我们想感谢温哥华的可视化分析（爆）研究所的成员，为项目提供意见和建议。特别是，我们要感谢约翰布赖恩费舍尔，莳萝，和大卫Darvill。我们还要感谢他们的支持和有益的讨论Kollmann实验室成员。这项工作是支持的，由国家过敏和传染病研究所，国立卫生格兰特N01 AI50023研究所的一部分;过敏原罗富国教育资助07 - A1A和07 - B2B的;和迈克尔史密斯健康研究基金会。 TRK是从宝来惠康基金广告事业在生物医学科学奖由加拿大卫生研究培训资助加拿大儿童健康临床医师科学家计划研究所与患病儿童基金，儿童与家庭研究所合作，在部分支持（不列颠哥伦比亚省），妇女和儿童健康研究所（艾伯塔），省和马尼托巴省儿童健康研究所。

1。探索为基础的分析，对抽穗<ol><li>你有一个数据集，你想探索在数据列之间的各种关系。 <ol><li> （图1）点击"连接到数据"导入数据集。选择数据源类型，并按照说明导入您的数据集。 </li><li> （图1）确保尺寸货架包含断然措施货架包含的列，而从您的数据集的数值列。有时一个主题标识符，如维列可能被列为衡量，如果它是numerated。右键单击它，改变它的尺寸，并选择"转换尺寸"。在图1中，从分类数据集，如刺激的浓度水平和主题标识符列是正确摆在货架尺寸，并正确措施货架上观察细胞因子的浓度。 </li><li> （图2）如果在分析过程中，需要计算的字段，只需右键单击在任一尺寸或措施货架，并选择"创建计算字段"。输入计算公式中结合函数或数学运算领域。图2显示了如何创建一个新的领域称为PFD功能，通过聚合PFD2，PFD3，和PFD4值&gt; 2。 </li><li> （图3）生成的2 - D在抽穗的情节，通常与维和利益的措施。由于界面提供了简单的拖动和拖放互动，维和度量的选择可以很容易地改变。行货架的尺寸通常是放置在列货架，并采取措施。在图3中，尺寸的货架包含刺激的浓度水平，措施货架包含观察到的细胞因子的浓度水平。 </li><li> （图3）为了区分由一个特定的层面，地方的可视化，文字，颜色或大小货架的尺寸标签，维度的性质而定。文本货架企图分化与所选维度提供的文本信息的标签，在可视化的数据。颜色和大小的货架会自动分辨颜色和大小的数据，在每个货架底部的滚动条可以调整。在图3中，放置在架子颜色的基因型，导致由不同颜色的三种基因型分离。 </li><li> （图3）过滤器在其他维度的具体列变量的可视化。这可以通过拖动到过滤器的架子尺寸标签。会出现一个窗口与列表中选择的值。选择的值将被留在可视化的唯一值。 </li><li> （图4），您可以与其他维度相结合可视化或措施，以产生一个矩阵的可视化。这可以通过拖放列货架多个层面，多种措施和行货架。图4包含两列的可视化矩阵列货架放置在两个不同的层面的例子。 </li><li>产生类似的可视化，右击当前工作表的左下角，并选择"重复表"。 </li><li>迭代分析的过程中，找出有趣的趋势，或离群，可以帮助您生成有关的数据的新假说。 </li></ol></li><li>您有多个来自同一源产生的数据集，并希望探讨这些数据集之间可能的连接。 <ol><li>放置在同一个来源，即两个电子表格放入同一工作簿中的多个数据集，允许抽穗通过相同的连接，这些数据集的连接。 </li><li> （图5）确保多个数据集相同的尺寸，选择连接的数据集，通过键值的逻辑加入。在图5中，有加入的五个核心价值:细胞类型，刺激的浓度水平，阶段或组，刺激，并受标识符。 </li><li>执行类似第1.1节的分析。 </li></ol></li></ol> 2。演示基于需求<ol><li>你知道数据之间的关系，但是你要能够快速生成一个列表在简报中使用的可视化。 <ol><li>按照协议第1.1节所述，相应的可视化和注释。 </li><li>使用导出功能，在抽穗产生的可视化图像。 </li></ol></li><li>您已经生成了一套强大的可视化形表的工作簿和要共享工作簿。 <ol><li>抽穗包装工作簿另存为工作簿和共享此文件。 </li><li>没有抽穗桌面的同事可以下载抽穗Reader打开包装的工作簿。抽穗Reader允许您的同事与您已创建的可视化交互。 </li></ol></li></ol> 3。代表性的成果<img src="/files/ftp_upload/2397/2397fig1.jpg" alt="图1" /> 截图图1。抽穗后从Excel文件demo.xls中导入一​​个名为NFKBIA电子表格。正确的尺寸和措施货架分类和数值数据，分别填入。 <img src="/files/ftp_upload/2397/2397fig2.jpg" alt="图2" /> 图2计算字段窗口调用创建一个特殊的计算字段使用在抽穗。左下角框的列表可以帮助确定可能的领域，并在右侧的列表中包含可以在公式中使用的功能的英文缩写。在这个例子中，我们要添加PFD4，PFD3和PFD2的值，以获得最终的价值，我们为PFD&gt; 2 <img src="/files/ftp_upload/2397/2397fig3.jpg" alt="图3" /> 图3。刺激的浓度水平与观察到的细胞因子浓度的可视化。可视化显示的刺激3M - 002的不同浓度水平对观察到的细胞因子TNF -α浓度的情节。线的颜色是指在NFKBIA在我们的先天免疫研究的个人基因的单核苷酸多态性的不同基因型。 <img src="/files/ftp_upload/2397/2397fig4.jpg" alt="图4" /> 图4。一个两列的可视化矩阵截图。我们生成一个两列的矩阵，以方便端侧两个刺激，3M - 003和LPS的反应比较。 x轴是两个不同的刺激的浓度水平，和y轴的地块计算字段的值，PFD&gt; 2。 <img src="/files/ftp_upload/2397/2397fig5.jpg" alt="图5" /> 图5。这些抽穗对话窗口，说明如何在不同的电子表格记录的数据连接。从不同的电子表格数据连接，可以通过这些使用逻辑加入键值条款相结合。 <table border="1"><tbody><tr><td></td><td colspan="10">可视化和分析工具</td></tr><tr><td>功能</td><td>抽穗</td><td>可见印花税</td><td> xmdvtool </td><td>与ggobi </td><td>星光</td><td> Gapminder </td><td> Visulab </td><td> InfoVis工具包</td><td> Geotime </td><td>启发</td></tr><tr><td>平行坐标图</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>无</td><td>是</td><td>是</td><td>无</td><td>无</td></tr><tr><td>散点图矩阵</td><td>是</td><td>无</td><td>是</td><td>是</td><td>是</td><td>无</td><td>是</td><td>无</td><td>无</td><td>无</td></tr><tr><td>尺寸减少</td><td>无</td><td>无</td><td>是</td><td>是</td><td>无</td><td>无</td><td>无</td><td>无</td><td>无</td><td>无</td></tr><tr><td>时间维度</td><td>是</td><td>是</td><td>无</td><td>是</td><td>是</td><td>是</td><td>无</td><td>是</td><td>是</td><td>无</td></tr><tr><td>地理空间尺寸</td><td>是</td><td>是</td><td>无</td><td>无</td><td>是</td><td>是</td><td>无</td><td>无</td><td>是</td><td>无</td></tr><tr><td>文本挖掘</td><td>无</td><td>无</td><td>无</td><td>无</td><td>是</td><td>无</td><td>无</td><td>无</td><td>无</td><td>是</td></tr><tr><td>直接操纵数据</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>无</td><td>是</td><td>无</td></tr><tr><td>过滤</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>无</td><td>是</td><td>是</td></tr><tr><td>可扩展到其它平台（如R） </td><td>是</td><td>无</td><td>是</td><td>是</td><td>无</td><td>是</td><td>无</td><td>是</td><td>无</td><td>无</td></tr><tr><td> CSV表格式</td><td>是</td><td>是</td><td>是</td><td>是</td><td>无</td><td>无</td><td>是</td><td>是</td><td>是</td><td>无</td></tr><tr><td> XML数据格式</td><td>是</td><td>无</td><td>无</td><td>是</td><td>是</td><td>无</td><td>是</td><td>是</td><td>是</td><td>无</td></tr><tr><td>可处理10000 +行</td><td>是</td><td>无</td><td>无</td><td>无</td><td>是</td><td>无</td><td>无</td><td>无</td><td>无</td><td>是</td></tr><tr><td>文档</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td><td>是</td></tr><tr><td>商用产品</td><td>是</td><td>无</td><td>无</td><td>无</td><td>是</td><td>无</td><td>无</td><td>无</td><td>是</td><td>是</td></tr></tbody></table> 表1列出的可视化分析工具和他们的一些特性。

