研究以预防失明，主任朱尔斯·斯坦眼科研究所，加州大学洛杉矶分校Bartly J. Mondino MD和拉米罗·拉米雷斯 - 索利斯，杰奎白，和珍妮Estabel的，威康信托基金会桑格研究所基因组校园。本研究符合ARVO声明眼科和视觉研究中使用动物。

1。在不固定的标本钝性分离：老鼠的眼睛剜除术<ol><li>拉开眼睑改善及接触后的地球（眼球）表面。 </li><li>将弯曲敷料镊子背后（下）的地球轨道（眼圈）。马哈詹Sharptip敷料镊子尖这一步更容易（见材料和​​试剂表）是一个自定义的仪器。 </li><li>关闭的镊子和把握的轨道结缔组织和视神经背后的全球，同时小心避免挤压全球。 </li><li>轻轻向上拉镊子和勇敢的眼球从眼眶。白线样组织是视神经。 </li><li>将在PBS中的眼睛。使用30号针头，使一个单一的穿刺卷绕紧接后方插入针1-2毫米到眼球的角膜缘。这允许固定剂，如戊二醛，否则无法穿透眼组织，直接连接进入眼睛。创作本穿刺伤口可能不是必要的，如果一种固定剂，如多聚甲醛或酒精，能够扩散到眼组织的利益。 </li><li>将整个眼睛马上进入固色剂浸泡固定。 </li></ol> 2。锐性剥离：老鼠的眼睛剜除以下灌注固定<ol><li>使用弯曲的科利柏镊子，保持眼睑远离地球。 </li><li>东方弯曲的Westcott解剖剪刀平行于地球，而针对朝后面的轨道。 </li><li>切固定的结缔组织，从下，内，优越，和侧面围绕着地球。 </li><li>将弯曲的镊子落后于世界各地，掌握结缔组织，而不按在地球上，并向前拉，enucleate的眼睛。由于灌注固定的轨道结缔组织僵硬，眼眶组织可能需要额外的切割，完全释放的globE。 </li></ol> 3。固定和包装运输必须遵循固定的组织机构和邮政服务的需求，包括适当的标签和包装。下面的协议是出货非传染性生物样品在室温下的一个例子。这需要3层包装，但没有A / B类生物物质或液态氮的标签。 <ol><li>到5毫升的玻璃闪烁瓶中至少3 mL的固色剂，固色剂缓冲，如果将眼睛固定短。发表在盖子上的小瓶中的跟踪号码和密封用Parafilm。 </li><li>将闪烁瓶到生物样品经认可的装运容器。在我们的研究所，例如，规定的容器内，需要三个层：将样品置于一个密封的容器，其中，吸收的中间层，和一个外保护层。 </li><li>成泡沫包装到一个应用程序包，然后将容器忙得不亦乐乎实验室海运集装箱以及适当的文件。 </li></ol> 4。代表性的成果组织学检查的眼睛可以进行苏木精和曙红染色，酶的表达检测，透射电子显微镜，和免疫组织化学的方法，包括由多种。浸泡在4％多聚甲醛固定后，组织包埋在石蜡切片切片机上。在高通量的表型，我们分析学生视神经部分样品，所有组织类型的眼睛（图1）。组织切片也审查lacZ的表达（图2），透射电子显微镜观察细胞器（图3），并与抗体（图4）的特定的分子的表达。 在我们的表型筛选的方法，这不是重要的是保持眼睛的方向与鼻（内眦）和时间（侧位片升眦）的两侧。有至少两个选项，如果眼睛的方向是必要的。