We thank Dr. Alexandre Benmerah (Institut Imagine Necker, Paris, France) for initial discussions on the experimental approach and Dr. Jamil Jubrail for reading the manuscript. Nad&#232;ge Kambou and Susanna Borreill are acknowledged for performing experiments with the method in our laboratory. This work was supported by grants from CNRS (ATIP Program), Ville de Paris and Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche M&#233;dicale (FRM INE20041102865, FRM DEQ20130326518 including a doctoral fellowship for FMA) to FN, and Agence Nationale de Recherche sur le SIDA et les h&#233;patites virales" (ANRS) including a doctoral fellowship for JM.

注意:使用Lifeact-mCherry质粒是纪尧姆蒙塔尼亚克博士，居里研究所，巴黎，17后产生的一种恩赐。 1.细胞和转染注意:RAW264.7巨噬细胞生长至在完全培养基分汇合（RPMI（罗斯韦尔园区纪念研究所）1640培养基中，10毫米的HEPES，1mM的丙酮酸钠，50μMβ巯基乙醇，2mM的L-谷氨酰胺和10％FCS（在100毫米的钢板胎牛血清））。他们被转染通过电编码荧光标记蛋白的质粒。例行约5 - 6×10 6个细胞用20微克或10微克质粒为每个转染或共转染的分别转染。注意，基于电穿孔或脂转染转染的其它装置可以用作替代方法。 <ol><li>刮细胞与细胞挺杆和通过上下吹打数次重悬在10ml培养基中。 </li><li>离心机细胞悬浮液300 XG，在RT在摆动角度转子5分钟。 </li><li>预热10的完全培养基中的溶液在37℃下在补充有10微克/毫升庆大霉素。 </li><li>离心后，弃去上清液和悬浮颗粒从电套件3毫升"洗涤液A"的。 </li><li>离心细胞悬浮液300 XG，在摆动角转子室温下5分钟。 </li><li>在此期间，在室温下制备的DNA混合物:120微升2×缓冲液B，20微克质粒DNA编码感兴趣的蛋白质，QSP 240微升H 2 O的</li><li>离心后，弃去全部上清。 </li><li>悬浮在该DNA混合物中的细胞沉淀和转制成4mm电试管。 </li><li>孵育在室温3分钟。 </li><li>在电穿孔250 V，900μF。 </li><li>立即重悬补充有庆大霉素在预热（37℃）的细胞完全培养基和板的米100毫米的菜。 </li><li>孵育转染的细胞在37℃，5％CO 2过夜。 </li><li>第二天早上，用10毫升无血清显微镜培养基（RPMI 1640无酚红，10毫米的HEPES，1mM丙酮酸钠，50μMβ巯基乙醇，2mM的L-谷氨酰胺）取代的完全培养基。 </li></ol> 2.调理作用红细胞注意:作为对巨噬细胞粒子目标的模型，绵羊红细胞（SRBCs）被使用。通常，使用？35毫米的玻璃底培养皿约7×10 6 SRBCs。 <ol><li>用100μl1×PBS（磷酸盐缓冲盐水）/0.1% BSA（牛血清白蛋白）和离心机（600 XG，4分钟）的洗SRBCs。 </li><li>离心后，弃去上清液并在/100μl的1×PBS中的0.1％BSA和离心机（600 XG，4分钟）重悬SRBCs。 </li><li>离心后，弃去上清液和/ 0.1％BSA中与兔IgG抗SRBCs悬浮SRBCs在1×PBS中在子凝集的浓度。用500微升含有抗体/ 5微升SRBCs的解决方案。 注:抗体的子凝集的浓度对应于没有诱导检测为形成网络的凝集的最低浓度。在一般情况下，该子凝集的浓度为8.2微克/毫升。 <ol><li>确定由一血凝试验，以opsonize的SRBCs所需IgG抗SRBCs的浓度。连续稀释IgG抗SRBCs（股票以13.1毫克/毫升）1/50之间 - 在微孔板1 / 25,600。加2×10 6 SRBCs在每个孔中。孵育在室温黑暗中板为几个小时。 </li></ol></li><li>孵育在室温下缓慢旋转30分钟。 </li><li>在1×PBS /洗涤两次如上所述0.1％BSA后，重悬在预热的无血清显微镜培养基中的IgG调理的SRBCs（IgG的SRBCs）（2毫升/皿）。 </li></ol>盖玻片的3聚赖氨酸涂层<ol><li>同时，治疗35毫米玻璃底培养皿用2ml 0.01％聚-L-赖氨酸，在室温下30分钟。 </li><li>用2毫升1X PBS的洗碗两次。 </li></ol> 4.玻璃底菜SRBCs的非共价固定<ol><li>倒入2毫升？35毫米的玻璃底菜SRBCs暂停。 </li><li>在2分钟到35 mm的玻璃底菜与500 XG摆动转子离心机。 </li><li>除去上清液并用2ml 1×PBS中/ 10％的BSA洗涤一次。 </li><li>孵育该颗粒30分钟，用2ml每皿1×PBS / 10％的BSA。 </li><li>用2毫升1X PBS的洗碗三次。 </li><li>替换1×PBS中与2ml预温（37℃）的无血清显微镜培养基。 </li></ol> 5.通过吞噬TIRFM可视化<ol><li> 显微镜 注意:使用配备有油浸物镜（N 100X，NA1.49），一个加热室用CO 2在显微镜下进行TIRFM，以及两个唱勒光子检测摄像机EMCCD（电子倍增电荷耦合器件）加上1.5X透镜。 <ol><li>的TIRF显微镜会话的前一天，在37ºC打开加热室，以允许显微镜阶段的均匀加热。 </li></ol></li><li> 临界角测定 注:ImageJ的颜色校准软件，用于处理TIRF图像流。 <ol><li>将含在显微镜下无血清培养基显微镜调理SRBC 35毫米的玻璃底菜。 </li><li>刮细胞，并加入他们之前重悬在中 - 在盘中（100 500微升）。 </li><li>使用"实时采集"软件控制的显微镜，并用491纳米和/或一个561纳米的激光来鉴定表达荧光标记蛋白的细胞进行激励。 </li><li>鉴定表达所述荧光标记蛋白的细胞。 </li><li>将其放置在场地中央，并获得500 IM年龄在一个激励波长，以不同的角度从0度开始到5度，与0.01º（ 图2A）的一个增量。的角度由显微镜系统自动改变。 </li><li>确定的临界角使得入射光在玻璃/介质界面全反射而产生损耗波。使用ImageJ色彩管理软件，通过点击"文件"打开图像序列中，"打开"，然后选择文件。 </li><li>具有均匀的荧光（ 图2B黄1）选择感兴趣区域（ROI）的区域，与矩形工具，在细胞。 </li><li>画出"Z轴轮廓"在下拉菜单和"情节Z轴配置文件"点击"图像"选项卡，"堆栈"上（ 图2B意味着与x轴的角度函数的ROI测量荧光强度红色2）。 注:临界角是导致马角在荧光急剧下降之前ximum荧光。 </li><li>在显微镜会话期间使用在x轴优于临界角的角度的任何值，以获得一个TIRF信号如实施例2.00（ 图2C）。 </li></ol></li><li> 收购 <ol><li>使用实时采集软件，设置采集与模块"协议编辑器"（ 图3A）的参数。 <ol><li>一个"循环"包括"多频道"采集获得从在TIRF区域感兴趣的蛋白质的荧光信号创建的协议。输入的TIRF角度（例如2.00），则曝光时间（例如50毫秒）和激光强度（例如50％）（图3B 1）。 </li><li>引进目标3微米TIRF区上方的"Z移动"来获得信号采集萤光中的"多渠道"的工具。输入低于T角他临界角（如实施例1.00），博览会的时间（50毫秒）和激光强度（50％）（ 图3B中，2和3）。 </li><li>接下来，添加以下目标3微米的"Z移动"，在TIRF区域（ 图 3B 4）返回。添加单元格的"快照"在明亮的光线的LED（发光二极管）（ 图3B 5）。 </li></ol></li><li>发现感兴趣的细胞与荧光标记蛋白表达的中等水平，通过在颗粒周围延伸质膜发起SRBC的吞噬作用。 </li><li>将其放置在场地中央。 </li><li>启动流媒体采集500 - 1000帧。在"循环计数"选项卡，输入"750帧"（ 图 3B 1）。 注:ImageJ的颜色校准软件，用于处理TIRF图像流。 </li><li>通过点击打开的Image J软件序列图像"文件"，"打开"，然后选择该文件。 </li><li>通过点击选项卡"图像"分开的通道，"Hyperstacks"下拉菜单，在"堆栈到hyperstack"功能。完成出现的窗口:订单:xyctz;通道（C）:信道数（例如:"2"，如果你有两个荧光染料）;切片（Z）:在Z轴的切片的数目（例如:"1"，如果在z轴没有运动）;帧（T）:每通道数除以图像的数量;显示模式:灰度。 注意:两个单独的图像序列被生成，对应于两个不同的通道。 </li></ol></li></ol>

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The authors have nothing to disclose.