<ol>
	<li>Thomas, J., Cook, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+the+Path:+The+Research+and+Development+Agenda+for+Visual+Analytics.">Illuminating the Path: The Research and Development Agenda for Visual Analytics.</a> , (2005).</li><li>Steed, C., Swan, J., Jankun-Kelly, T., Fitzpatrick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+Analysis+of+Hurricane+Trends+Using+Statistical+Processes+Integrated+with+Interactive+Parallel+Coordinates.">Guided Analysis of Hurricane Trends Using Statistical Processes Integrated with Interactive Parallel Coordinates.</a> IEEE Symposium on Visual Analytics Science and Technology. , 19-26 (2009).</li><li>Saraiya, P., North, C., Lam, V., Duca, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Insight-Based+Longitudinal+Study+of+Visual+Analytics.">An Insight-Based Longitudinal Study of Visual Analytics.</a> IEEE Transactions on Visualization and Computer Graphics. 12, 1511-1522 (2006).</li><li>Kollmann, T. R., Crabtree, J., Rein-Weston, A., Blimkie, D., Thommai, F., Wang, X. Y., Lavoie, P. M., Furlong, J., Fortuno, E. S., Hajjar, A. M., Hawkins, N. R., Self, S. G., Wilson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+innate+TLR-mediated+responses+are+distinct+from+those+of+adults.">Neonatal innate TLR-mediated responses are distinct from those of adults.</a> J. Immunol. 183, 7150-7160 (2009).</li></ol>