后摘除术，我们已经申请了光烧灼角膜的时间在三点钟位置，用手持烧灼设备。病变在组织学上是明显的，像组织的油墨在加工过程中不会丢失。缺点是，烧灼损害角膜，这可能是一个重要的组织检查。其他选项需要一个熟练的的解剖学家，可以识别眼外肌插入。在立体显微镜下，识别的劣质和优越的斜肌插入，马克劣势和优越的方面的全球3使用任一方向方法，重要的是作为右或左眼跟踪试样。总之，这将使全球的定位准确之前，切片。 <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="图1" /> 图1。小鼠眼组织学图像杨宗亮<html> R远程采集。鼠标的眼睛去核锐性剥离后，用4％多聚甲醛灌注固定。A.学生与苏木精和曙红染色显示视神经节保存的所有组织子结构。 ON，视神经。 （绿色比例尺= 500微米）B.较高的放大视图的正常视网膜显示其层状结构：RGC，视网膜神经节细胞，IPL，内丛状层; INL，内核层; OPL，外丛状层（ONL），外核层（感光细胞）（箭头）; IS，感光体的内段（箭头的头）; OS中，光感受器外段; RPE，视网膜色素上皮细胞，C，脉络膜。 （绿色比例尺= 50微米）C.与正常视网膜相比，这个标本是薄由于光感受器变性。 ONL，IS，OS是完全不存在的（箭头）。 （绿色比例尺= 50微米） “ALT =”图2“/&gt; 图2。lacZ的表达，在转基因小鼠中，眼睛摘除钝性分离和浸渍在1毫克/毫升X-gal的溶液（5mM的的K3Fe（CN）6，5mM的亚铁氰化钾（CN）6，2 mM MgCl2的，0.02％NP40 ，和0.1％脱氧胆酸钠）中过夜，在37℃下眼睛被固定在30分钟，然后切片截面2％formaldehyde/0.2％戊二醛。 X-gal的产品（蓝色）标签：A.纤毛上皮细胞，角膜上皮B. C.在视网膜视网膜神经节细胞（RGC）层。 INL，外核层（ONL），外核层。 <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="图3" /> 图3。透射电子显微镜的视网膜色素上皮细胞（RPE）。钝性分离后创造的穿刺伤口，眼睛浸泡在0.1M磷酸钠缓冲液在2.5％的多聚甲醛/ 2.5％戊二醛固定。眼睛是次生在1％锇textroxide的darily固定，逐渐脱水，和嵌入在Spurr树脂。组织在90处被切开，并放置到铜槽网格覆盖福尔瓦和成像使用透射电子显微镜。显微照片显示，光感受器外节的视网膜神经，视网膜色素上皮细胞内的黑色素体（M），和Bruch膜。 <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="图4" /> 图4。超氧化物歧化酶（SOD）的免疫组化标记（绿色）鼠标玻璃体摘除眼球钝性分离，并接受了4％多聚甲醛浸泡固定。他们被嵌入在石蜡切片用切片机。组织切片孵育，与多克隆SOD3抗血清1:50稀释。 SOD3表达检测使用的山羊抗兔IgG（H + L）的Alexa Fluor 488的共轭次级抗体。在GRE标签SOD3恩。