The experimental protocol described here proposes an unprecedented method to follow in real time and in living cells, with high-resolution, the formation of a phagosome and in particular its closure. Several technical aspects have to be discussed. Firstly, the assay is very sensitive to temperature. It is very important to check that the heating chamber is at 37 &#176;C and that all media, devices or cells are kept within the chamber to avoid temperature changes that could impair the efficiency of phagocytosis. We noticed that subtle temperature changes were sufficient to prevent phagosome formation. Secondly, the protocol described here allows a precise determination of the TIRF angle that will be used throughout the experiment. We believe that this constitutes a substantial advantage when performing TIRF experiments, allowing us to be confident when concluding about events near the plasma membrane. Thirdly, when working with transiently transfected cells, it is important to choose cells with moderate levels of expression of the proteins of interest. With the revolution of the genome-editing technology, it will be feasible to work with cells expressing proteins at native levels. Thus, quantitative fluorescence microscopy will be possible to explore the dynamics of major functions in living cells. 
With this assay, it is now possible to analyze precisely in time and space the different players involved in pseudopod extension and fusion. The forced orientation of the target to engulf allows better monitoring of extending pseudopods and the closure site, compared with phagosome formation imaging using a confocal microscope and a different experimental set up. The only caveat is the low output. Not all cells chosen on the basis of their fluorescence will phagocytose during the acquisition time. A potential improvement would be to have a patterned surface coated with target particles to increase the number of concomitant acquisitions. Using this method, we have dissected the spatio-temporal organization of F-actin dynamics and recruitment of recycling (VAMP3-positive) vesicles during phagosome formation 13. We observed recruitment of F-actin at the very tips of closing pseudopods, but no VAMP3-positive vesicles in this closing zone of the phagocytic cup. VAMP3 was only detected as a plasmalemmal diffuse signal. In contrast, VAMP3-positive vesicles were detected at the base of the forming phagosome, as visualized in the epifluorescence images. These results showed that F-actin polymerization occurs in the extending pseudopods and in their tips, while there is depolymerization at the base where the vesicular traffic is concentrated. This spatio-temporal dynamic of F-actin polymerization was proposed in seminal work reviewed in 2 and recently also confirmed in 12. 
Using the same experimental model, we recently showed that dynamin-2, which mediates the scission of endocytic vesicles, was recruited early and concomitantly with actin during phagosome formation and pseudopod extension. Dynamin-2 then accumulates at the site of phagosome scission. Acute treatment of cells with pharmacological inhibitors clearly showed that dynamin-2 plays a crucial role in phagosome scission 18. The unique possibility to monitor with high precision the closure step in phagosome formation allowed us to conclude the possible role of dynamin-2 in phagosome scission. 
The phagosome closure assay can be in principle adapted to any other phagocytic cell (dendritic cells, neutrophils), provided that they express fluorescently tagged proteins of interest. In addition, the method can be extended to other target particles like beads or bacteria. The size and shape of the different targets could also be varied to get further insight into the mechanism of phagosome formation and in particular the forces involved in phagosome closure. Following the localization of different molecular players in living phagocytosing cells using the phagosome closure assay described here opens avenues for better definition of the molecular mechanisms and machineries important for phagosome formation and closure.

