GFP -β-肌动蛋白的质粒慷慨提供韦恩奥尔博士，LSUHSC - S病理科，并在实验室贝基Worthylake博士，药理学教研室LSUHSC - NO扩增。这项工作是支持的国立卫生研究院（P20 RR - 018766）和美国心脏协会（05835386N）的赠款。

<ol>
	<li>Spindler, V., Schlegel, N., Waschke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+GTPases+in+control+of+microvascular+permeability.">Role of GTPases in control of microvascular permeability.</a> Cardiovasc. Res. 87, 243-253 (2010).</li><li>Wojciak-Stothard, B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases+and+the+regulation+of+endothelial+permeability.">Rho GTPases and the regulation of endothelial permeability.</a> Vascul. Pharmacol. 39, 187-199 (2002).</li><li>Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+pathways+in+enhanced+microvascular+permeability.">Signal transduction pathways in enhanced microvascular permeability.</a> Microcirculation. 7, 395-403 (2000).</li><li>Birukov, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+GTPases+in+mechanosensitive+regulation+of+endothelial+barrier.">Small GTPases in mechanosensitive regulation of endothelial barrier.</a> Microvasc. Res. 77, 46-52 (2009).</li><li>Duran, W. N., Sanchez, F. A., Breslin, J. W., Tuma, R. F., Duran, W. N., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcirculatory+Exchange+Function.">Microcirculatory Exchange Function.</a> Handbook of Physiology: Microcirculation. , 81-124 (2008).</li><li>Hu, Y. L., Chien, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+motion+of+paxillin+on+actin+filaments+in+living+endothelial+cells.">Dynamic motion of paxillin on actin filaments in living endothelial cells.</a> Biochem. Biophys. Res. Commun. 357, 871-876 (2007).</li><li>Borm, B., Requardt, R. P., Herzog, V., Kirfel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+ruffles+in+cell+migration:+indicators+of+inefficient+lamellipodia+adhesion+and+compartments+of+actin+filament+reorganization.">Membrane ruffles in cell migration: indicators of inefficient lamellipodia adhesion and compartments of actin filament reorganization.</a> Exp. Cell. Res. 302, 83-95 (2005).</li><li>Hotulainen, P., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fibers+are+generated+by+two+distinct+actin+assembly+mechanisms+in+motile+cells.">Stress fibers are generated by two distinct actin assembly mechanisms in motile cells.</a> J. Cell. Biol. 173, 383-394 (2006).</li><li>Pellegrin, S., Mellor, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+stress+fibres.">Actin stress fibres.</a> J. Cell. Sci. 120, 3491-3499 (2007).</li><li>Ballestrem, C., Wehrle-Haller, B., Imhof, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+in+living+mammalian+cells.">Actin dynamics in living mammalian cells.</a> J. Cell. Sci. 111, 1649-1658 (1998).</li><li>Helmke, B. P., Rosen, A. B., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+mechanical+strain+of+an+endogenous+cytoskeletal+network+in+living+endothelial+cells.">Mapping mechanical strain of an endogenous cytoskeletal network in living endothelial cells.</a> Biophys. J. 84, 2691-2699 (2003).</li><li>Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G., Verin, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Rho+GTPases+in+thrombin-induced+lung+vascular+endothelial+cells+barrier+dysfunction.">Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction.</a> Microvasc. Res. 67, 64-77 (2004).</li><li>van Nieuw Amerongen, G. P., van Delft, S., Vermeer, M. A., Collard, J. G., van Hinsbergh, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+RhoA+by+thrombin+in+endothelial+hyperpermeability:+role+of+Rho+kinase+and+protein+tyrosine+kinases.">Activation of RhoA by thrombin in endothelial hyperpermeability: role of Rho kinase and protein tyrosine kinases.</a> Circ. Res. 87, 335-340 (2000).</li><li>Moy, A. B., Blackwell, K., Kamath, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+histamine+and+thrombin+on+endothelial+barrier+function+through+actin-myosin+tension.">Differential effects of histamine and thrombin on endothelial barrier function through actin-myosin tension.</a> Am J Physiol Heart Circ Physiol. 282, H21-H29 (2002).</li><li>Wittman, T., Littlefield, R., Waterman-Storer, C., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+speckle+microscopy+of+cytoskeletal+dynamics+in+living+cells.">Fluorescent speckle microscopy of cytoskeletal dynamics in living cells.</a> Live Cell Imaging: A Laboratory Manual. , 184-204 (2005).</li><li>Rabut, G., Ellenberg, J., Goldman, R. D., Spector, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobleaching+techniques+to+study+mobility+and+molecular+dynamics+of+proteins+in+live+cells:+FRAP,+iFRAP,+and+FLIP.">Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP.</a> Live Cell Imaging: A Laboratory Manual. , 101-126 (2005).</li></ol>

