我们感谢吞噬体生物学实验室手稿的批判性阅读的成员。这项工作是由亥姆霍兹青年研究者补助（倡议和亥姆霍兹联合会的网络基金） 和优先方案，德国研究理事会（德意志研究联合会，DFG）的SPP1580资助。

在这个协议的动物保健和所有程序遵循的制度和国家指导方针。 准备是由“Π型”预先指示的准备工作。 1。桥梁养护管理的制备<ol><li>颈椎脱位进行小鼠的扑杀。 </li><li>从组织中取出股骨和胫骨，免费骨骼和修剪两端的骨髓的可访问性。 </li><li>保持骨骼在冰冷的PBS或冰冷的DMEM培养基（补充有100单位/毫升青霉素和100微克/毫升链霉素，置于冰上，直到下一个步骤。 注 ：进行下一个二类生物安全柜下面的步骤，以尽量减少污染的风险。 </li><li>用冷PBS +青霉素/使用26克（0.45mm）内针连接到1毫升注射器链霉素冲洗髓腔。 </li><li>丸粒细胞通过温和离心分离，在350×g离心10分钟，沉淀重悬在BMM MED鎓和板块在无菌微生物未涂覆培养皿。 <ol><li>在BMM培养基的制备<ul type="disc"><li> Dulbecco改良的Eagle培养基DMEM </li><li> 10％灭活FCS </li><li> 20％的小鼠成纤维L929细胞株培养上清（巨噬细胞集落刺激因子，M-CSF源） </li><li> 5％马血清</li><li> 2mM谷氨酰胺</li></ul></li><li>在L929细胞系培养基的制备; <ul type="disc"><li> RPMI（Roswell公园纪念研究所）1640 </li><li> 10％灭活FCS </li><li> 2mM谷氨酰胺</li></ul></li><li>的小鼠成纤维细胞L929细胞株培养上清准备; <ul type="disc"><li>汇合的L929细胞被分裂1:4，在175厘米培养2培养瓶中使用20毫升L929培养基和培养物上清液2天了48小时后收集，过滤并加入到BMM培养基</li></ul></li></ol></li><li>从孵育冲洗细胞股骨在培养箱中在37℃下在5％CO 2气氛中。 </li><li>每48小时培养的（最多72小时）后，吸出培养基并用新鲜BMM培养基更换，长达10天。超过95％的细胞呈阳性反应，CD14，流式细胞仪的测试。 CD14主要表达的巨噬细胞模式识别受体。通过测定细胞阳性该受体的百分比，已产生粘附桥梁养护的纯培养物。 </li></ol> 注 ：准备和培养细胞相应地，如果其他细胞类型的使用。 2。微球作为吞噬货物的涂层<ol><li>准备1毫升悬浮微珠，用400微升2.5％3微米羧基化聚苯乙烯微粒，或替代微粒。 </li><li>转让珠悬浮液到1.5 ml离心管，洗3次，用PBS，加入100微升500毫米2 - （N-吗啉）乙烷ulfonic酸的钠盐，MES缓冲液，在pH 6.7到胎圈芯块。 </li><li>溶于50微克小鼠IgG（多克隆IgG抗体）在50微升无菌DDH 2 O和添加到珠悬浮液。 </li><li>填写停牌长达1毫升无菌DDH 2 O（450微升）。 </li><li>孵育在旋转轮在室温15分钟，该溶液中。 </li><li>准备EDAC交联物，N-（3 - 二甲基氨基丙基）-N&#39;-乙基碳二亚胺盐酸盐的10毫克/毫升的浓度，并添加14微升的悬浮液珠。 注意 ：EDAC交联的氨基酸的IgG组与小珠的表面上的羧基和促进IgG固定在胎圈表面上。