吞噬作用是，随着识别和表面受体，其然后导致摄入材料的内化和降解材料的结合开始的主要细胞功能。而单细胞真核生物如模具粘菌和阿米巴使用吞噬的细菌喂食，高等生物进化与专业的细胞。巨噬细胞或树突状细胞是针对在各种组织和器官的病原体防御的第一线，并且通过抗原呈递和细胞因子的产生1-4激活适应性免疫系统的关键。在某些情况下吞噬作用可以由非专业的吞噬细胞， 例如，内皮细胞和上皮细胞中进行。这个过程是非常重要的开发过程中，并在成年后正常组织转化和重塑保持动态平衡。最后，专门吞噬细胞如支持细胞中的睾丸或视网膜色素上皮细胞是非常有效的吞噬细胞5。 一个吞噬体的形成，其中的微生物或细胞碎片的降解发生与吞噬受体的吞噬细胞的表面上的聚类开始。如Fc受体（FCR）或补体受体（CRS）以下调理受体集群下游信号事件已得到很好的特点。然而，也有许多非调理性受体，包括Toll样受体（TLRs），凝集素，甘露糖受体和清道夫受体。这些受体识别的粒子表面上的决定因素如甘露糖或岩藻糖残基，磷脂酰丝氨酸，和脂多糖1,6-9。 病原体或细胞碎片的识别包括结合和多种类型的吞噬受体，然后引起强烈的和短暂的肌动蛋白重塑的群集。在细胞内compartmen并行，焦胞吐TS有助于膜张力的释放而为大颗粒的有效细胞吞噬重要。导致肌动蛋白聚合和膜变形的信号事件在实验模型触发一个吞噬受体进行了解剖。期间的FcR介导的吞噬作用，存在由小GTP酶（外消旋，Cdc42的）调节的强烈的肌动蛋白聚合。在他们的下游效应，所述Wiskott-Aldrich公司综合征蛋白（WASP）导致了成核肌动蛋白微丝1,2,4,10的肌动蛋白相关蛋白2/3复合体（的Arp2 / 3）的激活。当地生产磷脂4,5二磷酸（PI（4,5）P 2）的为驱动伪足形成初始肌动蛋白聚合的关键。其转化为PI（3,4,5）P 3需要伪足扩展和吞噬体封闭11。几个途径有助于PI（4,5）P2的消失。首先磷脂酰肌醇磷酸基那支队SES（PIPKIs）从吞噬逮捕PI（4,5）P2的合成。其次，它可以被磷酸化并通过类消耗我PI3K激酶（PI3K）和PI（3,4,5）P 3 12转换。一种用于磷酸酶和磷脂酶作用也已在PI（4,5）P2水解和F-肌动蛋白去除在哺乳动物细胞和在盘基网柄 13,14吞噬期间暗示。磷脂酶C（PLC）δ水解的PI（4,5）P 2为二酰基甘油和肌醇1,4,5- 三磷酸盐。该PI（4,5）P2和PI（3,4,5）P 3磷酸OCRL（罗威oculocerebrorenal综合征）也被牵连的吞噬小体的形成。 F-肌动蛋白和其解聚的精确局部形成在空间和时间被严格调控，我们已经表明细胞内隔室的招募到本地传递OCRL磷酸酶，从而在吞噬杯的底部有助于当地肌动蛋白解聚是重要<sup&gt; 13,15。对于这一点，我们使用这里所描述的实验设置的。 对吞噬体闭合和膜断裂所需的机制和分子玩家仍定义不清，因为在可视化和监测吞噬体封闭的部位的困难。直到最近，观察到在该上其背面或在其两侧内在粒子固定或活细胞的吞噬能力，​​使得吞噬体闭合困难的部位的及时可视化。此外，固定方法可能会导致膜和偏见的伪足延伸和闭合的结果回缩。相比之下，我们已经建立并在这里描述的实验使我们能够可视化伪足延伸，并在活细胞13，吞噬关闭一步基于全内反射显微镜（TIRFM）16。该光学技术使用渐逝波在一个透明的界面，以激发在薄区域的荧光团，以便盖（盖玻片）和液体（细胞培养基）。激发深度的厚度为约100纳米的从固体表面，从而允许接近质膜分子事件的可视化。 TIRFM允许高的信号 - 背景比率，并且限制所收集的失焦荧光和细胞毒性由于细胞的照明。 趁着TIRFM，我们开发了"吞噬体闭合测定法"，其中盖玻片用聚赖氨酸活化，然后涂上的IgG调理的红血细胞（IgG的SRBCs）。然后表达的兴趣瞬时荧光标记蛋白的巨噬细胞被允许吞没的IgG-SRBCs。而细胞分离该非共价结合于玻璃表面上的目标粒子，所述伪足的尖端可观察并记录在TIRF模式。 TIRF收购相结合，与在落射荧光模式采集，移位上述阶段3微米，这允许相后吞噬杯的基部的ualization。的药理学药物，例如那些在此过程中抑制肌动蛋白或dynamin上加成，也可以在分子水平上，以进一步解剖过程。 在这里详细描述的协议为RAW264.7小鼠巨噬细胞系和调理粒子，但实际上，它可以适用于任何其他吞噬细胞，并与其他目标，如珠。这种方法将使监管更好的表征在时间和在各种吞噬过程中涉及伪足延伸和吞噬封闭分子球员空间。