没有利益冲突的声明。

GFP -肌动蛋白在活的内皮细胞成像使炎症刺激的反应的肌动蛋白细胞骨架的动态进行详细的分析。这种方法也可能是有益的借鉴以往的研究结果显示在细胞骨架重塑反应的物质力量，如剪应力 11 。此外，此方法允许的贡献的内皮细胞的活动，包括迁移，有丝分裂，形成间的路口和交界成熟，和屏障功能的维护的肌动蛋白细胞骨架动力学的详细评估。 在显示的数据，内皮细胞肌动蛋白细胞骨架的行为，可以观察治疗前后凝血酶。一直内皮细胞边缘的地方lamellipodia，观察nonconfluent和融合细胞单层形成，并随着时间的推移倒退。与凝血酶治疗短暂地打断lamellipodia形式ATION和营业额。凝血酶也引起细胞略有合同，与以前的报告中，凝血酶导致肌动蛋白应力纤维在12-14内皮细胞的形成和增加向心力紧张发展一致。然而，像这样的活细胞成像研究，现在能确定的应力纤维的起源。在HUVEC，大部分的应力纤维起源于细胞外围，类似横弧纤维细胞迁移8，9。这种方法比使用固定细胞的另一个优势是在单个细胞应力纤维的数量可以量化前后凝血酶治疗，消除选择实验组之间的偏见。 有了这个协议，我们评估在细胞边缘和肌动蛋白应力纤维的动态运动。要了解漂白后的内皮细胞肌动蛋白单体的动态，更先进的技术，如荧光恢复（FRAP）或荧光斑点显微镜复制（密克罗尼西亚）可用于15，16。此外，由于微血管内皮细胞可能代表了一种更好的模型微血管屏障功能，优化转染协议，有效地表达GFP -肌动蛋白在微血管内皮细胞代表一个逻辑的未来发展方向。 总之，生活的内皮细胞表达GFP -肌动蛋白的成像代表一个强大的工具，以确定如何内皮细胞肌动蛋白细胞骨架响应各类刺激。使用紧密融合的内皮单层的研究将有助于确定的动态结构，如肌动蛋白丰富lamellipodia和内皮屏障功能的横弧应力纤维的角色。此外，内皮细胞表达GFP -肌动蛋白或其他允许其他亚细胞结构的可视化的融合蛋白的活细胞成像将提供详细的时空信息需要了解的信令和结构机制的Determine屏障的完整性。

<table border="1"><tbody><tr><td colspan="4"> 1。林格5倍股 </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>氯化钠</td><td>电解二氧化锰</td><td> SX0420 - 3 </td><td> 35摹</td></tr><tr><td>氯化钾</td><td> JT贝克</td><td> 3040 </td><td> 1.75摹</td></tr><tr><td>氯化钙</td><td>西格玛</td><td> C - 3881 </td><td> 1.47摹</td></tr><tr><td>硫酸镁</td><td>西格玛</td><td> M - 9397 </td><td> 1.44摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 2。 MOPS缓冲 </td></tr><tr><td> 化学 </td><td>公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td> MOPS </td><td>西格玛</td><td> M3183 </td><td> 125.6摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 3。白蛋白的生理盐溶液（APSS） </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>林格股票（5X） </td><td> N / A </td><td> N / A </td><td> 200毫升</td></tr><tr><td> MOPS缓冲</td><td> N / A </td><td> N / A </td><td> 5毫升</td></tr><tr><td>磷酸钠</td><td>西格玛</td><td> S - 9638 </td><td> 0.168摹</td></tr><tr><td>丙酮酸钠&lt;/ TD&gt; <td>西格玛</td><td> P5280 </td><td> 0.22摹</td></tr><tr><td> EDTA钠盐</td><td>西格玛</td><td> ED2SS </td><td> 0.0074摹</td></tr><tr><td>血糖</td><td>西格玛</td><td> G7528 </td><td> 0.901摹</td></tr><tr><td>白蛋白，牛</td><td> USB </td><td> 10856 </td><td> 10克</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">调整pH至7.4，在37 °，然后放入高压灭菌瓶和储存在4℃的无菌过滤° C。 </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 4。 0.9％的生理盐水 </td></tr><tr><td> 化学 </td><td> 公司 </td><td> 产品编号 </td><td> 金额 </td></tr><tr><td>氯化钠</td><td>电解二氧化锰</td><td> SX0420 - 3 </td><td> 9摹</td></tr><tr><td>无菌过滤水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">进入蒸压瓶和商店的无菌过滤器，在4 ° C </td></tr><tr><td colspan="4"></td></tr><tr><td colspan="4"> 5。 1.5％的明胶溶液 </td></tr><tr><td>明胶来自猪皮肤</td><td>西格玛</td><td> G2500 </td><td> 15摹</td></tr><tr><td> 0.9％的生理盐水</td><td> N / A </td><td> N / A </td><td>置于1升</td></tr><tr><td colspan="4">温暖的解决方案，以37 ° C至充分溶解的明胶。虽然仍然温暖，无菌过滤器进入蒸压瓶和存储在4 ° C </td></tr></tbody></table>