这是珠吸收的关键，因此，溶液应新鲜配制。 </li><li>孵育悬浮液在旋转的车轮1小时在室温下。 </li><li>加14μl的EDAC交联物的孵育在一个旋转的车轮1小时在室温下将悬浮液。 </li><li>离心悬浮液以10,000 xg在微量离心2分钟。 </li><li>洗珠3倍于终止缓冲液（1％的Triton X-100在10mM Tris，pH值9.4），以停止该交联反应，通过再悬浮沉淀于终止缓冲液和纺丝溶液以10,000×g离心2分钟。 </li><li>洗涤珠3次在PBS中重悬沉淀在1ml PBS中。 可选：准备30-50微升的分装。 </li><li>存储IgG包被微珠悬浮液在4℃下不超过4周，绝不允许悬挂被冻结。 </li></ol> 注意 ：防腐剂如叠氮化钠以0.02％的终浓度（重量/体积）可以加入到悬浮液中，以防止细菌的生长和污染。在这种情况下，其使用前的珠子洗涤必须执行，以避免防腐剂的细胞毒性作用。离心该悬浮液的等分试样以10,000×g离心2分钟，并通过重新洗珠悬浮于PBS中的沉淀。重复洗涤3次。 3。葡聚糖探针储备溶液的制备<ol><li>溶解德克萨斯红70000千道尔顿的葡聚糖（Dex70kD）在PBS中于20毫克/毫升，并储存在-20℃下，按照制造商的说明。 注意 ：保持库存在-20℃可保存数月，或于4℃下几个星期，看厂家的文档。 </li><li>通过在约1毫克/毫升它在完全DMEM细胞培养基中稀释（DMEM，10％FCS，2mM的L-谷氨酰胺）（几倍浓缩溶液将稀释至准备用于从库存的实验葡聚糖溶液的20微克/毫升加入到细胞之前最终工作浓度）。 </li><li>使用超声水浴下超声葡聚糖等分试样5分钟使其均匀化溶液和溶解的聚集，可能以后导致工件生成成像期间。 </li><li>离心机在最大克为5分钟，在微量离心从溶液中除去任何不溶团块或污染物。 </li><li>小心地将上清液移至新管中，同时避免了沉淀在试管底部并稀释至20微克/毫升的完全DMEM细胞培养基中的最终工作浓度。 </li><li>过滤灭菌使用0.2μm的注射器过滤器安装在该溶液中。 </li></ol> 4。葡聚糖预压<ol><li>种子细胞在活细胞成像玻璃底菜和孵育至少12小时（37℃，5％CO2气氛），以确保依恋和驯化。 注意 ：有，让每盘进行多个实验（最多四个车厢）可分割的玻璃底菜。 </li><li>如在协议3的介绍准备葡聚糖的DMEM液。预热的DMEM溶液的制备葡聚糖，使用水浴在37℃。 </li><li>吸介质次添加葡聚糖溶液至细胞小心和孵育在37℃，5％CO 2的气氛中2-8小时进行摄取。 注意 ：计划和优化的基础上，用于和重复实验时，严格保持此期间探针和细胞类型的协议。这是必不可少的，以便内吞途径的葡聚糖积累的轮廓，从而再现性的实验。 </li><li>洗涤细胞3倍与预热的完全DMEM培养基，吸出培养基，并仔细冲洗细胞用新鲜培养基。 </li><li>在5％CO 2气氛中培养细胞以完全DMEM培养基为4-12小时，在37℃。 </li><li>用预热的完整过滤的无酚红的DMEM培养基洗细胞一次。 </li><li>为了获得最佳的成像效果，改变成像开始之前预热完成滤波的无酚红的DMEM培养基中至少30分钟。酚红可能引起的荧光信号淬灭，增加背景噪声和从而降低图像的对比度和清晰度。 </li></ol> 注意 ：设置显微镜和预先摄像设置，根据基于探针与细胞中所使用的类型预先计划的和优化的协议。 5。