LPE是苏格兰高级临床研究员和承认的首席科学家，的办公室（SCD/03）的支持。这项工作是由威康信托基金会资助计划LPE（089930）。 NARG是由威康信托计划资助（080088）和设备补助金（075470）（DeltaVision的），并通过一个FP7-2007-2013格兰特（HEALTH-F2-2010-260338-ALLFUN）。我们想感谢阿伯丁大学的成像设备，尤其是凯文·麦肯齐，有益的支持和建议。

1。C.白色念珠菌的生长和条件<ol><li>准备SC-URA琼脂平板上，通过加入6.9克无氨基酸的酵母氮碱，1毫升1 M NaOH溶液，10毫升1％（重量/体积）腺嘌呤hemisulphate盐和20克技术琼脂（先前11中详细描述）。化妆用蒸馏水，H 2 O的体积至900毫升高压灭菌器，允许琼脂冷却，但还不够巩固，然后在无菌条件下，添加50毫升无菌的40％D-葡萄糖和50毫升无菌4％SC-URA辍学。混合，倒入培养皿中，并留下琼脂平板上，冷却和固化。商店琼脂平皿上，在5℃下直至使用。 </li><li>数C。白色念珠菌血清型的菌株CAI4 + CIp10从甘油保存于-80°C到SC-URA琼脂平板上。 </li><li>孵育板在30°C至菌落形态和存储在5°C。 </li><li>文化单一C. SC-URA在5毫升培养基（配方在1.1，但没有技术的琼脂）和白色念珠菌殖民地孵育过夜30℃，200 rpm到生成固定相C.白色念珠菌 。 </li></ol> 2 C.白色念珠菌染色异硫氰酸荧光素（FITC） <ol><li>加入10微升C.白色念珠菌 990微升PBS（pH 7.4）中，并过夜培养，使用血球计进行细胞计数。 </li><li>为了帮助可视化C.吞噬作用测定期间的白色念珠菌 ，染色剂1×10 8 C。白色念珠菌在0.05M碳酸盐-碳酸氢盐缓冲液（pH9.6）在室温下在黑暗中10分钟，用1毫克/毫升FITC。 </li><li>要除去未结合的FITC，洗C.白色念珠菌在1毫升1×PBS中，离心机在3000×g下5分钟，去除上清液，沉淀重悬在1ml 1×PBS。重复3次。 </li><li>重悬浮沉淀，在1×10 6细胞/μl，以1×PBS。 </li></ol> 3。 J774.1小鼠巨噬细胞系的制备<ol><li>保持在75厘米2组织J774.1巨噬细胞在DMEM培养基的培养瓶中补充有10％（V / V）胎牛血清（FCS），200 U / ml青霉素/链霉素和2mM的L-谷氨酰胺，37℃，5％CO 2的 。原发性巨噬细胞的制备12，13中详细描述的其他地方。 </li><li>刮J774.1细胞从组织培养烧瓶中，并转移到一个50毫升Falcon管中。在600×g下离心5分钟，得到的细胞沉淀。 </li><li>去除上清，重悬沉淀在10ml预先温热的补充的DMEM培养基中。计数细胞，用血细胞计数板1×10 6 J774.1巨噬细胞在2ml补充的DMEM培养基，在35毫米的玻璃为基础的成像菜。孵育过夜，在37℃，5％CO 2。 </li><li>成像前，取代补充的DMEM培养基中，用2毫升预加热补充CO 2独立的介质（有10％（V / V）胎牛血清（FCS），200 U / ml青霉素/链霉素和2mM的L-谷氨酰胺） 1的μMLysoTracker红DND-99。</li></ol> 4。活细胞视频显微细胞吞噬功能测定<ol><li>显微镜的选择将取决于什么是本地可用，但在显微镜设置将需要包括一个倒置的阶段，环境室，加热至37℃，并激发/发射过滤器的所选择的污渍（FITC和TRITC）。 </li><li>打开显微镜上的加热器在实验前，留出足够的时间对环境的控制腔升温到37°C。室温度稳定所需的时间会有所不同，不同的显微镜设置。 </li><li>显微镜和电脑，打开和加载的图像处理软件。安装盘上的显微镜载物台的成像和调整的重点，找到J774.1巨噬细胞。优化TRITC和DIC图像的外观，通过调整透射光的百分比和曝光时间。 </li><li>取出成像菜和添加3×10 6 FITC染色C.白色念珠菌为t他的菜。记录的时间，C.白色念珠菌的菜。菜的阶段，并优化如有必要，FITC图像的外观。如果需要的话，设置了积分榜。 </li><li>开始成像时，所有的点都聚焦和渠道进行了优化。捕获FITC，TRITC和DIC的图像，每分钟6小时。 </li></ol>

