这项工作是由国家法新社​​倒拉RECHERCHE由欧洲研究理事会。

检测工作在各种不同的格式，但是我们建议缩减样品的体积，节省试剂，特别是CCF4-AM。因此，下面的协议被描述为固定的HeLa细胞或THP-1巨噬细胞样细胞在96孔格式，然后活的HeLa细胞在35毫米的玻璃底菜。该试剂盒可以适用于其他病原体。除了​​志贺氏菌感染，我们描述了检测THP-1细胞吞噬不同M Y cobacterial株。 重要的是，包括所有的试剂和缓冲液，洗涤缓冲液，CCF4-AM装载后需要丙磺舒的存在下，在1 mM。丙磺舒是一种阴离子通道的抑制剂促进胞浆保留CCF4的协议的各个步骤。应该获取固定的样品在实验后的几个小时内，因为的CCF4信号的长的时间周期并不稳定。 1。 CCF4含量固定的样品使用痢疾flexneri（96孔板） <ol><li>准备过夜培养的细菌。接种细菌培养（M90T AFAI）野生型和突变株（BS176 AFAI）8毫升的胰蛋白酶的酪蛋白大豆肉汤（TCSB），的补充了氨苄青霉素（50微克/毫升）中，在摇床上于37℃培养过夜感染前1天。 </li><li>电镀感染细胞。种子HeLa细胞感染前1天在96孔板的密度为5×10 3个细胞每孔，在含10％胎儿小牛血清（FCS）和1％青霉素-链霉素，在5％CO 2培养箱中 ，终体积为100μl。 THP-1巨噬细胞样细胞的情况下，完成细胞接种密度为5×10 4个细胞/孔感染前2天，和30 nM的佛波醇肉豆蔻酸乙酸酯（PMA）的含10％的RPMI培养胎牛血清（FCS），1％青霉素 - 链霉素和0.05 mM的2 - 巯基乙醇。 </li><li>准备细菌的亚文化。接种过夜细菌培养在TCSB补充了50μg/ ml的氨苄青霉素的摇床中于37℃下2.5小时（OD600 = 0.3〜0.4）1/100稀释的。 </li><li>载入细胞CCF4-AM。准备CCF4-AM装载混合，终体积为25微升每孔含有1 mM的丙磺舒（Sigma公司），1.5μMCCF4-AM（6μM在THP-1的情况下）和1.25微升加载溶液B（100毫克/毫升聚醚F127表面活性剂在醋酸DMSO/0.1％CCF4-AM LiveBlazer装载套件一起提供Invitrogen公司）在EM缓冲液（120 mM氯化钠，氯化钾7毫米，1.8毫米氯化钙 ， 氯化镁 0.8毫米，5毫米葡萄糖，的25mM HEPES，在pH为7.3）。一次，用PBS清洗细胞，CCF4-AM装载2.5小时，在黑暗中在室温下混合，每孔中加入25μl。 </li><li>准备细菌感染。自旋为1毫升细菌的亚文化，以9,000 rpm的转速下，持续1分钟。洗一次用500μlPBS中，自旋在每分钟9000转的1分钟，补充有10微克/毫升多聚-L-赖氨酸（Sigma公司）和40μg/ ml的β-内酰胺于500μlPBS重悬马斯（Sigma公司）。一个旋转的轮子上在室温下温育10分钟后，洗一次，用500μl的PBS重悬细菌于500μl的EM缓冲液/ 1mM丙磺舒的。 </li><li>细胞感染和固定。洗细胞一次，加入150μl的PBS / 1 mM的丙磺舒，制备最终体积为100μl的EM缓冲液/ 1mM丙磺舒的细菌重新悬浮于10微升混合，并分发到各孔中。在黑暗中温育15分钟后，在室温下，切换板到37℃，1小时（90分钟的THP-1的情况下），用150微升PBS / 1 mM的丙磺舒洗和修复用50μl多聚甲醛4％ / 1毫米丙磺舒，在黑暗中10分钟。然后，用150微升PBS / 1毫米丙磺舒，30微升10μM的细胞核染料DRAQ5（Biostatus）孵育30分钟，用150微升PBS / 1毫米丙磺舒，并留下样品100μlPBS中，丙磺舒/ 1毫米。 </li><li>固定样本的采集设置。使用（一）倒落射荧光显微镜进行收购配合20倍（0.5的数值孔径（NA）2.1工作距离（WD））或40倍（0.75，0.72 NA WD）N-计划的空中目标或（ii）利用共聚焦显微镜用10倍的目标。进行荧光成像，激发波长为405纳米（Semrock FF01-387/11-25）和排放检测（Semrock FF02-447/60-25）通过450纳米和535纳米的过滤器（Semrock，FF01-520/35- 25）使用曝光时间5毫秒（透射光），1000毫秒（535纳米）和500毫秒（450纳米）。使用下面的曝光时间:240毫秒（450纳米）和360毫秒（535纳米）的405 nm激光（870μW），640毫秒（DRAQ5）（2560μW）的640 nm激光共聚焦成像。 </li><li>收购后分析。随后，图像分析的计算机算法，可以为每个单独的细胞的荧光信号的自动评分。的MetaMorph 7.1或阿卡贝拉软件，比如可以用来创建一个脚本用于测量450nm和535nm的发射信号之间的比例（可根据要求提供）。值得注意的是，在收购耦合到自动显微镜的焦点校准装置的使用可以同时获得几十个不同位置的多口井。 </li></ol> 2。 CCF4化验现场样品痢疾杆菌 （35毫米玻璃底格式，MatTek） <ol><li>准备过夜培养的细菌。如前面所述继续操作。 </li><li>电镀感染细胞。种子的HeLa细胞在35毫米的玻璃底培养皿（10 MatTek毫米微孔，团MatTek），感染1天之前的密度为2×10 5个细胞/皿，终体积为2ml。 </li><li>准备细菌的亚文化。