加入IgG包被磁珠<ol><li>平衡的预先制备1％珠悬浮液至室温等分和超声处理在超声水浴2-3分钟。 </li><li>加1-6微升1％的珠悬浮到下一个二类生物安全柜的完整的滤波无酚红DMEM培养基，以尽量减少污染（一个很好的起点浓度为1μL/ 2平方厘米的菜面）的风险。 注 ：此处桥梁养护接种在25,000个细胞/孔和6微升1％的库存珠悬浮液加入到培养基中的菜。这大致等于15:1珠/细胞的比例。的比例要调整到用于和胎圈材料的细胞的种类;岛Ë。乳胶珠具有密度低，不沉底很快，而聚苯乙烯珠下沉，并提出与贴壁细胞接触得更快，从而在更大的数字。 注：根据不同的实验，它可能是优势，选择将提前观察到的区域。珠悬浮液然后可以只添加到玻璃底菜的特定区域，因此增加观测有利的摄取事件的概率。 </li><li>致密云的珠可以轻轻吹打悬浮液在玻璃底培养皿中扩散。 </li><li>等到珠沉到玻璃底培养皿的底部和大致与所述贴壁吞噬细胞的同一平面上。 </li><li>专注于感兴趣区（ROI）的区域，并启动时间推移成像。 </li></ol> 注意 ：这可能是必要的微调焦点而成像过程中，根据所使用的成像系统，其站稳定性和所观察到的细胞的移动性。但是，请注意，这可能使不可能的，吞噬体的强烈的焦点偏离平面作为重新定位的结果的正确分析。 6。成像按照下面的最佳成像质量的一般准则; <ol><li>使用共焦成像系统，如激光扫描或旋转盘片的系统。 注 ：徕卡TCS SP5 AOBS激光扫描共聚焦显微镜的徕卡LAS AF软件控制，并配备了一个环境控制室使用这里的时间推移视频成像。 </li><li>优化影像设置，如扫描速度，放大倍率，分辨率等 。的方式，以允许对一个帧的每7-20秒的速率，取决于所使用的系统。 注 ：在这里，一个200赫兹的扫描速度，扫描仪2倍变焦和2 - 3倍的512×512像素RESU分辨率的平均线lted在3-5秒/ RAME之间的录制时间。一帧中每10秒的速率来记录时间推移电影与至少120分钟的持续时间。 </li><li>使用基于所使用的成像系统和探头（S）适当的激发/发射设置。 <ol><li>优化设置，以防止过度的光漂白。 注 ：在这里，德州红葡聚糖70KD很兴奋，使用半导体泵浦（561纳米）激光发射光由徕卡混合检测器（路政署）在600-650 nm的在610 nm处的发射峰检测。激光强度必须适应信号的强度，并保持尽可能低，以减少光致漂白的荧光基团（S）和所述激光对细胞照片毒性效应。通常，为了平衡扫描速度，行平均，扫描分辨率，帧间隔和成像的持续时间，以获得适当的和稳定的信号强度，并捕获珠吸收和吞噬体成熟亲的完整持续时间是很重要的塞斯。 </li></ol></li><li>包括亮场（BF）信道用于观察珠子如果它们不共轭的荧光团。 注意 ：除了将德克萨斯红通道，BF通道被用于可视化的珠子，相同的DPSS激光器被用作光源和信号与一个光电倍增（PMT）检测器进行检测。 注意 ： 请确保采集数据要被整理时，套用相同的录制设置。最重要的参数是：激发激光强度，探测器的设置，采集设置，放大倍率和帧间隔。没有使用相同的帧间隔将需要一个曲线平均的步骤，因为观测数据点不重叠（例如整理的观察与收购在每7秒，另一组与帧画面，在每12秒帧）。这会增加所需的数据评估的步骤，而且需要用曲线平均功能的附加软件，如OriginLab&#39;源流专业统计软件（ <a href="http://www.originlab.com/">http://www.originlab.com/</a> ）。 </li><li>最好，保存在用过的成像系统的本机文件格式的时间推移视频，并避免出口压缩格式。 </li></ol> 注 ：在这里，时间推移电影被保存在本机徕卡LAS AF格式的文件，然后将其直接导入在斐济开（LIF）。斐济是能够从使用附带生物格式导入插件，在大多数显微镜制造商读取本机文件格式。以JPG或TIFF或作为一个AVI容器格式的视频文件导出时间推移电影文件中的图像序列是没有必要或建议。除了从原始观测保持最佳的图像质量，避免有损压缩算法（如JPEG），使用原生显微镜文件格式确保了该文件的元数据（时间标记，放大，激光和采集强度，探测器的设置等）保存并提供给斐济。但是，如果由于某种原因，时移视频文件需要分析不同的格式导出，确保使用未压缩的文件格式，如TIFF图像系列和独立的RGB颜色（红，绿，蓝）通道已经在这步骤，斐济往往不能成功地从其他颜色通道中导出的文件分开BF通道。此外，请务必保留原始显微镜的文件作为参考。 7。的时间推移电影分析<ol><li>使用ImageJ的，斐济，或提供一个类似物信号关联分析能力的任何其他软件。 注 ：在这里，斐济被用于定时短片的分析。斐济是一个包含一个图像处理软件包与重组工具菜单和附加原生的插件，可在免费下载的ImageJ的分布<a href="http://fiji.sc">http://fiji.sc</a> 。在这个教派所描述的过程离子是描绘在图2中 。 </li><li>点击“文件”，“打开”，然后选择该文件，或通过拖放文件到斐济打开斐济文件。 </li><li>选择以下选项; <ol><li>堆栈观看：查看堆栈与Hyperstack， </li><li>颜色选择：色彩模式彩色的， </li><li>拆分通道分离为单独的窗口：斯普利特通道（用于记录每个RGB颜色通道的一个单独的窗口将被打开）。 </li></ol></li><li>在情况下的图像系列是被使用，进入“文件”，“导入”，“图像序列”，然后选择包含目标图像序列文件夹中的所有图像文件加载一系列斐济。 <ol><li>设置过滤器，并在一系列装载选定的图像，例如条件; <ol><li>图像的数目：多少帧将被加载。 </li><li>启动图像：这形象是该系列中的起始帧。 </li><li>增加：之间的增量帧被加载（每帧，每第二帧， 等等 ）。 </li><li>文件名中包含：使用文件名（例如，通过设置“C001”，这是多么正常的“通道1”的图像分色多通道时间推移视频后，被标记）过滤特定的通道。 </li></ol></li></ol></li><li>进入“分析”，“设置比例...”，选中复选框“环球”，然后点击“点击清除水垢”删除缩放。 注意 ：该步骤允许基于像素的不同放大倍率的已被用于各种试验台是要评估的情况下的图像的分析。如果放大率设置一直保持不变，原生显微镜的文件直接导入到斐济，这一步可以跳过，因为斐济能理解并使用从显微镜文件的元数据的放大倍率和规模的数据。 </li><li>设置测量参数s由去“分析”，“设置测量”，并勾选下列方块：“区”，“标准偏差”，“集成密度”，“显示标签”和“平均灰度值”。 </li><li>重定向到包含从探针是被测量并用“OK”信号的颜色通道。 </li><li>去其中的测量是应该开始，并且通过点击窗口的上边缘选取BF通道窗口框架。 </li><li>使用“椭圆选择工具”，选择感兴趣区域（ROI）， 即一个圆形的投资回报率的内在珠上。