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作者有没有透露。 NG是无限制的资金支持基础研究吉利德科学公司（Gilead Sciences）的收件人。 LE担任顾问，葛兰素史克公司在早期药物开发工作。

这里使用视频显微镜活细胞的方法研究巨噬细胞的吞噬能力。视频显微镜提供了多种附加层的信息进行分析。的一个基本优点是，摄取的数据在整个6小时观察期间的任何时间点，可以产生（从一个单一的实验）。更重要的是，所描述的方法使差分分析的各个阶段的吞噬作用。我们已经表明，例如，C 的整体摄取变动白色念珠菌的糖基化和巨噬细胞细胞系和原发性巨噬细胞的形态发生的突变体可以是一个后果，在巨噬细胞迁移的变化建立了细胞-细胞接触一次向靶细胞或吞噬率7。 有一些陷阱，以避免当进行这些实验。首先，这是非常重要的，以确保稳定的环境条件下，在整个实验的多项式dure。这是最好的实现中的环境的腔室，它被设置在开始实验之前，几个小时的实验条件。视频质量高度依赖于复杂的模块，使用红外激光器自动保持的样品的机械或热的变化，从而消除了手动对焦校正需要z位置，而不管。 鉴于扩展性的实验中，重要的是要限制激光曝光和相关联的光漂白及经光的影响，这可以最小化通过采用高度的荧光标记物，有利于低曝光时间。 FITC染色这里所描述的协议，快速和可靠的。作为FITC染色是一个非常光明的，稳定的，低的曝光时间是必需的，这使得FITC延长时间推移电影的理想选择。然而，我们经常使用各种不同的目标污渍，包括Calcofluor白和PKH染料标记的有机体。 在这项研究中，在1分钟的时间间隔超过6小时的吞噬功能测定图像被抓获。图像之间的时间间隔，可以调整根据调查过程下。例如，时间间隔进行查处时，可以降低快速过程，如小泡运输。显微镜和摄像头规格的最小时间间隔将被限​​制。有一些注意事项时，要牢记调节定时。首先，减少快照之间的时间间隔将意味着可以少点成像和文件的大小将大大增加。相反，增加的图像间隔太多会使的电影失去连续性。 这种方法可以应用原则上学习其他病原体和吸收的宿主细胞死亡。例如，我们最近的研究表明骨髓来源的巨噬细胞从sialoadhesin的基因敲除小鼠表现出极大地减少唾液酸化空肠弯曲菌的结合和吞噬。但是，目标的大小是一个重要的决定因素的可行性，减少靶细胞的大小，图像分析变得越来越困难。先进的图像分析软件是必不可少的快速视频显微镜分析，这应该加上适当的生物信息学的支持，方便的生成算法研究单个细胞和整个细胞群的迁移。活细胞视频显微镜在复杂的图像分析软件的迁移和单个细胞-巨噬细胞白念珠菌相互作用分钟按分钟分析相结合，提供了独特的复杂性C.白色念珠菌的巨噬细胞的吞噬作用ES。有巨大的潜力，以扩大这些方法来研究其他病原体和吞噬细胞（树突状细胞，嗜中性粒细胞），并更详细地或更多的生理，如上皮细胞和内皮细胞层的表面上的图像细胞 - 细胞相互作用的3D视频显微镜。这种技术的下一代工具在宿主 - 病原体相互作用的研究，将有助于生成详细的空间和时间信息，广泛的动态过程。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>无氨基酸的酵母氮碱</td><td> Formedium </td><td> CYN0402 </td></tr><tr><td>氢氧化钠</td><td>西格玛</td><td> S5881-500G </td></tr><tr><td>腺嘌呤hemisulphate盐</td><td>西格玛</td><td> A3159-25G </td></tr><tr><td>琼脂技术（第3号） </td><td> Oxoid公司</td><td> LP0013 </td></tr><tr><td> D-葡萄糖</td><td>西格玛</td><td> G7021-1KG </td></tr><tr><td> SC-URA辍学</td><td> Formedium </td><td> DSCK102 </td></tr><tr><td> FITC </td><td>西格玛</td><td> F7250-1G </td></tr><tr><td>纯碱</td><td> BDH </td><td> 301215M </td></tr><tr><td>碳酸氢钠</td><td> BDH </td><td> 102475W </td></tr><tr><td> PBS </TD&gt; <td> Gibco公司</td><td> 18912-014 </td></tr><tr><td> DMEM </td><td>龙沙</td><td> BE12-614F </td></tr><tr><td> FCS </td><td> Life Technologies公司</td><td> 10106169 </td></tr><tr><td>青霉素/链霉素</td><td> PAA </td><td> P11-010 </td></tr><tr><td> L-谷氨酰胺</td><td>龙沙</td><td> BE17-605E </td></tr><tr><td> DND-99 LysoTracker红， </td><td> Invitrogen公司</td><td> L7528 </td></tr><tr><td> 35毫米的玻璃菜</td><td> PAA </td><td> PAA12305160X </td></tr></tbody></table>

