Name: visualizing the early stages of phagocytosis
Uploaded: 2017-02-03T12:30:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

我们感谢温迪三文鱼和凯克设施的尼基·沃森在麻省理工学院的Whitehead研究所成像。

1. DC2.4和RAW 264.7细胞系的制备注:巨噬细胞样细胞系RAW 264.7和树突状细胞系DC2.4都是鼠源，并使用下述条件下生长的细胞。 <ol><li>生长在DMEM的RAW 264.7细胞（Dulbecco氏最小的Eagle培养基），补充了10％灭活胎牛血清（FBS）的在37℃在具有5％CO 2的加湿培养箱。 </li><li>生长DC2.4细胞与RAW 264.7细胞的相似的条件，除了补充有L-谷氨酰胺代替DMEM使用RPMI（罗斯韦尔园区纪念研究所）培养基。 </li></ol> 2.荧光共轭颗粒的制备:包被抗体白色念珠菌 ，酵母多糖，珠<ol><li> 成长白色念珠菌 <ol><li>从冷冻甘油贮存接种白色念珠菌菌株SC5314 25 YPD补充有乌里（2％细菌蛋白胨，1％的溶液酵母提取物，2％葡萄糖，并在30℃下过夜80微克/毫升尿苷）。 注意:避免增长的念珠菌培养一个多外径12的600。 </li><li>取1毫升的白色念珠菌和自旋下来以2,000 xg离心在室温下5分钟。 </li><li>吸出上清液并重新悬浮用1毫升的PBS沉淀。 </li><li>收集通过离心沉淀在2000 xg离心在室温下5分钟。 </li><li>重复步骤2.1.4。 </li><li>计算白色念珠菌的感染复数（MOI）的关系的多重到吞噬细胞的数量，并与吞噬作用测定法进行如下的第3节的OD描述的1 600相当于每毫升2.5×10 7个白色念珠菌 。 注:念珠菌-BFP的产生在参考13进行说明。 </li></ol></li><li> 酵母聚糖和IgG包被珠子的制备 注:酵母聚糖是含β葡聚糖来自酿酒酵母来源真菌碳水化合物颗粒制剂。酵母聚糖已知唤起在巨噬细胞和树突状细胞的炎性信号，并且已经在吞噬作用的研究13，14，15广泛使用。 <ol><li>短暂涡旋包含微粒的管中，并分装足以实验到1.5毫升管的量。 注意:通常我们使用1:10的比例（为一个小区，加10酵母多糖颗粒或IgG包被的珠子）。 </li><li>加入1ml冰冷PBS中，并通过以10,000×g下离心1分钟收集所述颗粒。 </li><li>吸出上清液，重悬在1ml冰冷的PBS沉淀。 </li><li>重复步骤2.2.2-2.2.3 注意:继续执行步骤2.2.5或管保存在4℃直至使用。 </li><li>超声处理10秒在超声浴超声波仪的微粒（120伏，50-60千赫），以避免一纽约颗粒聚集。 </li></ol></li></ol> 3.吞噬注:吞噬作用是一个复杂的过程，与通过的PRR的相互作用与颗粒的表面上的配体的吞噬细胞的细胞表面上的颗粒的结合开始。结合之后是肌动蛋白的组装，并在接触的部位及其相关的蛋白质，并吞噬杯的形成。随后的肌动蛋白拆卸发生在吞噬体与导致颗粒的完全吞没。下面我们描述吞噬的不同阶段。 <ol><li> 结合分析 注:此实验的目的是监测吞噬的第一步涉及对目标颗粒的配位体和吞噬细胞的受体之间的相互作用。 <ol><li>种子约5×10 4个细胞中的腔室滑动，使得它们在实验的时间的60-70％汇合。 Alternatively，在盖玻片上板的细胞（在24或12孔格式）或玻璃底96孔板。孵育细胞在37 5％的CO 2℃培养箱过夜。 注:细胞可以通过血细胞计数器或类似的技术来计算。 </li><li>放置在10℃的室中滑动（或板）10分钟。 </li><li>轻轻地从每孔用吸管或抽吸删除网上平台。 注意:执行此步骤仔细作为一个腔玻璃罩系统的每孔小;很容易扰乱细胞用移液管的尖端或吸气的强气流。 </li><li>用细胞培养基混合荧光标记的颗粒（如上所述制备）。添加念珠菌 -BFP（以10的MOI），荧光共轭酵母多糖或乳胶珠-兔IgG-FITC（每个细胞10个粒子）细胞中。使用200μl的媒体的终体积为一个腔室以及，以确保所有的细胞都包括在内。 </li><li>降速在277室的幻灯片XG的在10℃下3分钟。 注意:此离心步骤有助于提高微粒与细胞之间的接触，并进行同步的装订处理。 </li><li>紧接在室滑动用5％的CO 2转移到37℃培养箱5分钟。 </li><li>取下孵化室幻灯片，吸媒体。 </li><li>洗细胞用冰冷的PBS 3倍。 注意:检查未结合的颗粒，用光学显微镜的存在下，必要时，用PBS洗涤多次。它从井除去未结合的粒子在分析过程中，以减少背景噪声是必不可少的。 </li><li>通过添加在室温下30分钟温育在PBS中稀释500μl的4％低聚甲醛（PFA）固定细胞。 