确保挑选紧紧地装到所述胎圈在BF通道为可见的外边缘。 注意 ：当使用一台PC，使用选择工具，以确保一个圆形的投资回报，同时按住Shift键。 </li><li>开始测量优化胎圈的全部吞没前几帧完成并注意帧，其中一个完全形成的新生珠吞噬小体的形成，吞噬杯被关闭。这应该是相对的高炉通道清晰可辨。 </li><li>去“分析”，然后点击“测量”执行测量，其结果将显示在一个单独的窗口标题为“结果”。 </li><li>转到下一帧，调整投资回报率的情况下珠已经移动的位置，并重复测量。结果将被添加到该列表中的结果窗口中，每个分析帧可以基于在“标签”列中的值来识别。 注意 ：使用快捷键（如“M”为衡量标准）可显著加快评估进程;看到快捷键的列表和菜单中的“插件”下的指定服装的人。 </li><li>在完成所有相关的帧的测量后，结果可以被复制到电子表格应用程序，如MS Excel中。列“IntDen”描绘了intensitie在那只测量被重定向到信道所选择的区域。 </li></ol> 8。漂白校正根据所使用的探针和成像设置，可能会发生光漂白。根据其程度，这可能强烈地影响了评价的结果。但是，这种影响可以部分通过应用变革的相等但方向相反率测量值校正。如果存在的话，光漂白系数可通过测量随时间变化的信号强度降低，在整个帧或该帧的具体裁剪区域，相关于每个吞噬体的分析的时间范围内进行计算。这个过程示于图3和图4。 <ol><li>在斐济的“插件”菜单中，使用“时间序列分析”插件来确定，一般跌幅在整个帧，或在一段时间的投资回报率特异性探针的信号强度（ 图3）。 注 ：时间序列分析仪插件可供下载的<a href="http://rsbweb.nih.gov/ij/plugins/time-series.html">http://rsbweb.nih.gov/ij/plugins/time-series.html</a> ，如果不是已经存在的插件菜单。 </li><li>暗算时间的测量（以秒为单位）在MS Excel中，只对对应于分析吞噬的帧的帧。 </li><li>应用一个线性趋势线（ 图4A）的曲线图;负斜率的减小信号表示的光致漂白效应的存在。 </li><li>从分析的吞噬小体（ 图4B和4C）应用逆转斜率值： 修正信号强度（SI）=原SI +（以秒为单位的时间*反向坡） </li></ol> 注 ：每单分析吞噬体将一个记录的时间推移电影中有一个特定的时间跨度（从完整的摄取帧）;光bleac兴必须重新计算该特定跨度和将只对从该特定吞噬体校正测量值。 9。数据评估<ol><li>整理数据，并计算在适当的软件漂白校正的值的平均值和统计误差。 </li><li>剧情以适当的形式的评估数据。 </li></ol> 注 ：在这里，科学统计和绘图软件的GraphPad棱镜（ <a href="http://www.graphpad.com/scientific-software/prism/">http://www.graphpad.com/scientific-software/prism/</a> ）使用。 Prism软件能够生成并画出平均曲线与相应的错误指示灯，只要所有的数据具有相同的帧速率（即所有都在相同的时间间隔，比如一帧每隔10秒与收购）。 注意 ：绝对的SI值相同experim的重复中宽变化耳鼻喉科集将介绍非常大的统计误差和呈现数据无法使用。这可能是情况下，如果协议中， 如成像设置，没有举行不同的实验重复要被整理中严格相同。