现代生物医学研究的高通量技术的问世，LED需要一个更有效的方法，分析研究数据的爆炸。可视化分析（VA）是通过交互式可视化界面（1​​）便利的分析推理的科学。在弗吉尼亚州的做法，恢复在分析人类的分析师手中的权力，违背传统的方法来检测电脑图案。可视化分析已在各个领域，如国防科研（1）和飓风的趋势（2），用于研究。到目前为止，有VA在生物学上的应用（3）只有几个例子。?...

facilitating the analysis of immunological data with visual analytic techniques

可视化分析（VA）已成为一种新的方式来分析大型数据集通过交互式可视化显示。我们证明了在弗吉尼亚州的生物数据集的分析方法的实用性和灵活性。在免疫学的这些数据集的例子包括流式细胞仪，Luminex公司的数据，和基因分型（如单核苷酸多态性）的数据。相反，传统的信息可视化方法，VA恢复在手中的分析师的分析权力，让分析师进行实时数据探索的过程。我们选择了在弗吉尼亚州的软件称为抽穗后评估了几个弗吉尼亚州的工具。在视频演示，使用的方法称为配对分析，两种类型的分析任务分析和数据集之间进行了论证。配对分析，在弗吉尼亚州的定义，是一个分析的方法，其中一个VA工具专家一个领域的专家在分析端侧。该领域的专家是谁理解数据的意义，并要求所收集的数据可能解决的问题之一。然后创建工具专家，以帮助寻找可能回答这些问题的数据模式的可视化。之间的假设生成和数据的快速可视化显示滞后时间短是一个VA方式的主要优势。

Watch this Scientific Journal Video about Facilitating the Analysis of Immunological Data with Visual Analytic Techniques at JoVE.com

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

可视化分析（VA）是一种新的交互方式分析数据的方法。在这段视频中，我们讨论的高通量生物实验所带来的数据超载的问题，并提出为解决这样的问题的弗吉尼亚州。视频演示和分析使用的是弗吉尼亚州的工具，称为抽穗的免疫学数据集之间的。

促进免疫学数据分析与视觉分析技术

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

facilitating-analysis-immunological-data-with-visual-analytic

Research

JoVE Journal

Immunology and Infection

10.0K 次观看.  University of British Columbia. 可视化分析（VA）是一种新的交互方式分析数据的方法。在这段视频中，我们讨论的高通量生物实验所带来的数据超载的问题，并提出为解决这样的问题的弗吉尼亚州。视频演示和分析使用的是弗吉尼亚州的工具，称为抽穗的免疫学数据集之间的。

视频: Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

科研

免疫与感染

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

ExCYT: A Graphical User Interface for Streamlining Analysis of High-Dimensional Cytometry Data

With the advent of flow cytometers capable of measuring an increasing number of parameters, scientists continue to develop larger panels to phenotypically explore characteristics of their cellular samples. However, these technological advancements yield high-dimensional data sets that have become increasingly difficult to analyze objectively within traditional manual-based gating programs. In order to better analyze and present data, scientists partner with bioinformaticians with expertise in analyzing high-dimensional data to parse their flow cytometry data. While these methods have been shown to be highly valuable in studying flow cytometry, they have yet to be incorporated in a straightforward and easy-to-use package for scientists who lack computational or programming expertise. To address this need, we have developed ExCYT, a MATLAB-based Graphical User Interface (GUI) that streamlines the analysis of high-dimensional flow cytometry data by implementing commonly employed analytical techniques for high-dimensional data including dimensionality reduction by t-SNE, a variety of automated and manual clustering methods, heatmaps, and novel high-dimensional flow plots. Additionally, ExCYT provides traditional gating options of select populations of interest for further t-SNE and clustering analysis as well as the ability to apply gates directly on t-SNE plots. The software provides the additional advantage of working with either compensated or uncompensated FCS files. In the event that post-acquisition compensation is required, the user can choose to provide the program a directory of single stains and an unstained sample. The program detects positive events in all channels and uses this select data to more objectively calculate the compensation matrix. In summary, ExCYT provides a comprehensive analysis pipeline to take flow cytometry data in the form of FCS files and allow any individual, regardless of computational training, to use the latest algorithmic approaches in understanding their data.

ExCYT is a MATLAB-based Graphical User Interface (GUI) that allows users to analyze their flow cytometry data via commonly employed analytical techniques for high-dimensional data including dimensionality reduction via t-SNE, a variety of automated and manual clustering methods, heatmaps, and novel high-dimensional flow plots.

超 cyt: 一种用于简化高维细胞学数据分析的图形用户界面

With the advent of flow cytometers capable of measuring an increasing number of parameters, scientists continue to develop larger panels to phenotypically ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

在小鼠小腿上应用一致的按摩样扰动，并监测由此产生的肌肉压力变化

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

原子力显微镜罐体功能化,具有单T细胞或单粒子免疫单细胞力光谱

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...