注意:此步骤需要使用4％PFA的这是高度有毒，腐蚀性，以及潜在的致癌物质。适当的谨慎在使用此液体时，要采取。而且，要保护样本的光。 </li><li&gt;通过加入500微升的PBS进入室清洗细胞并轻轻吸去溶液。 </li><li>重复3.1.9步骤两次。 注:在这一步骤中，填充每个小瓶删除以前的PBS之后。 </li><li>通过用在PBS中500μl的50mM的NH 4 Cl作为10分钟孵育停止固定。 </li><li>如在步骤3.1.10中描述，用PBS洗涤细胞两次。 </li><li>在室温下加入250微升（在PBS中0.1％皂甙，0.2％BSA）中的结合缓冲液30分钟通透细胞。 </li><li>通过加入荧光共轭毒伞素（:在结合缓冲液500稀释1）染色F-肌动蛋白。孵育细胞，在室温下1小时。 </li><li>如在步骤3.1.10所述洗涤细胞三次，用PBS。 </li><li>捕获使用共聚焦显微镜（63X目标，402 nm和488 nm激光）的图像和使用ImageJ 16分析图像。 注意:一个成功的粒子结合的特征是吞噬铜的存在下p在所在的部位微粒接触的吞噬细胞（ 图1）。请参阅步骤3.2.5。 </li></ol></li><li> 吞噬作用摄取测定 注意:此实验的目的是监测由使用共聚焦显微镜（ 图2）的吞噬细胞荧光标记颗粒的完全内化。 <ol><li>种子约5×10 4个细胞中的腔室滑动，使得它们在实验的时间的60-70％汇合。可替代地，在盖玻片上板的细胞（在24或12孔格式）或玻璃底96孔板。孵育细胞在37 5％的CO 2℃培养箱过夜。 注:细胞可以通过血细胞计数器或类似的技术来计算。 </li><li>保持腔室载玻片（或板）在10℃下10分钟。 注:冷却是必要的，以便阻止细胞内吞作用以及不必要的吞噬作用，并随后到具有一个同步吞噬作用。 </li><li> PERFORM的步骤从3.1.3至3.1.5 </li><li>传送腔室滑动到5％的CO 2 37℃培养箱，孵育不同的时间点。修正后的30，60和90分钟后感染的细胞。观察白色念珠菌开始形成，60分钟后感染菌丝;吞噬细胞开始90分钟后8显示细胞死亡（pyroptosis）。 注意:这是一个建议，并且取决于细胞类型和所用的微粒的优化是必需的。 </li><li>执行步骤从3.1.7至3.1.15。分析使用ImageJ 16个图像。 注意:一个成功的摄取的特征是吞噬细胞的细胞质内的颗粒的完全内化（ 图2）。与细胞表面标记物染色的吞噬细胞如鬼笔环肽有助于描绘细胞的轮廓。它也必须按顺序执行的z堆栈来确定微粒是否绑定到细胞表面或并发症etely内化。 </li></ol></li><li> 吞噬的实时成像 注:在这个协议中，我们描述了利用共聚焦显微镜来捕捉使用荧光酵母多糖结合吞噬的不同阶段。我们使用的树突状细胞系DC2.4稳定表达mCherry标记的F-肌动蛋白8，一种生物传感器，显示丝状肌动蛋白的活细胞中的分布。由于吞噬作用是一种肌动蛋白驱动的过程中该细胞的实时成像不仅使我们能够捕获F-肌动蛋白网络的移动和动力学，但也可视化的活细胞内的不同的吞噬活动（ 图3，电影1）。 <ol><li>种子在室滑动约5×10 4个细胞的浓度，使它们在实验的时间的60-70％汇合。孵育细胞在37 5％的CO 2℃培养箱过夜。 注:细胞可以用血球或计数类似的技术。 </li><li>保持室载玻片在冰上10分钟。 注:冷却是必要的，以阻止不需要的吸收，并且有一个同步吞噬作用。 </li><li>执行步骤从3.1.3至3.1.5 </li><li>预暖显微镜阶段至37℃并达到平衡，用5％ 的 CO 2的室中。等待温度和CO 2 30-45分钟开始成像之前稳定。 注:由于CO 2和温度水平是吞噬作用至关重要，它在显微镜加热器转动实验之前以平衡环境室是重要的。 </li><li>放置装有将细胞在37℃和5％CO 2平衡的显微镜培养器外壳的腔室滑动。 </li><li>执行实时成像17,18。 注:对于激光功率（增益，针孔等 ）的设置可以在根据第的类型而改变Ë显微镜和荧光探针使用。因此，我们建议咨询显微镜的手册最佳条件为您实时成像17,18。 <ol><li>使用共聚焦激光扫描显微镜（和相关联的软件），用于实时成像。使用63X 1.4NA油浸物镜与艾里1-1.1单位针孔。 </li><li>要启动成像，使用明场找上了玻片的代表区域。接下来，通过点击软件上的位置选项选择字段。为了检测GFP在田径1在582 nm的分束器使用的过滤器设置为488纳米的激光，检测488和582纳米之间的波长。 </li><li>在第2道中选择使用的光束分离器在578纳米和578和纳米之间600检测的波长滤波器，用于mCherry（RFP）设置最佳的。使用488 nm和578 nm激光50％激光功率和600-700增益输出。获得的图像，每隔30秒，共90分钟。 </li></ol></li></ol></li></ol>