live-cell video microscopy of fungal pathogen phagocytosis

吞噬清除病原真菌和微生物，更一般地，可以认为包括四个不同的阶段：（ⅰ）吞噬细胞迁移的病原体是所在的站点;（ii）确认病原体相关的分子模式（PAMPS）通过模式识别受体（PRRS）;吞没的（ⅲ）的吞噬细胞的细胞膜结合的微生物，及（iv）处理成熟的吞噬体和消化摄入的粒子的吞噬细胞内。研究，评估其全部吞噬1，2，3，4，5的信息，但被限制的，因为它们通常不会打破过程分解成迁移，吞噬和吞噬体的成熟，可能受到影响的差异。此外，这些研究评估摄取作为一个单一的事件，而不是作为一个连续的动态过程。最近，我们已经开发了先进的活细胞成像技术，并结合这两个基因的功能分析病原体和宿主细胞创造一个跨学科平台的先天免疫细胞的功能和致病机理进行了分析。这些研究揭示了小说方面的吞噬作用，只能观察系统的时域分析人类巨噬细胞和真菌病原体和更普遍的传染性微生物的分子和细胞之间的相互作用。例如，我们已经开始定义如下：（a）该细胞表面的组件所需的每个阶段的过程中识别，吞噬和杀真菌细胞1，6，7，8（b）如何表面几何形状影响巨噬细胞吞噬和杀灭酵母和菌丝细胞的效率;及（c）如何吞噬导致的巨噬细胞，细胞周期和行为的改变。 相反，单一的时间点快照，活细胞的视频显微镜使作为共同研究各种各样的宿主细胞和病原体ntinuous序列在漫长的时间段，提供广泛的动态过程，包括细胞迁移，复制和小泡运输的时间和空间信息。在这里，我们详细描述如何准备主机和真菌细胞，并进行视频显微镜实验。这些方法可以提供未来的研究与其他吞噬细胞和微生物的用户指南。

