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>

<ol>
	<li>Flannagan, R. S., Jaumouille, V., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+phagocytosis.">The cell biology of phagocytosis.</a> Ann Rev Pathol. 7, 61-98 (2012).</li><li>Swanson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+cups+into+phagosomes+and+macropinosomes.">Shaping cups into phagosomes and macropinosomes.</a> Nat Rev Mol Cell Biol. 9, 639-649 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+comparative+innate+immunity:+learning+on+the+fly.">Phagocytosis and comparative innate immunity: learning on the fly.</a> Nat Rev Immunol. 8, 131-141 (2008).</li><li>Niedergang, F., Bradshaw, R. A., Stahl, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Cell Biology. 2, 751-757 (2016).</li><li>Poon, I. K., Lucas, C. D., Rossi, A. G., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+clearance:+basic+biology+and+therapeutic+potential.">Apoptotic cell clearance: basic biology and therapeutic potential.</a> Nat Rev Immunol. 14, 166-180 (2014).</li><li>Aderem, A., Underhill, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+phagocytosis+in+macrophages.">Mechanisms of phagocytosis in macrophages.</a> Annu. Rev. Immunol. 17, 593-623 (1999).</li><li>Underhill, D. M., Goodridge, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+during+phagocytosis.">Information processing during phagocytosis.</a> Nat Rev Immunol. 12, 492-502 (2012).</li><li>Underhill, D. M., Ozinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+microbes:+complexity+in+action.">Phagocytosis of microbes: complexity in action.</a> Annu Rev Immunol. 20, 825-852 (2002).</li><li>Canton, J., Neculai, D., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scavenger+receptors+in+homeostasis+and+immunity.">Scavenger receptors in homeostasis and immunity.</a> Nat Rev Immunol. 13, 621-634 (2013).</li><li>Freeman, S. A., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+receptors,+signal+integration,+and+the+cytoskeleton.">Phagocytosis: receptors, signal integration, and the cytoskeleton.</a> Immunol Rev. 262, 193-215 (2014).</li><li>Scott, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4,5-bisphosphate+hydrolysis+directs+actin+remodeling+during+phagocytosis.">Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis.</a> J Cell Biol. 169, 139-149 (2005).</li><li>Schlam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase+enables+phagocytosis+of+large+particles+by+terminating+actin+assembly+through+Rac/Cdc42+GTPase-activating+proteins.">Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins.</a> Nat Commun. 6, 8623 (2015).</li><li>Marion, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF-kappaB+Signaling+Protein+Bcl10+Regulates+Actin+Dynamics+by+Controlling+AP1+and+OCRL-Bearing+Vesicles.">The NF-kappaB Signaling Protein Bcl10 Regulates Actin Dynamics by Controlling AP1 and OCRL-Bearing Vesicles.</a> Dev Cell. 23, 954-967 (2012).</li><li>Loovers, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+phagocytosis+in+Dictyostelium+by+the+inositol+5-phosphatase+OCRL+homolog+Dd5P4.">Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4.</a> Traffic. 8, 618-628 (2007).</li><li>Deschamps, C., Echard, A., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+and+cytokinesis:+do+cells+use+common+tools+to+cut+and+to+eat?+Highlights+on+common+themes+and+differences.">Phagocytosis and cytokinesis: do cells use common tools to cut and to eat? Highlights on common themes and differences.</a> Traffic. 14, 355-364 (2013).</li><li>Johnson, D. S., Jaiswal, J. K., Simon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12,+Unit+12.29:+Total+internal+reflection+fluorescence+(TIRF)+microscopy+illuminator+for+improved+imaging+of+cell+surface+events.">Chapter 12, Unit 12.29: Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events.</a> Curr Protoc Cytom. , (2012).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Marie-Anais, F., Mazzolini, J., Herit, F., Niedergang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin-actin+cross-talk+contributes+to+phagosome+formation+and+closure.">Dynamin-actin cross-talk contributes to phagosome formation and closure.</a> Traffic. 17 (5), 487-499 (2016).</li></ol>

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 notice...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, &#8805;98%&#160;</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 4mm</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>100mm 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&#160;</td>
			<td>TILL Photonics</td>
			<td></td>
			<td>&#160;Oil-immersion objective&#160;(N 100x, NA1.49.),&#160; heating chamber with CO2, 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&#160;</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium GLUTAMAX&#160; Supplement</td>
			<td>Life technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, no phenol red (10x500 ml)</td>
			<td>Life technologies</td>
			<td>11835-105</td>
			<td>Warm in 37&#176;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&#176;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。 <p class="jove_content" fo:keep-together....

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

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

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

免疫与感染

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

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

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

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

使用双光子显微镜在受感染的肺部的嗜中性粒细胞吞噬和NET形成的直接观察

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

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

利用荧光蛋白可视化和定量<em&gt;衣原体</em&gt;液泡生长动态的活细胞

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

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 ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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 ...

"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

探索更多视频

此视频中的章节