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作者什么都没有透露。

在下面的部分我们将讨论所提出的方法及其局限性的关键步骤。此外，我们将连接一些与他们的解决方案中常见的问题，提出可能的修改，并考虑我们的方法的优点，以及合适的配套方法，这种方法。 的珠子，包括IgG的胎圈表面上的耦合的制备方法，是至关重要的，应该避免使用unfresh制剂。在增加的，它是同样重要的是葡聚糖是从冷冻股票（稀释和消毒）新鲜每次实验前准?...

专业的吞噬细胞，包括巨噬细胞，发挥着关键的免疫系统。除了 ​​作为防御的第一线中的先天免疫系统，它们也激活适应性免疫通过其信令和抗原中发挥关键作用呈递作用1-3。虽然专业吞噬细胞的功能是多方面的，吞噬体成熟是专业吞噬细胞4,5的杀菌和抗原处理功能的关键支柱。经受体介导的吞噬和吞噬靶，如细 ​​菌的吸收，新生的吞噬体经过一个复杂的和精心策划的互动和交流与内吞网络的舱室和其他一些细胞器6的序列。所述化合物的性质交换与这些细胞的实体，以及调节和融合事件的定时决定了管腔吞噬体环境和phagosOMAL膜组成，因此7，成熟的吞噬体8的命运。 吞噬体成熟的生理意义是由各种细胞内的病原体采用从逃跑，逮捕或颠覆吞噬体成熟9多样化战略例证。大多数这些策略的直接或间接阻止吞噬体成熟过程的最后和关键的阶段：融合吞噬体与晚期胞内体/溶酶体区室，因而具有大量的水解酶和一个成熟的吞噬体7的抗菌因素，8,10。因此，这最后的和关键的一步分析，可以为我们提供强有力的指标有关的吞噬体的成熟的状态，如果自然的生理状态是正面或负面的根据正在使用的特定的实验设置的影响。 晚期内体/溶酶体S是通常被认为是内吞途径的终端室，通过各种存在的定义，并区别于早期的内吞车厢，特定阶段，标记分子。例如水解酶或膜成分，例如溶酶体相关膜蛋白（灯）等等11。终端内吞区室，此后简称为溶酶体，也可作为消化的最后阶段的主要位置处的吞噬/内吞途径12的端部。以这种方式，不易消化的探针可以通过内吞作用和巨胞饮作用从细胞外环境被加载，并通过内吞途径到溶酶体，在那里它下面定义的摄取和追逐的循环后累积13,14输送。荧光团共轭的探针，用于荧光显微镜，诸如有机聚合物的不易消化葡聚糖通常用作endocyticprobes 15-17。 <p clasS =“jove_content”&gt;在该方法中，我们描述了IgG包被的微珠的利用率作为吞噬货物通过分析交付溶酶体腔内容到吞噬体，调查吞噬体成熟。通过使用荧光标记的葡聚糖探针在活的骨髓来源的巨噬细胞（BMM）和延时视频成像与激光共聚焦扫描显微镜溶酶体的易检测的标记，我们按照货物配送的动态过程分成吞噬体与高时间分辨率。然后，我们将介绍如何收集时间推移成像数据可以用开源及可自由查看的图像分析软件，斐济（或ImageJ的）进行评估，统计分析采用Microsoft Excel和使用GraphPad Prism软件14,18呈现。 按照我们的方法的描述，类似的实验，可以使用修改后的设置和因素，探讨其在吞噬体成熟的影响进行设计;几乎任何OT她的类型附着的初级或细胞系吞噬细胞，功能实验包括遗传损失基因敲除和敲除体，突变体或融合蛋白，吞噬靶，微珠涂覆化合物，核内体/溶酶体探针和因素，例如一个表达细胞因子，化学抑制剂，或siRNA等等都可以应用这种方法的基本方法的所有变量。 我们定义该方法的如下目标与基本要求： <ul type="disc"><li>目标的方法：启用直接，定量和定性的观察吞噬体成熟和分析生活中的吞噬细胞高时间分辨率。 </li><li>吞噬货物：一个适当包衣微粒或生物靶标。在这里，我们用IgG包被通过的Fc-γ受体介导的吞噬作用容易内化3微米的球形聚苯乙烯微粒。另外，任何微生物或涂层麦克风可用于roparticle为其中吞噬受体是存在于细胞中。 </li><li>吞噬细胞：表达适当的吞噬受体粘附吞噬细胞。在这里，我们用鼠标主要桥梁养护管理的丰富表达的Fc-γ受体。可替换地，树突状细胞（DC），单核细胞或吞噬上皮细胞或其遗传突变或敲除变体也可以使用。 </li><li>溶酶体货物：一个荧光标记的溶酶体探针。这里，德克萨斯红偶联70000千道尔顿的葡聚糖（Dex70kD）用作溶酶体的腔货物。可替换地，一个细胞区室或细胞器，或用于吞噬体性能如酸度或降解能力的适当的探针的任何荧光检测的标志物，可用于研究吞噬体成熟的进展。 </li><li>影响因素：例如，信号分子，化合物，基因敲下来，或突变蛋白可能的瞬时表达。在这里，我们已经锻件1使用这样的一个因素为简单起见，但对于最近的一个例子与突变体蛋白的表达和敲低影响因素请参阅Kasmapour 等人 18，可以影响细胞吞噬吞噬体或情况和迁移率和/不限因子或与吞噬体成熟交互的车厢可能被使用。 </li></ul>

