1。准备脂类<ol><li>为了连结蛋白双层上层单张，我们使用的是镍 2 +螯合血脂，锚聚组氨酸标签的重组蛋白。某些类型的细胞可能会坚持非特异性Ni 2 +的涂层双层，但T细胞通常不这样做，使这种方法为研究T细胞活化的最佳。 </li><li>脂质双分子层的解决方案是由准备解决20％的镍2 +螯合脂质和磷脂1-2毫升氯仿-甲醇在玻璃管的适当数额的80 ％。 <ol><li>溶剂蒸发温暖（30-37 ° C）水浴下氮气流。这通常需要大约15分钟。 </li><li>删除任何剩余的溶剂，在高真空冷冻干燥机使用90分钟。 </li></ol></li><li> 2％N -辛基-葡萄糖苷的Tris缓冲液，它创建了一个解决方案洗涤剂/磷脂混合胶束的洗涤剂溶解在脂类。最后磷脂浓度应为0.4毫米。要形成脂质体，透析删除的清洁剂和形式脂质体对3的Tris -生理盐水变化的解决方案。我们平时工作与卷1毫升的顺序与光谱/ POR 6毫米直径的2＃管切断与一个约10 kDa的。我们仔细排除任何气泡s时夹紧油管。我们通常是无菌过滤器的这种解决方案和清洁的条件下做透析，使用70％乙醇消毒手套进行处理。 </li><li>这个解决方案应该是清澈的。它是存储在氩气，以防止氧化。如果溶液混浊，那么多壁囊泡已经产生，您将需要重新开始。 </li></ol> 2。确定多少ICAM - 1和MHC存入平面双层<ol><li>为了设立了实验，这是首先要确定多少ICAM - 1和MHC在一定浓度的可溶性蛋白沉积的Ni 2 +的螯合剂双层表面积。要做到这一点，存款双层直径5微米的玻璃珠，孵育条件下，蛋白质溶液的玻璃珠，流细胞成像，并宣读了一个流immunofluorometric检测结合蛋白密度。 </li><li> FITC标记的抗体与荧光素每分子已知的数字是用来检测表面结合的蛋白质。荧光素标准珠用于校准流量microfluorimeter。 </li><li>我们将创造条件近似那些抗原呈现细胞ICAM - 1和200molecules/μm20.2-20我的EK - MCC91 - 103复杂molecules/μm2。 </li></ol> 3。清洁形成平面双层玻璃盖玻片<ol><li>平面双层自发形成的脂质体融合玻璃，但只有当玻璃是正确的清洁。 </li><li>食人鱼工作时，我们穿全身防护服，包括酸围裙和重，耐酸笞刑。小心地从有机废物分开的浪费和妥善处置。 </li><li>要准备的酸食人鱼溶液，添加75毫升浓硫酸干燥的烧杯中，并精心添加25毫升30％过氧化氢。该解决方案，迅速加热到大于100 ° C。 </li><li>沉浸在15分钟的解决方案的干燥盖玻片。从溶液中取出，并在一分钟，每边纯化水冲洗。使用真空源纯净水排水干的盖玻片。允许的Piranha溶液冷却，并加入到食人鱼废物的容器，这是第松散通风柜标记，左。当所有的脂质体，盖玻片和蛋白质，有准备，免疫突触的形成。 </li></ol> 4。成型的双层<ol><li>要形成双层，我们的实验室使用Bioptechs FCSII商会，集成式加热系统的优点。 FCSII系统是在一个不锈钢底座，夹具一起与一个0.5毫米的垫片，用于加热的铟锡氧化物涂在一个表面的一个microaqueduct幻灯片，与对方的流体槽，的微流体歧管，无粉尘0.25毫米定义的流室和食人鱼高度的垫片，清洗40毫米的圆形盖玻片上形成的双层。 </li><li>虽然赖以形成的双层，盖玻片，需要高度的亲水性和清洁，microacqueduct幻灯片实际工作更好，如果它的疏水性。使得microaqueduct疏水性达到1％，在室温下60分钟的人血清白蛋白与治疗，然后用纯净水清洗，并完全干燥后，这个调节过程，以及使用之间。在此过程中留下的变性蛋白质的涂层允许1μL脂质体混悬液，形成一个圆形，hemispherica升下降大于0.25毫米高。 </li><li>组装流动细胞和形成平面双层，放置气歧管，管指示向上，后一个平坦的表面。然后，放置在0。 5毫米的垫片孔定位在流体管，确保它是坐在平坦。轻轻地放到垫片microaqueduct一面朝上的流体通道，并确保席位平而不申请任何下调的压力，直到验证，在幻灯片与微管的孔对齐。躺在一个长方形的削减microaqueduct幻灯片的顶部，使通道一字排开的流体通道和没有大的空气空间的无粉尘的0.25毫米的垫圈。 </li><li>放置到5 × 1μL脂质体混悬液滴上表面滑动的模式，这样每下降之间的中心到中心的距离是2.5毫米microaqueduct。 5脂质体下降，可能有不同的的成分，但在这种情况下，我们只会形成双层膜，无镍2 +螯合血脂和10％的镍2 +螯合血脂。 </li><li>一旦这些下降到位，小心地将盖玻片一项议案表面上。到基地钳尽快。脂质体下降，应与盖玻片接触。这种接触不应该被打破，因为双层有可能已经形成;任何中断可能会导致有缺陷的双层膜。 </li><li>重要的是要记住microacqueduct幻灯片，以帮助定位显微镜的盖玻片上的几个小墨点，以纪念双层位置。等待20分钟，以确保该系统已平衡。传输所有的解决方案，用20 ml注射器连接软管约20厘米和3路阀。由短管长度活塞的另外一个端口连接到流通池，以尽量减少之间的活塞和流动池的死体积。 HEPES缓冲液含有MgCl 2的 2毫米，1毫米氯化钙2，5毫米的葡萄糖和1％的人血清白蛋白的填充注射器。 </li><li>总理管和活塞，所以有没有气泡。连接流细胞，并推动通过一项议案5毫升，一边看，以确保无气泡在双层传递。现在，你有你自己的双层。 </li></ol>此视频文章支持“猎人收集和返回：免疫突触和Kinapses的变形虫运动的主题变奏曲<a href="http://arjournals.annualreviews.org/action/doSearch?action=runSearch&amp;type=advanced&amp;result=true&amp;prevSearch=%2Bauthorsfield%3A%28Dustin%2C+Michael+L.%29" target="_blank">“迈克尔L。</a>达斯汀<a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.24.110707.175226">，细胞和发育生物学的</a>年度审查，24卷， 2008。

