 到细胞的细胞质中。此外, UCNP 固定化的 bioeffector 需要 upconverted 光来激活。因此, 光学仪器需要进一步的修改来测量、可视化和利用转换光。在这项工作中, 提供一个笼 PKA-UCNP 复合物的细胞使用显微注射和以下基本光谱学和显微镜修改的近红外活化将详细介绍。

<table border="0" cellpadding="0" cellspacing="0" width="985">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:431px;">Reagent</td>
			<td style="width:243px;"></td>
			<td style="width:119px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris(hydorxymethyl)aminomethane</td>
			<td>Sigma</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOPS</td>
			<td>Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>31434</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride</td>
			<td>Sigma</td>
			<td>12636</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Yttrium acetate&#160;hydrate</td>
			<td>Sigma</td>
			<td>326046</td>
			<td>Y(C2H3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thulium(III) acetate hydrate</td>
			<td>Alfa Aesar</td>
			<td>14582</td>
			<td>Tm(CH3CO2)3 &#183; xH2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ytterbium(III) acetate tetrahydrate</td>
			<td>Sigma</td>
			<td>326011</td>
			<td>Yb(C2H3O2)3 &#183; 4H2O</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Octadecene</td>
			<td>Sigma</td>
			<td>O806</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid</td>
			<td>Sigma</td>
			<td>364525</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol&#160;</td>
			<td>macron</td>
			<td>304168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Sigma</td>
			<td>30620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium fluoride</td>
			<td>J.T.Baker</td>
			<td>69804</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IGEPAL&#160;CO-520</td>
			<td>Sigma</td>
			<td>238643</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyclohexane</td>
			<td>J.T.Baker</td>
			<td>920601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amomonium hydroxide (28%-30%)</td>
			<td>J.T.Baker</td>
			<td>972101</td>
			<td>Ammonia</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetraethyl orthosilicate (TEOS)</td>
			<td>Sigma</td>
			<td>8658</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DL-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D0632</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-hydroxymaleimide (NHM)</td>
			<td>Sigma</td>
			<td>226351</td>
			<td>PKA activity blocking reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prionex protein stabilizer solution from hog collagen</td>
			<td>Sigma</td>
			<td>81662</td>
			<td>Protein stabilizer solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-nitrobenzyl bromide (NBB)</td>
			<td>Sigma</td>
			<td>107794</td>
			<td>PKA caging reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-(4-Chlorophenylthio)adenosine 3&#8242;,5&#8242;-cyclic monophosphate sodium salt</td>
			<td>Sigma</td>
			<td>C3912</td>
			<td>8-CPT-cAMP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyruvate Kinase/Lactic Dehydrogenase enzymes from rabbit muscle</td>
			<td>Sigma</td>
			<td>P0294</td>
			<td>PK/LDH</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine 5&#39;-triphosphate disodium</td>
			<td>Sigma</td>
			<td>A2387</td>
			<td>ATP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#946;-NADH reduced from dipotassium</td>
			<td>Sigma</td>
			<td>N4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphoenolpyruvate</td>
			<td>Sigma</td>
			<td>P7127</td>
			<td>PEP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coomassie Protein Assay Reagent, 950 ml</td>
			<td>Thermo Scientific</td>
			<td>23200</td>
			<td>Bradford assay reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">cAMP-dependent protein kinase</td>
			<td>Promega</td>
			<td>V5161</td>
			<td>PKA activity control</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pET15b-PKACAT plasmid</td>
			<td>Addgene</td>
			<td>#14921</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pKaede-MC1 plasmid</td>
			<td>CoralHue</td>
			<td>AM-V0012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline (PBS), pH 7.4</td>
			<td>Thermo Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose, pyruvate</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Leibovitz L-15 Medium</td>
			<td>Biological Industries</td>
			<td>01-115-1A</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>ACROS</td>
			<td>416785000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Nucleus staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa 594-phalloidin</td>
			<td>Invitrogen</td>
			<td>A12381</td>
			<td>F-actin staining dye</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5(6)-Carboxyfluorescein</td>
			<td>Novabiochem</td>
			<td>8.51082.0005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5(6)-Carboxytetramethylrhodamine&#160;</td>
			<td>Novabiochem</td>
			<td>8.51030.9999</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce Coomassie (Bradford) Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CelluSep T4 Tubings/Nominal filter rating MWCO 12000-14000 Da</td>
			<td>Membrane Filtration Products, Inc.</td>
			<td>1430-33</td>
			<td>Dialysis membrane</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millex-HV Syringe Filter Unit, 0.45 &#181;m, PVDF, 13 mm, gamma sterilized</td>
			<td>EMD Milipore</td>
			<td>SLHVX13NL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dynamic Light Scattering/Zetapotential Zetasizer nano-ZS</td>
			<td>Malvern</td>
			<td>M104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transmission Electron Microscope</td>
			<td>JEOL</td>
			<td>JEM-1400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Spectrophotometer</td>
			<td>Agilent Technologies</td>
			<td>10075200</td>
			<td>Cary Eclipse&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV-Vis Spectrophotometer</td>
			<td>Agilent Technologies</td>
			<td>10068900</td>
			<td>Cary 50&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Microscopy</td>
			<td>Olympus</td>
			<td>IX-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">950 nm longpass filter&#160;</td>
			<td>Thorlabs</td>
			<td>FEL0950</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">850 nm dichroic mirror shortpass</td>
			<td>Chroma</td>
			<td>NC265609</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RT3 color CCD system</td>
			<td>SPOT</td>
			<td>RT2520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Illumination</td>
			<td>PRIOR</td>
			<td>Lumen 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">980nm Infra-red diode laser</td>
			<td>CNI</td>
			<td>MDL-N-980-8W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV LED Spot Light Source</td>
			<td>UVATA</td>
			<td>UVATA-UPS412</td>
			<td>With a UPH-056-365 nm LED at 200 mW/cm2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal pile sensor</td>
			<td>OPHIR</td>
			<td>12A-V1-ROHS</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td>Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PC-10 Needle puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MANOMETER Digital pressure gauge</td>
			<td>Lutron</td>
			<td>PM-9100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">One-axis Oil Hydraulic Micromanipulator</td>
			<td>Narishige</td>
			<td>MMO-220A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heraeus Fresco 17 Centrifuge, Refrigerated</td>
			<td>Thermo Scientific</td>
			<td>75002421</td>
			<td></td>
		</tr>
	</tbody>
</table>