在这里，我们显示出有代表性的结果为C.白色念珠菌的吸收在6小时的活细胞视频显微镜实验的小鼠巨噬细胞株J774.1。 图1是一个具有代表性的活细胞的视频显微镜电影，示出C 的吞噬念珠菌的小鼠J774.1巨噬细胞。视频显微镜使活细胞内的C.白色念珠菌是可视化而不需要染色外部念珠菌。然而，在这些实验中，C。白色念珠菌是染色使用的pH值敏感的染料FITC（绿色），骤冷过程中的巨噬细胞吞噬体酸化，并促进念珠菌已内在化（ 图1）的确认。为了进一步援助可视化的摄入C.白色念珠菌，巨噬细胞染色使用的红色荧光染料LysoTracker红色DND-99，这污渍酸性舱室和作为吞噬体成熟的非特异性的标志物。图2是从活细胞的视频显微镜实验的静止图像，并示出了变化的数目C 的白色念珠菌个人J774.1巨噬细胞摄入。在这个实验中，82％J774.1巨噬细胞吞噬的至少一个C。白色念珠菌细胞在6小时的吞噬作用测定结束。在图3中的数量的内化C.白色念珠菌细胞每巨噬细胞的报道。的平均数C.白色念珠菌每巨噬细胞为3.4，但有趣的巨噬细胞可以摄取16真菌细胞， 图4是一个静止图像的序列，从有代表性的活细胞的视频显微镜实验示出了巨噬细胞吞噬C.白色念珠菌细胞与巨噬细胞和巨噬细胞最终裂解，菌丝生长， 图4示出视频显微镜使吞没的靶细胞后的事件的分析， 即数据可为き产生lling的巨噬细胞C.白色念珠菌菌丝在有关摄入的靶细胞的数量和形态发生的，以及如何巨噬细胞杀涉及可以实时研究吞噬体成熟。 图1。活细胞的吞噬作用C.视频显微镜电影念珠菌的小鼠J774.1巨噬细胞。巨噬细胞染色用红色荧光染料LysoTracker红色的DND-99，和C。染色用FITC（绿色） 白色念珠菌 。巨噬细胞和 C。 白色念珠菌共培养6小时的期间在37℃下在完全独立的CO 2 -介质。捕获的图像，在1分钟的时间间隔6小时。巨噬细胞建立与 C的细胞-细胞接触，可以看出白色念珠菌，随后是C 的吸收白色念珠菌为巨噬细胞的吞噬体。箭头指向为 C。 白色念珠菌之前J774.1巨噬细胞的吞噬。此视频还显示，FITC的荧光是quenc的的HED以下吞噬C.白色念珠菌 。比例尺，10微米。 <a href="https://www.jove.com/files/ftp_upload/50196/50196fig1.avi" target="_blank">点击这里观看电影</a> 。 <img alt="图2" src="/files/ftp_upload/50196/50196fig2.jpg" /> 图2代表活细胞视频显微镜静止图像表示在由个别的巨噬细胞吞噬的白色念珠菌的数目变化。巨噬细胞（染色使用LysoTracker红色DND-99）共同培养与FITC染色C.白色念珠菌 （绿色）。存在偏差的情况的数目C 的白色念珠菌吞没（箭头旁边的数字表示吞没白色念珠菌的数目），和C 中白色念珠菌形态（ 即酵母的诗句菌丝）。比例尺，20微米。 <img alt="图3" src="/files/ftp_upload/50196/50196fig3.jpg" /> 图3图形示出的 C数白色念珠菌摄入每J774.1小鼠巨噬细胞的吞噬功能检测结束6小时。这些数据代表2个独立实验。一共有100个巨噬细胞计数从每个实验的3个领域，共600名个人的巨噬细胞。 <img alt="图4" src="/files/ftp_upload/50196/50196fig4.jpg" /> 图4。这一系列的图片，从有代表性的活细胞视频显微镜实验显示巨噬细胞（M）和C.白色念珠菌 （C）之前，在识别过程中（A，B），期间和之后的吸收（C，D）C.白色念珠菌菌丝内的巨噬细胞（E，F），这可能会导致在巨噬细胞裂解（G）的继续增长。其他的巨噬细胞被招募到破裂的部位，并试图摄取公布的C.白色念珠菌 （H）。是染色使用红色荧光染料LysoTrac的的巨噬细胞KER红色DND-99 和 C。染色用FITC（绿色） 白色念珠菌 。需要注意的是FITC染色淬火后，C.白色念珠菌 J774.1巨噬细胞（C）的吸收。比例尺，10微米。

Watch this Scientific Journal Video about Live-cell Video Microscopy of Fungal Pathogen Phagocytosis at JoVE.com

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

我们描述的方法对活细胞视频显微镜<em&gt;白色念珠菌</em&gt;巨噬细胞的吞噬作用。这些方法使巨噬细胞迁移的特定阶段的分析，识别，吞噬和吞噬小体的成熟和揭示小说方面的吞噬作用。

活细胞视频显微镜病病原菌的吞噬作用

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

live-cell-video-microscopy-of-fungal-pathogen-phagocytosis

Research

JoVE Journal

Immunology and Infection

38.8K 次观看.  University of Aberdeen. 我们描述的方法对活细胞视频显微镜白色念珠菌巨噬细胞的吞噬作用。这些方法使巨噬细胞迁移的特定阶段的分析，识别，吞噬和吞噬小体的成熟和揭示小说方面的吞噬作用。

视频: Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

科研

免疫与感染

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

功能研究和过继转移实验小鼠骨髓嗜中性粒细胞的分离，纯化及贴标

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.