supported planar bilayers for the formation of study of immunological synapses and kinapse

支持的平面双层功能强大的工具，可以用来模拟在免疫突触的分子间的相互作用。为了模仿淋巴细胞和抗原提呈细胞之间的相互作用，我们用Ni2 +的螯合血脂重组细胞粘附和MHC蛋白质锚定与聚组氨酸标签双层上层单张。准备血脂，治疗会形成的双层玻璃表面，然后形成了一个专门的包含流量将增加淋巴细胞的细胞室的双层平面双层生成。然后，双层与倪和他的标签的重组蛋白被控加入。最后，淋巴细胞注射到流通池和TIRF显微镜可用于图像的突触的形成和机制，控制T细胞的运动，受体分拣网站，和受体的退化网站。

Watch this Scientific Journal Video about Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse at JoVE.com

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

支持的平面双层功能强大的工具，可以用来模拟在免疫突触的分子间的相互作用。在这里，我们显示锚定细胞粘附蛋白来调节突触的形成脂质bilyer上单张和可视化突触的形成，使用TIRF显微镜的方法。

形成免疫突触和Kinapse研究的支持平面双层膜

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

supported-planar-bilayers-for-formation-study-immunological-synapses

Research

JoVE Journal

Biology

12.5K 次观看.  New York University - NYU. 支持的平面双层功能强大的工具，可以用来模拟在免疫突触的分子间的相互作用。在这里，我们显示锚定细胞粘附蛋白来调节突触的形成脂质bilyer上单张和可视化突触的形成，使用TIRF显微镜的方法。

视频: Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

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

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

调查的基本免疫机制，器官移植排斥反应

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

分离纯化果蝇</em磁性微球的排序>外周神经元

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

固体支持基于膜的电

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

直接成像诱导ER钙有针对性的酯酶染料装载（TED）

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

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 ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

肿瘤细胞球体的高通量图像分析：一个用户友好的软件应用来测量球体的大小自动准确

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...