该研究是由威康信托基金和脑膜炎英国。 HBMEC细胞系博士KS金。共焦成像和电子显微镜，英国布里斯托尔大学的欧胜生物成像设施。我们还要感谢他们的帮助和建议黄志光Leard，马克博士杰普森（英国布里斯托尔大学），艾伦先生蒂利（珀金埃尔默）。

1。免疫议定书“ 播种，感染及免疫组织化学染色下面的过程，需要合适的安全级别的组织培养和微生物实验室设施。 第1天 A.感染靶细胞的制备<ol><li>盖玻片50％汇合时（〜1.5x10 4细胞/厘米2）（直径16毫米）的种子HBMECs放入12孔板（3.8 厘米 2的增长领域/） 。 辅以15％热灭活（56 ° C，30分钟）胎牛血清，谷氨酰胺2毫米，1毫米丙酮酸钠，100 U / mL青霉素/链霉素，1％（V / V）的MEM非必需氨基酸的生长培养基：RPMI 1640酸溶液和1％的MEM维生素的解决方案。 </li><li>文化在夜间（O / N）在37 ° C，在5％ 二氧化碳培养箱。 </li></ol> B.细菌培养<ol><li>发展利益的应变O / N（16-18小时）所需的介质，例如脑心浸液（BHI）辅以10％的加热马血琼脂平板在37℃，5％CO 2的气氛 4 。 </li></ol>第2天 A.细菌（ 脑膜炎奈瑟菌 ）暂停的制备<ol><li>使用10μL的文化循环，在2 mL PBSB（钙和镁的PBS）暂停过夜的细菌培养。 </li><li>允许大量细菌总量，以解决离开悬浮站立在室温（RT）为5分钟。 </li><li>互不干扰沉淀，转移到无菌管中的悬浮液1毫升（股票接种）。 </li><li>估计在股市接种的细菌数量，加等分（20-50μL），含有1％SDS 0.1M氢氧化钠1ML卷管，轻轻混匀solubilise。 </li><li>测量确定的吸光度在260nm（A260）解决方案的核酸含量。在我们的手中，A260 1对应5X10 8菌落形成单位/毫升（CFU / ml为）。这可以验证准备了一系列的股票在PBSB接种稀释，电镀琼脂平板和O / N后的潜伏期菌落计数。 </li><li>在感染媒介稀释股票接种等分[（2％补充M199 decomplemented（56℃，30分）正常人体血清（NHS）]以获得所需的细菌感染的靶细胞密度。 <ol><li>在我们的实验室，200-300每靶细胞中的细菌感染的比率是经常使用的。 </li></ol></li></ol> B.细胞培养感染<ol><li>洗净的盖玻片培养的靶细胞的3倍，与汉克的介质，以消除任何抗生素的跟踪。 </li><li>用新鲜配制的菌悬液（如上所述）为3到8小时在37 ° C感染细胞，在5％的CO 2 。 </li><li>感染期的结束，洗涤细胞，用PBS三次，并固定在500μL的2％多聚甲醛（PFA）RT或O / N为30-45分钟，4 ° C。 <ol><li>多聚甲醛固定在上面所示的浓度和时间被认为是适合细菌和细胞形态的保护。 </li></ol></li><li>洗涤后的多聚甲醛，permeabilise 0.1％PBS稀释为10分钟Triton X - 100的的孵化，多聚甲醛固定细胞。 </li><li>用PBS清洗样品的3倍。 </li><li>进行免疫染色或，或者，留在500μL1％BSA / PBST O / N的样本在4 ° C。 </li></ol>第3天免疫染色染色细胞内的细菌和α-辅肌动蛋白可以按顺序执行，或同时使用适当的小学和中学抗体如下，所有的程序可以进行12孔板。 <ol><li>座含500微升3％permeabilised细胞的盖玻片，在室温为30-60分钟BSA / PBST（PBS含0.05％Triton X一100） </li><li>用PBS洗涤后，转移到一个新的干好每盖玻片的12孔板。 </li><li>对细菌和α-辅肌动蛋白的抗体;八十至100μL每盖玻片抗体是足够的，如果添加仔细盖盖玻片表面。 1h后在室温下孵育。 <ol><li>我们用兔多克隆抗体对α-辅肌动蛋白（Abcam公司），同时对脑膜炎奈瑟菌（实验室提出）和鼠单克隆抗体（mAb） 。在一些实验中，抗α-辅肌动蛋白，对肌动蛋白或波形的单克隆抗体（Sigma公司）。 </li></ol></li><li>在孵化结束，加200μL的PBS井，电梯盖玻片，并在一个新的以及含有500μL的PBS的地方。 </li><li> 5分钟后，取出吹打PBS和再加入新鲜的PBS。重复两次。将盖玻片传输到新干的好。 </li><li>添加适当的辅助在1％BSA / PBST稀释的不同荧光标记抗体，在黑暗中1H在室温下孵育。 <ol><li>对于细菌检测，我们使用抗兔免疫球蛋白结合，以TRITC和α-辅肌动蛋白和其他细胞骨架蛋白检测，我们使用抗鼠免疫球蛋白偶联要么FITC（Sigma公司）的Alexa Fluor 488 - （Invitrogen公司）。 </li></ol></li><li>孵化结束，洗净，在步骤4和5。 </li><li>计数器0.6微克/毫升的PBS的DAPI（脱氧核糖核酸染色）染色5分钟在室温下，在黑暗中。 </li><li>用PBS冲洗。 </li><li>摩盖玻片（细胞朝下）与安装介质中的幻灯片（E G. Mowiol或Vectashield） </li><li>商店在黑暗中，在4 ° C。 </li><li>显微镜下观察样品。 </li></ol> 2。激光共聚焦显微镜（CLSM） 为了观察和捕捉细胞内的细菌和细胞骨架元素的图像，我们使用immunolabelled样本和使用的Leica SP5的AOBS共聚焦激光扫描显微镜徕卡DM I6000的倒置epifluorescence显微镜拍摄的图像。使用一个63X NA 1.4油浸目标和与徕卡软件的过程中收集的所有图像。 CLSM程序： <ol><li>要开始的CLSM程序，添加浸油下降的目标，并把标本幻灯片，在显微镜舞台上，盖玻片的一面朝下，。 </li><li>显微镜设置一个可视化模式，并使用显微镜的眼片的利益方面找到。 </li><li>使用徕卡软件，选择XYZ采集模式。 </li><li>选择512 × 512格式（帧大小）。 colocalisation研究，具有更高的分辨率，更准确的形象;牢记在显微镜的分辨率极限。然后选择双向X模式，这将增加扫描速度，并有助于减少光漂白。 </li><li>设置顺序扫描设置。点击“SEQ”功能，并选择扫描模式之一。我们使用“线”模式之间。 </li><li>选择激光束根据荧光标记的二级抗体的DAPI（蓝色），488纳米的Alexa Fluor 488（绿色）和为TRITC为561 nm（红色）：405纳米。激活光电倍增管（PMT），1，2和3，分别。调整光电倍增管的设置检测到正确的发射波长。 </li><li>设置顶部（“开始”）和Z -堆叠或系列的底部（“结束”）。接下来，设置所需的“Z -步长”。 <ol><li> 脑膜炎奈瑟菌是一种双球菌（图1G），因为每个球菌有一个直径大约0.5微米，0.20微米的Z -步长被选为增强扫描每个球菌至少两次的概率。这也是步长内的最佳分辨率为0.1 - 0.2微米。 </li></ol></li><li>最终的扫描参数设置选择3平均线，以提高信噪比。 </li><li>通过点击“开始”，双重或三重染色的图像得到不同波长的顺序扫描，以消除不同生色团之间的串扰。 </li><li>对于两个荧光colocalisation的指示，选择“叠加”功能合并到一个单一的形象选择的渠道，例如，当两个Alexa的488（绿色）和TRITC（红色）荧光colocalise，黄色覆盖的形象出现在“ 。 </li><li>编译Z -堆叠或串联使用的可视化可能colocalisation需要一个二维图像的建设的“最大投影”功能。每个光部分的分析，可以得到更详细的分析colocalisation。 </li><li> Z -堆叠或系列收购后，处理您的数据，以获得一个细胞内各种要素的本地化（图1E）的可视化的正交形象。 </li></ol> 3。量化的Colocalisation 共焦扫描显微镜图像的统计分析与Volocity软件（即兴创作，珀金埃尔默）。该软件提供专门设计colocalisation分析曼德斯等人 （1993）5的一个工具。 Colocalisation数字荧光成像方面可谓是相同的体素（像素体积）每个通道中的位置的信号检测。这两个通道是从同一样品的面积（Volocity用户指南）采取两种不同的荧光图像。 Volocity软件使用下面描述的定量Colocalisation分析（即兴创作，珀金埃尔默）进行统计分析。 定量Colocalisation分析<ol><li> CLSM图像使用Volocity软件创建一个库。 </li><li>选择从顶部栏的形象“扩展的重点”。此工具将编译Z -堆叠在一个二维图像进行分析。 </li><li>选择“的共存”的工具。要分析这两个渠道应该具有相同的颜色深度。 </li><li>选择的区域，它要进行量化。设置阈值，以消除任何背景。 </li><li>选择“产生共定位”的创建colocalisation输出。 Colocalisation生成统计先前选择的地区的利益。 </li><li>选择曼德斯系数R（重叠系数）和我（colocalisation系数）。 <ol><li>曼德斯系数不敏感，染色，因为它们是对总像素强度归的强度，因此他们可受聘当一个抗原的染色比对方强。 </li><li>据曼德斯5,6重叠系数R代表colocalisation真实程度，即colocalise总像素数相比的像素数量。 </li><li>另一方面，colocalisation系数，我介绍的不太丰富的基团（在这种情况下，细菌，红色）的更丰富的基团（在这种情况下，α-辅肌动蛋白，绿色）的荧光贡献，即红色像素数重叠的红色像素的总数相比，与绿色像素。 </li><li>曼德斯“系数介于1和0，1 colocalisation高，0没有，但他们可以容易解释的百分比表示。 </li></ol></li><li>出口统计值数据演示的Excel文档。 </li></ol> 4。代表性的成果 OPC表达脑膜炎奈瑟菌和α-辅肌动蛋白细胞内的本地化共焦成像人类大脑与以上的α-辅肌动蛋白和牛，这似乎是在3小时感染（未显示）微血管内皮细胞的实验不太频繁表示colocalisation描述3日和8小时的牛感染与感染8小时的文化相比，（图1自动对焦）。 α-辅肌动蛋白与OPC -表示脑膜炎球菌的一个显而易见的colocalisation观察每次都在重复实验&gt; 5。 colocalisation统计分析，使用几个共聚焦图像进行了上文所述。总体而言，在HBMEC感染OPC表示，脑膜炎球菌，25％的绿色（α-辅肌动蛋白）和红色（NM）像素的重叠，得到（图2B，重叠系数R）。相比之下α-辅肌动蛋白，其中标签化的细菌和肌动蛋白或波形进行实验，观察与肌动蛋白，但偶尔colocalisation与波形中是罕见的（Figure. 2B）。 使用我的系数，其中考虑到每个基团的相对丰度的数据进行了分析。我是一个更丰富的信号（在这种情况下，绿色，α-辅肌动蛋白），每次发生较丰富的信号（红色，细菌在这种情况下）的发生频率的措施。这项措施显示了一个惊人的水平化的脑膜炎球菌（图2A和C）附近的α-辅肌动蛋白发生。 <img src="/files/ftp_upload/2045/2045fig1.jpg" alt="图1" /> 图1。共聚焦激光扫描显微镜来评估细胞内相互作用的 N与α-辅肌动蛋白的脑膜炎 。啊。被感染上盖玻片种植聚合内皮单层OPC -表达（AF）N脑膜炎 。 8 h后，非粘附的细菌洗掉，细胞固定与多聚甲醛和0.1％的Triton X - 100的permeabilised。随后，细菌和染色α-辅肌动蛋白（α-辅肌动蛋白;细菌，红色，绿色）如上所述。 交流。牛（A）或α-辅肌动蛋白（二）的位置显示XY图像的领域之一。 （c）中的叠加图像显示的几个地区，其中橙黄色的色彩似乎暗示colocalisation。在箭头（A）和（b）显示的α-辅肌动蛋白积累的高度大约有细菌发生的地区。 D.光的夹层感染HBMEC单层表明围绕位于细胞内的细菌细胞的基本的colocalisation。 再次，这colocalisation是不是由于意外接近α-辅肌动蛋白，α-辅肌动蛋白染色一般在本地区为低。 E和F三维图像处理上述感染HBMEC单层。一个斜的顶面（五）视图显示附着细菌染成红色（红色箭头），而对血管内皮细胞（黄色箭头）的基底表面的一些细菌，是明显的橙/黄色。基底的位置，可以更清楚地看到在（F）的，这是上月底，新征跨节。 G. 北路一负染电子显微镜图像脑膜炎显示出其主要diplococcal。每个球菌直径约为0.5微米。 <img src="/files/ftp_upload/2045/2045fig2.jpg" alt="图2" /> 图2。 HBMEC细胞α-辅肌动蛋白，肌动蛋白和波形蛋白的位置和分布。 答：感染的HBMEC单层视为描述在上面的传说，但除了α-辅肌动蛋白，一些盖玻片使用过程类似于α-辅肌动蛋白的肌动蛋白或波形的检测。如上所述，α-辅肌动蛋白集中在几个内在细菌（白色箭头）。波形蛋白和肌动蛋白没有colocalise细菌可观的水平。律师代表20微米。 B.＆C系数R和我的价值，获得超过三个实验使用Volocity软件如上所述。