study of the actin cytoskeleton in live endothelial cells expressing gfp-actin

微血管内皮细胞中起着重要的作用，作为一种选择性渗透的阻隔液体和溶质。内皮细胞之间的粘附路口调节内皮细胞的通透性，许多研究已表明肌动蛋白细胞骨架，以确定交界的完整性 1-5的重要贡献。皮层肌动蛋白带被认为是维护稳定路口1，2，4，5的重要。相反，肌动蛋白应力纤维被认为是内皮细胞内产生的向心力紧张，削弱路口 2-5 。这一理论已被内皮细胞治疗与已知增加内皮细胞通透性的炎症介质，然后修复细胞和标签显微镜观察F - actin的研究基础上。然而，这些研究提供了肌动蛋白细胞骨架的作用非常有限的理解，因为固定细胞的图像只提供快照没有时间约肌动蛋白的结构5动态的信息。 活细胞成像允许的决定内皮屏障的完整性机制的研究肌动蛋白细胞骨架的动态性质纳入。这种方法的主要优点是各种炎症刺激内皮细胞中的肌动蛋白结构的影响，可在同一组的活细胞前和治疗后评估，消除潜在的偏见，观察固定标本时可能出现的的。的GFP -β-肌动蛋白的质粒转染人脐静脉内皮细胞（HUVEC）的盖玻片上生长汇合。汇合的人脐静脉内皮细胞肌动蛋白GFP的定时拍摄的图像前后此外，引起炎症介质的内皮屏障的完整性时间依赖性变化。这些研究使流体的肌动蛋白细胞骨架的变化的序列视觉观察到内皮贡献升屏障的破坏和恢复。 我们的研究结果一致显示，本地​​，肌动蛋白丰富lamellipodia内皮细胞的形成和营业额。也可以观察到肌动蛋白应力纤维的形成和运动。一个当地lamellipodia的形成和成交量的频率分析前和治疗后炎症刺激，可记录kymograph分析。这些研究提供了可以用于发现以前未知的分子机制的内皮屏障的完整性，维护重要内皮细胞的肌动蛋白细胞骨架的动态性质的重要信息。

Watch this Scientific Journal Video about Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin at JoVE.com

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

显微成像可以表达GFP -肌动蛋白的内皮细胞骨架结构的动态变化特征。这种方法与使用固定标本的技术，在之前的相同细胞的肌动蛋白细胞骨架的时空变化提供了详细的评估，期间和之后，各种生理，药理，或炎症刺激。

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

study-actin-cytoskeleton-live-endothelial-cells-expressing-gfp

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

Unit 1

The Cytoskeleton I: Actin and Microfilaments

SCIENTISTS IN ACTION

27.1K 次观看.  Louisiana State University Health Sciences Center. 显微成像可以表达GFP -肌动蛋白的内皮细胞骨架结构的动态变化特征。这种方法与使用固定标本的技术，在之前的相同细胞的肌动蛋白细胞骨架的时空变化提供了详细的评估，期间和之后，各种生理，药理，或炎症刺激。

视频: Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

F - actin的动力学通过荧光活细胞成像斑点显微镜（密克罗尼西亚）

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) 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 ...