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The authors declare no conflicts of interest.

专业的吞噬细胞如巨噬细胞和树突细胞，吞噬和消除侵入的病原体，因此使吞噬宿主防御系统的一个重要组成部分。在此过程中吞噬细胞进行大量的膜重组和骨架重排在其细胞表面8，19，20。为了更好地了解这个动态的过程，吞噬作用的不同阶段的可视化是必不可少的。在这里，我们描述了用于监测吞噬的各个步骤的基于显微?...

尽管连续暴露在病原体如细菌，病毒和真菌，我们的身体是配备了能够提供保护，免受感染的免疫机制。先天免疫系统是防御的第一线对抗入侵的病原体，主要依赖于识别和内外国目标吞噬细胞。 吞噬作用是一种进化上保守的细胞过程，包括不想要的颗粒小于0.5微米更大的吞噬。吞噬细胞表达的广泛的免疫受体（也称为模式识别受体，模式识别受体）在细胞表面，使他们能够识别病原体相关分子模式（PAMP）存在于前吞没3病原体。病原体结合后跟受体聚类在细胞表面并触发吞噬杯的形成。这将导致在突出左右的肌动蛋白驱动的膜重塑目标，最终包围它和捏断以形成离散的吞噬体的液泡2,5。吞噬体成熟，然后通过后续的融合与后期内涵体和溶酶体形成吞噬溶酶体酸化6。 虽然吞噬作用被描述为受体介导的肌动蛋白和驱动事件，这个过程也依赖于组成质膜脂质，如磷酸肌醇（PIS）和鞘脂7,8的时空修饰。而肌动蛋白聚合是通过在吞噬杯的基磷酸肌醇-4,5-二磷酸（PI（4,5）P 2）的局部积累决定的，肌动蛋白解聚取决于（PI（4,5）P的转换2到磷酸肌醇-3,4,5-二磷酸（PI（3,4,5）P 3）3,9。这两种修改是因为前者导致围绕目标伪足而后者使得能够在吞噬细胞10的胞质溶胶下沉颗粒的成功扩展必要的。 具有吞噬能力的细胞是专业的吞噬细胞，如同巨噬细胞/单核细胞，粒细胞/嗜中性粒细胞，和树突细胞（DC）或外行吞噬细胞，如成纤维细胞和上皮细胞11。由所有吞噬细胞进行吞噬作用起着组织维持和重塑中发挥中心作用，在由专业的吞噬细胞进行吞噬作用是负责对病原体的先天和适应性免疫反应的协调。专业的吞噬细胞不仅吞噬和杀死病原体，但也存在抗原的适应性免疫系统的淋巴样细胞。这有助于促炎性细胞因子的释放和淋巴样细胞的接合，因此导致感染12成功封锁。 常规生化技术一直在获得关于吞噬期间不同的细胞过程，如翻译后修饰和蛋白质之间的各种高亲和力关联的分子机制的知识的工具。然而，很难获得关于使用常规生化方法吞噬活动的空间和时间的动态信息。活细胞成像不仅使我们能够监视在时间敏感的方式的细胞事件，但也使我们在单细胞水平上获得的信息。这里，我们描述的方法来研究吞噬的不同阶段，以及spatiotemporally使用共聚焦显微镜分析的全过程。