<table><tbody><tr><td>Anti-sheep red blood cells IgG</td><td>MP Biomedicals</td><td>55806</td><td></td></tr><tr><td>Bovine Serum Albumin heat shock fraction, pH 7, &amp;ge;98%&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cuvettes 4 mm</td><td>Cell project</td><td>EP104</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo Fischer Scientific</td><td>14190-094</td><td>Room temperature</td></tr><tr><td>Electrobuffer kit</td><td>Cell project</td><td>EB110</td><td></td></tr><tr><td>100 mm TC-Treated Cell Culture Dish</td><td>Corning</td><td>353003</td><td></td></tr><tr><td>Gene X-cell pulser</td><td>Biorad</td><td>165-2661</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1397</td><td></td></tr><tr><td>Glass Bottom Dishes 35 mm uncoated 1.5</td><td>MatTek corporation</td><td>P35G-1.5-14-C Case</td><td></td></tr><tr><td>iMIC</td><td>TILL Photonics</td><td></td><td>Oil-immersion objective (N 100X, NA1.49), heating chamber with CO&lt;sub&gt;2&lt;/sub&gt;, a camera single photon detection EMCCD (Electron Multiplying Charge Coupled Device) and a 1.5X lens</td></tr><tr><td>Poly-L-Lysine Solution 0.1%</td><td>Sigma</td><td>P8920-100ml</td><td>Dilution at 0.01% in water&amp;nbsp;</td></tr><tr><td>RPMI 1640 medium GLUTAMAX&amp;nbsp; Supplement</td><td>Life technologies</td><td>61870-010</td><td></td></tr><tr><td>RPMI 1640 medium, no phenol red (10 x 500 ml)</td><td>Life technologies</td><td>11835-105</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Sheep red blood cells (SRBCs)</td><td>Eurobio</td><td>DSGMTN00-0Q</td><td>Conserved in Alsever buffer at 4 &amp;deg;C before use</td></tr></tbody></table>

"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.

在这个手稿所述的实验系统在图1中示意性地表示。表达融合于荧光标签目的蛋白质转染RAW264.7巨噬细胞被置于接触的IgG调理的绵羊红细胞（SRBCs），该是非共价地固定上盖玻片。巨噬细胞可以从盖玻片分离SRBC吞噬它。所使用的TIRF显微镜允许伴随采集从TIRF区域对应于萤光模式的伪足和信号的尖端一个Z偏移大于3微米后的信号。 如在图2中这确保从质膜低于100nm的区域的清洁的TIRF信号描述有必要确定用于全内反射荧光的临界角。 图3表示acquisiti的发展通过一个被称为"协议编辑器"模块参数包含在现场采集软件。该模块允许用户创建，他们被送到显微镜，将执行过程之前管理工作流。在收购结束时，用户收集了TIFF流。 如图4所示，RAW264.7中的巨噬细胞"吞噬体封闭法"与质粒（ 如 Lifeact-mCherry，纪尧姆蒙塔尼亚克博士，居里研究所，巴黎的一种礼物，之后转产生的代表活细胞TIRFM电影17）按照肌动蛋白聚合。巨噬细胞瞬时表达质粒被允许吞没绑定到盖玻片IgG的SRBCs。作为伪足的前端分别结合在玻璃盖玻片围绕一个SRBC，在TIRF区域中检测到F-肌动蛋白环（顶面板），该逐步变窄，直到其关闭。并行地，模糊F-肌动蛋白移上述（底部面板）对应于解聚在吞噬杯的底部的阶段为3μm后通过荧光信号来检测。采集3分钟后，将SRBC被完全内化，如用透射光（面板上的右侧）确认。 因此，这种方法可以被用于正确区分发生在伪足的非常末端并在吞噬杯的底部发生的分子事件的分子事件。 <img alt="图1" src="/files/ftp_upload/54470/54470fig1.jpg" /> 图1."吞噬体封闭测定"通过TIRFM分析的示意图 。吞噬体闭合测定是使用巨噬细胞瞬时表达一种或两种荧光标记的蛋白进行的。巨噬细胞上沉积的IgG调理的SRBCs非共价地固定在多聚赖氨酸COA特德盖玻片。影像会记录在TIRF模式检测萤光模式吞噬体封闭和现场检测吞噬杯的底部。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54470/54470fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54470/54470fig2.jpg" /> 图2:TIRFM的临界角的测定。 （A）使用"实时采集"，表达荧光标记的蛋白质的细胞被置于场（区1中红色）的中间。与TIRF堆栈选项，图像在一个激励波长被收购，以不同的角度从0°开始最多5°，0.01°（在红色区域2）的增量。（B）中的图像的序列是使用ImageJ打开色彩管理软件和AR平均荧光强度感兴趣egion（ROI）的曲线，显示使用"栈"和"PlotZ轴轮廓"（红色区域1）的x轴的角度的函数。（C）的荧光的峰值在图上的x位置对应于临界角:1.98°（黑色虚线）。可以用这个角度后的任何价值。作为一个例子，2.00°可以选择（红色线）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54470/54470fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54470/54470fig3.jpg" /> 图3. 使用实时采集模块的工作流过程:"协议编辑器"。 （一）在"议定书编辑器"窗口，创建工作流画布。（B）该协议包括的行动，该显微镜将决定重复的次数"循环"由用户。作为一个例子:750（1）。一个循环包括:"多渠道"收购与在TIRF模式的兴趣荧光蛋白的激光激发。作为例子:激光波长491强50％-TIRF角度2.00（2）;客观的"Z移动"3微米（3）; "多渠道"收购落射荧光模式（4）客观的"Z移动"回TIRF区域（5）和一个"快照"，在透射光（6）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54470/54470fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54470/54470fig4.jpg" /> 图4. F-肌动蛋白积累截至吞噬体封闭。封闭吞噬体实验的网站使用点 RAW264.7细胞瞬时表达质粒Lifeact-mCherry（纪尧姆莫博士惠赠进行ntagnac，居里研究所，巴黎，17后生成）。质粒信号在TIRF区域（顶部）和在表面荧光模式（底部）获得的。红色箭头表示在TIRF区域肌动蛋白的积累。在3分钟透射光图像呈现，表明内在SRBC。比例尺，10微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54470/54470fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="电影1" src="/files/ftp_upload/54470/54470mov1.jpg" /> 电影1:F-肌动蛋白的累积在伪足的提示和在吞噬体封闭的站点，而这是使用的RAW264.7巨噬细胞瞬时表达的质粒进行吞噬杯吞噬体闭合测定法的基本清除。使用TIRFM在TIRF区域（顶部）的质粒成像和在表面荧光模式（底部）进行替代立法院在37℃3分钟内每50毫秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54470/Movie1.mp4" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM at JoVE.com