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内在约束力的可能性OPC - colocalisation细菌的检查和3和8小时的潜伏期后，感染的细胞的细胞骨架蛋白α-辅肌动蛋白表达脑膜炎奈瑟菌进行了探讨使用HBMEC。共聚焦显微镜，可以证明，与α-辅肌动蛋白的脑膜炎奈瑟菌colocalisation 。值得注意的是，虽然细菌在3小时内化，有小colocalisation与α-辅肌动蛋白，在这个时间点。与细胞骨架蛋白的细菌协会似乎需要一个较长时期细胞内居住为8 h后感染期，大量的细菌α-辅肌动蛋白，显然在密切的联系。 α-辅肌动蛋白是一种多功能蛋白，细菌与细胞骨架元素的相互作用，可以对目标细胞的功能，这是当前研究的课题有重大影响。 如上所述colocalisation量化要求细致的样品制备。应特别注意标本固定，封锁期和抗体稀释。最好的信噪比，每个小学和中学抗体应在初步实验中滴定，以确定最佳浓度。根据我们的经验，安装介质Mowiol产生更好的图像。

1。激光共聚焦显微镜（CLSM）： 莱卡SP5的共焦成像系统：该系统使用一个组合AOTF（声光可调谐滤波器）和AOBS（声光分束器），简化了特定波长的激发。 2，软件： 徕卡共聚焦软件LCS，徕卡显微系统，德国。 Volocity 5，即兴，珀金埃尔默，美国。

visualisation and quantification of intracellular interactions of neisseria meningitidis and human &alpha;-actinin by confocal imaging

脑膜炎奈瑟菌 （Nm的，脑膜炎双球菌）OPC蛋白表面表达不可或缺的外膜蛋白，它可以作为人类上皮细胞和血管内皮细胞的粘附和有效invasin。我们已经确定了OPC，OPC的过程，需要先绑定到vitronectin，如整合素配体的主要受体，并通过这些细胞表达的受体 1的内皮表面的整合。这个过程导致细菌入侵血管内皮细胞的2。最近，我们观察到3人体细胞的整个细胞裂解液中发现了一个100kDa蛋白OPC互动。最初，我们观察到这种相互作用时宿主细胞蛋白电泳分离和抹杀硝化棉覆盖OPC - Nm的表达。互动是直接的，不涉及中间分子。通过质谱，我们建立了α-辅肌动蛋白的蛋白质的身份。由于没有表面表达的α-辅肌动蛋白上找到任何的八个细胞线研究，并作为OPC与血清导致细菌进入到靶细胞的存在的内皮细胞的相互作用，我们研究的两个相互作用的细胞内蛋白质的可能性。对于这一点，体外培养的人脑微血管内皮细胞（HBMECs）感染与OPC -表示长时间的牛和内在的细菌和α-辅肌动蛋白的位置，通过共聚焦显微镜检查。我们观察到细胞骨架蛋白，这是相当大的后8小时内的细菌内部化牛colocalisation时间依赖性增加。此外，定量成像软件的使用，使我们获得了与α-辅肌动蛋白和其他细胞骨架蛋白的牛colocalisation相对措施。在这里，我们提出了一个可视化和量化后进入人体血管内皮细胞的细菌进入细胞内蛋白质的细菌colocalisation的协议，虽然过程也适用于人类的上皮细胞。

Watch this Scientific Journal Video about Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human Î±-actinin by Confocal Imaging at JoVE.com

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

脑膜炎奈瑟菌 （NM），革兰氏阴性人类特有的呼吸道病原体，人类α-辅肌动蛋白结合。在这里，我们提出了一个可视化的细菌colocalisation后细菌进入到人脑微血管内皮细胞（HBMECs）的α-辅肌动蛋白与细胞内的协议。

胞内相互作用的可视化和量化脑膜炎奈瑟菌和人类α-辅肌动蛋白共聚焦成像

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

visualisation-quantification-intracellular-interactions-neisseria

Research

JoVE Journal

Immunology and Infection

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

18.0K 次观看.  University of Bristol, UK. 脑膜炎奈瑟菌 （NM），革兰氏阴性人类特有的呼吸道病原体，人类α-辅肌动蛋白结合。在这里，我们提出了一个可视化的细菌colocalisation后细菌进入到人脑微血管内皮细胞（HBMECs）的α-辅肌动蛋白与细胞内的协议。

视频: Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

科研

免疫与感染

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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.

一种用于丝状真菌菌落细菌生物膜的定性多尺度分析的新方法利用共聚焦和电子显微镜

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

激光内在人肺组织中的新型细菌特异探针的光学筛选

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...