<table><tbody><tr><td>&amp;beta;-Mercaptoethanol</td><td>AppliChem</td><td>A1108</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Cell Signaling Technology&amp;nbsp;</td><td>9998</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dilactate)</td><td>ThermoFisher</td><td>D3571</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>ThermoFisher</td><td>BP-231-1</td><td></td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s Minimal Eagle&amp;rsquo;s medium)</td><td>Gibco</td><td>11965</td><td></td></tr><tr><td>PBS, 1x (Phosphate- Buffered Saline)</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>Fetal Bovine serum</td><td>Sigma Aldrich</td><td>12003C</td><td></td></tr><tr><td>FITC-coupled IgG-coated latex beads</td><td>Cayman</td><td>500290</td><td></td></tr><tr><td>L-Glutamine 200 mM (100x)</td><td>ThermoFisher</td><td>25030081</td><td></td></tr><tr><td>Paraformaldehyde Solution (4% in PBS)</td><td>Affymetrix</td><td>19943 1 LT</td><td></td></tr><tr><td>Penicillin-streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>&lt;a href=&quot;https://www.fishersci.com/shop/products/gibco-penicillin-streptomycin-10-000-u-ml-3/15140122&quot;&gt;15-140-122&lt;/a&gt;</td><td></td></tr><tr><td>Phalloidin-Alexa Fluor 488</td><td>ThermoFisher</td><td>A12379</td><td></td></tr><tr><td>RAW 264.7 cells</td><td>ATCC</td><td>TIB-71</td><td></td></tr><tr><td>RPMI (Roswell Park Memorial Institute)</td><td>Gibco</td><td>61870</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>S7900</td><td></td></tr><tr><td>Trypan blue solution (0.4% (w/v) in PBS)</td><td>Corning cellgro</td><td>MT25900CI</td><td></td></tr><tr><td>Trypsin-EDTA (1x) (0.05%)</td><td>ThermoFisher</td><td>25300054</td><td></td></tr><tr><td>Tween 20 Surfact-Amps Detergent Solution</td><td>ThermoFisher</td><td>&amp;nbsp;85114</td><td></td></tr><tr><td>Zymosan-Alexa Fluor 594</td><td>ThermoFisher</td><td>Z23374</td><td></td></tr><tr><td>Chambered 1.0 Borosilicate Coverglass system (8 chambers)</td><td>ThermoFisher</td><td>155361</td><td></td></tr><tr><td>Glasstic slide 10 with grids</td><td>Hycor</td><td>87144</td><td></td></tr></tbody></table>