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

没有利益冲突的声明。

大多数转基因小鼠中存在的不检查眼睛的实验室。我们的视频技术，说明了一个简单的，标准化的方法，用于远程手术技巧转移到优化组织收购，从实验室经验少用眼。此视频技术可以帮助克服在高通量的表型，这是由于非标准化组织收集和固定方法，防止有意义的比较表型和分子生物学研究的专业网站的使用数量有限的一个主要缺陷。任何新的远程实验室和技术人员，建立和保持质量控制，重?...

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>弧形修整镊子</td><td> STORZ眼药水</td><td> E1408 </td><td></td></tr><tr><td>马哈詹Sharptip敷料镊子</td><td> STORZ眼药水</td><td> E1406（SP7-64520 REF） </td><td></td></tr><tr><td>弯曲韦斯科特剪刀</td><td> STORZ眼药水</td><td> E3321 WH </td><td></td></tr><tr><td> 15°BD海狸显微叶片</td><td>贝克顿 - 迪金森</td><td> 374881 </td><td></td></tr><tr><td> 0.22精细Castroviejo的缝合钳</td><td> STORZ眼药水</td><td> E1805 </td><td></td></tr><tr><td> 0.12科利柏钳</td><td> STORZ眼药水</td><td>一百三十二分之二</td><td></td></tr><tr><td> 30号针头</td><td>贝克顿 - 迪金森</td><td> 305128 </td><td></td></tr><tr><td>生化危机梅勒</td><td>费舍尔</td><td> 03-523-4 </td><td></td></tr><tr><td>封口膜</td><td>费舍尔</td><td> 13-374-10 </td><td></td></tr><tr><td>玻璃闪烁瓶</td><td>惠顿</td><td> 4500413033 </td><td></td></tr><tr><td> PBS，pH值7.4 </td><td> Invitrogen公司</td><td> 70011-044 </td><td></td></tr><tr><td> 16％的多聚甲醛</td><td>电子显微镜科学</td><td> 15700 </td><td></td></tr><tr><td> 2.5％的多聚甲醛/ 2.5％戊二醛的0.1M磷酸钠缓冲液中</td><td>电子显微镜科学</td><td> 15700和16300 </td><td>混合实验室</td></tr><tr><td> 50％的戊二醛</td><td>电子显微镜科学</td><td> 16300 </td><td></td></tr><tr><td> 0.25％福尔瓦， </td><td>电子显微镜科学</td><td> 15810 </td><td></td></tr><tr><td>警察每插槽电网</td><td>电子显微镜科学</td><td> M2010-CR </td><td></td></tr><tr><td> 4％的四氧化锇</td><td>电子显微镜科学</td><td> 19140 </td><td></td></tr><tr><td>反SOD3抗体</td><td> Abcam公司</td><td> Ab21974 </td><td></td></tr><tr><td>山羊抗兔的Alexa Fluor 488 </td><td> Invitrogen公司</td><td> A11070 </td><td></td></tr><tr><td>斯珀尔的嵌入树脂</td><td>电子显微镜科学</td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

老鼠的眼睛是一个重要的遗传模型的平移人类眼科疾病的研究。在转基因小鼠在人类的致盲性疾病，如黄斑变性，光感受器变性，白内障，青光眼，视网膜母细胞瘤，糖尿病性视网膜病变已概括。1-5大多数转基因和基因敲除小鼠的实验室研究已产生非眼科疾病，但器官系统之间的遗传保护，许多相同的基因也可能在眼发育和疾病发生中发挥作用。因此，这些老鼠的眼睛发现新的基因型与表型的相关性是一个重要的资源。由于这些老鼠分散在世界各地，它是难以获取，维护和​​表型以高效，成本效益的方式。因此，大多数高通量眼表型屏幕被限制在检查现场，眼科专业知识，需要几个地点的眼睛在活体小鼠6-9本实验室所开发的另一种方法是远程组织收购，可用于转基因小鼠的眼睛在或大或小规模的调查方法。基于视频的手术技术转移，组织固定和运输的标准化程序允许任何实验室收集整个眼睛突变的动物，并将它们发送的分子和形态学表型。在这个视频文章中，我们提出了远程表型分析技术enucleate和转让不固定，并灌注固定鼠标的眼睛。

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

的解剖技术说明组织固定，进行表型的高通量筛选老鼠的眼睛剜除术。

老鼠的眼睛剜除术的远程高通量的表型

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.1K 次观看.  University of Iowa. 的解剖技术说明组织固定，进行表型的高通量筛选老鼠的眼睛剜除术。

视频: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

一种高通量自动化的平台，制造蛋白质治疗的细胞系发展

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

科研

医学

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

在自觉的，奔放的小鼠正常血糖高胰岛素 - 夹钳

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

遥控磁导航导管定位准确，实时和心脏电生理消融程序

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

高通量顺序酶联免疫吸附试验的急性移植物抗宿主病的生物标志物的验证

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

对专为递送治疗因素的神经保护策略成人干细胞高通量表征

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

良性前列腺增生的外科治疗: 钬激光前列腺摘除术 (计画)。

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

穿刺诱导的虹膜新生血管虹膜虹膜小鼠模型的建立

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.