<table border="0" cellpadding="0" cellspacing="0" width="610">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagles Medium without phenol red (D-MEM)</td>
			<td>PAA, Austria</td>
			<td>E15-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>PAA, Austria</td>
			<td>A15-151</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>PAA, Austria</td>
			<td>M11-004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>PAA, Austria</td>
			<td>H15-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>PAA, Austria</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr>
			<td>WillCo-dish&#174; Glass bottom dish (35 mm &#8709;, #1.5)</td>
			<td>WillCo Wells, Netherlands</td>
			<td>GWSt-3522</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLviewTM glass bottom dish, 35 mm</td>
			<td>Greiner Bio one, Germany</td>
			<td>627871</td>
			<td>Alternative: Available as segmented</td>
		</tr>
		<tr>
			<td>Carboxylated latex microspheres</td>
			<td>Polysciences, USA</td>
			<td>#09850</td>
			<td>Available in different diameters, we used 3 &#956;m</td>
		</tr>
		<tr>
			<td>Polystyrene microparticles</td>
			<td>Kisker Biotech, Germany</td>
			<td>PPs-3.0COOH</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>ROTH, Germany</td>
			<td>305.1.3</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse whole IgG</td>
			<td>Rockland, USA</td>
			<td>010-0102</td>
			<td></td>
		</tr>
		<tr>
			<td>EDAC crosslinker</td>
			<td>Sigma-Aldrich, USA</td>
			<td>E6383-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM Tris</td>
			<td>Sigma-Aldrich,USA</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ml syringe Omnifix -F</td>
			<td>B.Braun, Germany</td>
			<td>9161406V</td>
			<td></td>
		</tr>
		<tr>
			<td>26&#160;G needle (0.45*25 mm)</td>
			<td>B.Baun, Germany</td>
			<td>4657683</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>PAA, Austria</td>
			<td>E15-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco, USA</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MES hydrate</td>
			<td>Sigma-Aldrich, USA</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#956;M syringe mounted filter</td>
			<td>Sartorius, Germany</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of phagolysosome biogenesis in live macrophages

吞噬细胞去除和消除其吞噬体入侵的微生物发挥先天免疫系统的重要作用。吞噬体成熟是复杂而严格监管的过程中，其中一个新兴的吞噬体通过与各种细胞器和车厢在细胞质中精心策划的互动经历了激烈的变革。这个过程，这是对吞噬细胞的吞噬体赋予其溶菌和杀菌性能的生理功能所必需的，高潮在融合吞噬体与溶酶体并且被认为是成熟为吞噬体的最后和关键的阶段吞噬溶酶体的生物合成。在这份报告中，我们描述了一个活细胞成像为基础的方法对溶酶体的动态过程的定性和定量分析，以吞噬小体内容交付，这是吞噬溶酶体生物发生的一个标志。这种方法使用IgG包被微珠作为一种模式phagocytosis和荧光标记的葡聚糖分子作为管腔溶酶体货物感，为了使用时间推移成像和激光扫描共聚焦显微镜遵循lysosmal含量的动态传递到吞噬体中实时直播巨噬细胞。在这里，我们详细描述了后台，准备步骤和一步一步的实验装置，以便在其他实验室容易和精确部署此方法。我们描述的方法很简单，功能强大，而且最重要的是，可以很容易地适应学习在不同的系统，并根据不同的实验设置，如利用各种吞噬细胞类型，功能丧失的实验，不同探头的吞噬体相互作用和成熟，以及吞噬颗粒。

正确的制剂的细胞和珠子成像是至关重要的。 图1显示了播种，探针装载和在该方法中初始成像步骤，根据我们对桥梁养护管理优化的协议的轮廓。它是必不可少的，因此，该协议参数进行测试和优化取决于细胞和探针要使用的类型。 另外的抗体包被的珠的预加载的细胞后，重要的是，视场被选择的方式，以提供最大数目的潜在摄取事件的最大数量的细胞成为可?...

Watch this Scientific Journal Video about Study of Phagolysosome Biogenesis in Live Macrophages at JoVE.com

Study of Phagolysosome Biogenesis in Live Macrophages

在现场研究巨噬细胞吞噬溶酶体生源

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

study-of-phagolysosome-biogenesis-in-live-macrophages

Research

JoVE Journal

Immunology and Infection

14.3K 次观看.  Helmholtz Centre for Infection Research. 在现场研究巨噬细胞吞噬溶酶体生源

视频: Study of Phagolysosome Biogenesis in Live Macrophages

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 ...

科研

免疫与感染

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

在小鼠体内的巨噬细胞的枯竭和重建

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

巨噬细胞通过二维电泳蛋白组学分析

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...