visualizing the early stages of phagocytosis

哺乳动物身上配备，帮助自己从病原体的入侵防御机制各层。免疫系统的专业吞噬细胞-如中性粒细胞，树突细胞，和巨噬细胞-保留来检测和通过吞噬1清除这样侵入的病原体的固有能力。吞噬作用涉及在细胞表面2,3膜重组和肌动蛋白重塑的精心策划的事件。吞噬细胞成功地内化和消灭外来分子，只有当吞噬的所有阶段都得到满足。这些步骤包括识别与通过模式识别受体驻留在细胞表面的病原体（的PRR）的结合，通过肌动蛋白富集膜突起（伪足）的形成吞噬杯包围颗粒，并随后吞噬溶酶体成熟吞噬体的断裂那结果在杀死病原体3,4。 吞噬作用的不同阶段的成像和定量是工具阐明此细胞过程的分子机制。本稿件报告的方法来研究细胞吞噬功能的不同阶段。我们描述了一个基于显微镜的方法来可视化和量化的结合，吞噬杯的形成和颗粒的吞噬细胞内化。由于当吞噬细胞受体的先天上遇到一个目标粒子大于0.5微米，我们在座的试验包括使用致病性真菌白色念珠菌和其他微粒，如酵母多糖和IgG涂层珠的配体发生吞噬。

基于显微镜对监测吞噬不同阶段的方法。通过DC2.4细胞各种荧光颗粒的吞噬过程中不同的事件被示出。使用此处描述的技术，我们研究在吞噬作用的早期阶段鞘脂的作用。为了这个目的，DC2.4树突状细胞在Sptlc2，催化在鞘脂生物合成途径的第一个和限速步骤的酶的基因缺失，被使用。相比于野生型细胞，Sptlc2 - / - DC2.4细胞已显著减少鞘脂包括神经酰胺，鞘磷脂和葡糖8的水...

Watch this Scientific Journal Video about Visualizing the Early Stages of Phagocytosis at JoVE.com

Visualizing the Early Stages of Phagocytosis

Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 &#181;m including zymosan and IgG-coated beads.

可视化吞噬的早期阶段

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune ...

visualizing-the-early-stages-of-phagocytosis

Research

JoVE Journal

Biology

16.5K 次观看.  Oregon Health & Science University. Here we describe a microscope-based technique to visualize and quantify the early cascades of events during phagocytosis of pathogens such as the fungi Candida albicans and particulates that are larger than 0.5 µm including zymosan and IgG-coated beads.

视频: Visualizing the Early Stages of Phagocytosis

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

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 ...

科研

免疫与感染

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

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 ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up ...

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (M&#934;) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study M&#934; phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance M&#934; phagocytosis of non-opsonized zymosan of both naive and IFN-&#947; treated M&#934;. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Presented here is a protocol to quantify and produce dynamic images of mesenchymal stem cell (MSC) mediated regulation of macrophage (M&#934;) phagocytosis of non-opsonized yeast (zymosan) particles that are conjugated to a pH-sensitive fluorescent molecule.

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics ...

Real-Time Imaging of Macrophage Phagocytosis of IgG-Opsonized Red Blood Cells

Source: Horsthemke, M., et al. <a href="https://app.jove.com/v/57566">Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages.</a> J. Vis. Exp. (2018)
This video demonstrates an assay to monitor independent macrophage-mediated red blood cell (RBC) phagocytosis events. Mouse immunoglobulin G (IgG)-opsonized human RBCs are incubated with mouse macrophages and observed under a confocal microscope. The RBCs are engulfed by macrophages via a phagocytic cup formation and targeted for lysosomal degradation.

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

Source: Horsthemke, M., et al. Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages. J. Vis. Exp. (2018)
This video demonstrates an assay to monitor ...

可视化吞噬的早期阶段

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活细胞视频显微镜病病原菌的吞噬作用

在现场研究巨噬细胞吞噬溶酶体生源

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

由成比例的荧光显微镜测量吞噬体pH值

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

小鼠巨噬细胞吞噬的延时3D 成像

使用共聚焦显微镜研究利什曼原虫的吞噬作用

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

可视化吞噬的早期阶段

摘要

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高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

由成比例的荧光显微镜测量吞噬体pH值

"吞噬体封闭试验"可视化吞噬体形成的三个维度利用全内反射荧光显微镜（TIRFM）

成像中性粒细胞吞噬体和细胞质使用比例测量pH指示剂

小鼠巨噬细胞吞噬的延时3D 成像

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

间充质干细胞调节巨噬细胞吞噬作用;定量和成像

IgG 调导红细胞巨噬细胞吞噬作用的实时成像

使用共聚焦显微镜研究利什曼原虫的吞噬作用