"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 ...

phagosome-closure-assay-to-visualize-phagosome-formation-three

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

6.4K 次观看.  Université Paris Descartes, Sorbonne Paris Cité. 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.

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

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 ...

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 ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.

Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

生物学

Visualizing the Early Stages of Phagocytosis

The mammalian body is equipped with various layers of mechanisms that help to defend itself from pathogen invasions. Professional phagocytes of the immune system &#8212; such as neutrophils, dendritic cells, and macrophages &#8212; retain the innate ability to detect and clear such invading pathogens through phagocytosis1. Phagocytosis involves choreographed events of membrane reorganization and actin remodeling at the cell surface2,3. Phagocytes successfully internalize and eradicate foreign molecules only when all stages of phagocytosis are fulfilled. These steps include recognition and binding of the pathogen by pattern recognition receptors (PRRs) residing at the cell surface, formation of phagocytic cup through actin-enriched membranous protrusions (pseudopods) to surround the particulate, and scission of the phagosome followed by phagolysosome maturation that results in the killing of the pathogen3,4.
Imaging and quantification of various stages of phagocytosis is instrumental for elucidating the molecular mechanisms of this cellular process. The present manuscript reports methods to study the different phases of phagocytosis. We describe a microscope-based approach to visualize and quantify the binding, phagocytic cup formation, and the internalization of particulate by phagocytes. As phagocytosis occurs when innate receptors on phagocytic cells encounter ligands on a target particle bigger than 0.5 &#181;m, the assays we present here comprise the use of pathogenic fungi Candida albicans and other particulates such as zymosan and IgG-coated beads.

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 ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

<meta charset="utf8"></head><body>活荧光、细胞褶皱的逆向成像和巨胞饮作用

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

活荧光、细胞褶皱的逆向成像和巨胞饮作用

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...

"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）

摘要

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由成比例的荧光显微镜测量吞噬体pH值

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

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

可视化吞噬的早期阶段

活荧光、细胞褶皱的逆向成像和巨胞饮作用

活荧光、细胞褶皱的逆向成像和巨胞饮作用