Name: "phagosome closure assay" to visualize phagosome formation in three dimensions using total internal reflection fluorescent microscopy (tirfm)
Uploaded: 2016-08-26T15:00:00.000+00:00
Duration: 10 min 7 s
Description: 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

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><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。 <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

Universidad de Cartagena UDEC

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

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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 ...

Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic Microtubules and Associated Proteins

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. Total-internal-reflection-fluorescence (TIRF) microscopy has a high signal-to-background ratio, but it suffers from photobleaching and photodamage of the fluorescent proteins. Label-free techniques such as interference reflection microscopy (IRM) and interferometric scattering microscopy (iSCAT) circumvent the problem of photobleaching but cannot readily visualize single molecules. This paper presents a protocol for combining IRM with a commercial TIRF microscope for the simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro. This protocol allows for high-speed observation of MAPs interacting with dynamic microtubules. This improves on existing two-color TIRF setups by eliminating both the need for microtubule labeling and the need for several additional optical components, such as a second excitation laser. Both channels are imaged on the same camera chip to avoid image registration and frame synchronization problems. This setup is demonstrated by visualizing single kinesin molecules walking on dynamic microtubules.

We present a protocol for implementing interference-reflection microscopy and total-internal-reflection-fluorescence microscopy for the simultaneous imaging of dynamic microtubules and fluorescently labeled microtubule-associated proteins.

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

Several techniques have been employed for the direct visualization of cytoskeletal filaments and their associated proteins. ...

生物化学

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

神经科学

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

生物工程

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 ...

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro&#160;microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an &#34;eat-me&#34; signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Neurodegenerative diseases are associated with dysregulated microglia functions. This article outlines an in vitro assay of phagocytosis of neuroblastoma cells by iPSC-macrophages. Quantitative microscopy readouts are described for both live-cell time-lapse imaging and fixed-cell high-content imaging.

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

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

TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Source: Marie-Ana&#239;s, F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54470">&#34;Phagosome Closure Assay&#34; to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM).</a> J. Vis. Exp. (2016).
This video demonstrates a high-resolution total internal reflection fluorescence (TIRF) microscopy technique for real-time visualization of phagosome formation and closure during macrophage-mediated phagocytosis of IgG-opsonized red blood cells (RBCs) attached to the surface of a glass bottom dish. The macrophages extend pseudopodia around the RBCs and engulf them inside the phagosomes, detaching them from the glass surface.

"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

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

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

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

小鼠巨噬细胞吞噬的延时3D 成像

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

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

基于 TIRF 显微镜的吞噬体形成和闭合可视化

"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

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

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

同步干涉反射和全内反射荧光显微镜，用于动态微管和相关蛋白质的成像

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

小鼠巨噬细胞吞噬的延时3D 成像

iPSC-巨噬细胞死亡神经母细胞的体外定量成像检测

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

基于 TIRF 显微镜的吞噬体形成和闭合可视化

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术