an integrated system to remotely trigger intracellular signal transduction by upconversion nanoparticle-mediated kinase photoactivation

转换纳米粒子 (UCNP) 介导的活化是一种新的方法远程控制 bioeffectors 与更少的光和更深的组织渗透。然而, 现有的仪器在市场上是不容易兼容的转换应用。因此, 修改商业上可用的仪器是这项研究的关键。本文首先阐述了传统的 fluorimeter 和荧光显微镜的改进, 使其与光子转换实验相兼容。然后, 我们描述了一个近红外线 (近红外) 触发笼蛋白激酶催化亚基 (PKA) 固定在 UCNP 复合物的合成。还报告了显微注射和近红外活化程序的参数。在笼 PKA-UCNP 微量成 REF52 成纤维细胞后, 近红外辐射明显优于传统的紫外线照射, 有效地触发了活细胞中的 PKA 信号传导通路。同时, 正、负控制实验证实, PKA 诱导的应力纤维分解通路是由近红外辐射引起的。因此, 使用蛋白质修饰的 UCNP 提供了一种创新的方法, 远程控制 light-modulated 细胞实验, 其中直接暴露在紫外线必须避免。

在图 1中说明了笼式酶 UCNP 结构的设计。PKA 酶第一反应与 2-硝基苄溴生成一个非活性笼 PKA, 然后静电固定在 UCNP 表面。UCNPs 发出 upconverted 光, 因此 photolytically 在胱氨酸199和胱氨酸343上切割邻硝基苄基基团, 生成激活的 PKA。TEM 图像和布拉德福德化验证实, PKA 和笼 PKA 被固定在 UCNPs 表面, 并在紫外 UCNP 检测后的笼 PKA 活化溶液的激酶活性 (<strong class="xfig"...

Watch this Scientific Journal Video about An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation at JoVE.com

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

在这个协议中, 笼状蛋白激酶 a (PKA), 细胞信号转导 bioeffector, 被固定在纳米粒子表面, 微量进入胞, 并激活由 upconverted 紫外线光近红外线 (近红外) 照射, 诱导下游应力纤维解体的胞。

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

an-integrated-system-to-remotely-trigger-intracellular-signal

Research

JoVE Journal

Bioengineering

7.4K 次观看.  Academia Sinica. 在这个协议中, 笼状蛋白激酶 a (PKA), 细胞信号转导 bioeffector, 被固定在纳米粒子表面, 微量进入胞, 并激活由 upconverted 紫外线光近红外线 (近红外) 照射, 诱导下游应力纤维解体的胞。

视频: An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

在胎儿肺细胞的机械传导的实验系统研究

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

o 型血管哺乳动物细胞产生均匀集料的轨道晃动培养

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Generating a Fractal Microstructure of Laminin-111 to Signal to Cells

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.

We describe three methods to generate Ln1 polymers with fractal properties that signal to cells differently compared to unpolymerized Ln1.

生成层粘连蛋白-111的分形微观结构以向细胞发出信号

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.