如前面所述继续操作。 </li><li>载入细胞CCF4-AM。准备加载CCF-AM终体积为2毫升，每盘混合如前所述。在安装之前，一次用PBS洗细胞。 </li><li>准备细菌感染。如前面所述继续操作。 </li><li>本收现场样品离子的设置。收购执行时间为60分钟，每90秒倒落射荧光显微镜使用20倍（0.5 NA，2.1 WD）或37℃加热室内部的40倍（0.75 NA 0.72 WD）N-计划的空中目标。进行荧光成像，激发波长为405纳米（Semrock FF01-387/11-25）和排放检测（Semrock FF02-447/60-25）通过450纳米和535纳米的过滤器（Semrock，FF01-520/35- 25）使用曝光时间5毫秒（透射光），200毫秒（535纳米）和100毫秒（450纳米）。 </li><li>细胞感染和现场采集。一次洗涤细胞2毫升PBS / 1毫米丙磺舒，加2毫升EM /丙磺舒1毫米，在舞台上的显微镜安装盘，开始采集。 6分钟后（时间4点），把收购搁置，加入250μl细菌重悬细胞上，然后重新启动收购。 </li><li>收购后分析。电影可以是可视化用的MetaMorph或ImageJ的。 450/535 nm的强度比每个单个双电池可以使用ImageJ获得。如前所述，在收购耦合到自动显微镜的焦点校准装置使用，允许根据时间推移获得多个不同的位置。 </li></ol> 3。 CCF4含量固定的样品使用 分枝杆菌（96孔格式） <ol><li>准备细菌。增长 中旬日志相分枝杆菌菌株在7H9含有白蛋白葡萄糖过氧化氢 ​​酶（ADC），在30°C（ 分枝杆菌 ）或37℃（ 卡介苗和结核分枝杆菌）。 </li><li>电镀感染细胞。铠THP-1巨噬细胞在感染前2天培养与PMA的RPMI 30 nM的含10％胎牛血清（FCS），1％青霉素-链霉素和0.05 mM的2 -巯基乙醇中，在5％CO 2培养箱中 ，终体积为加入100μl5×10 4个细胞/孔的密度。 </li><li>准备细菌感染。衡丰东部时间培养，通过注射器之前，超声处理，过滤，用PBS洗涤，重悬，以避免结块。确定各菌株的浓度通过OD600测量和感染THP-1细胞以MOI为1:1，在30℃（ 分枝杆菌 ）或37℃（适用于牛分枝杆菌 BCG和结核分枝杆菌）的RPMI培养基中，用5％的的CO 2。 2小时后，去除培养基，用PBS洗3次，并添加完整的新鲜培养基为1〜2天（ 分枝杆菌的情况下），或者3〜7天（ 卡介苗和结核分枝杆菌的情况下）。 </li><li>中CCF4-AM和固定细胞。一次，用PBS清洗细胞，并准备CCF4-AM装载混合像先前描述的最终体积为每孔25微升，使用CCF4-AM的最终浓度为6μM。载入中发生在室温下在黑暗中前2小时，洗涤150微升PBS / 1毫米丙磺舒和修复通报BULLETIN使用50微升4％多聚甲醛丙磺舒30分钟，在黑暗补充户型1毫米。然后，用150微升PBS / 1毫米丙磺舒，并留下样品100μlPBS中，丙磺舒/ 1毫米。 </li><li>收购的设置和采集后的分析。按如前所述，在第一段使用痢疾杆菌 。 </li></ol>补充协议荧光法进行，以检查在表面的细菌的β-内酰胺酶的活性。这项工作应为每个所描述的测定中使用的细菌菌株。为此目的，洗涤后的细菌接触，用100nM的CCF4-AM（Invitrogen公司），50微克/毫升猪酯酶肝提取物的（σ）在1ml的PBS中，在37℃1小时，在黑暗中。可溶性内酰胺酶的1毫克/毫升（Invitrogen公司），可以用来作为阳性对照。然后，发射在405nm的激发使用PTI Quantamaster的荧光计和1毫升的夸脱执行扫描Ž比色皿。 FRET的损失在535 nm和450nm的峰值高的外观的可视化后，细菌除了CCF4探头。

<ol>
	<li>van der Goot, F. G., Gruenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-endosomal+membrane+traffic.">Intra-endosomal membrane traffic.</a> Trends Cell Biol. 16, 514-521 (2006).</li><li>Raposo, G., Marks, M. S., Cutler, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosome-related+organelles:+driving+post-Golgi+compartments+into+specialisation.">Lysosome-related organelles: driving post-Golgi compartments into specialisation.</a> Curr. Opin. Cell Biol. 19, 394-401 (2007).</li><li>van der Wel, N., 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=M.+leprae+translocate+from+the+phagolysosome+to+the+cytosol+in+myeloid+cells.">M. leprae translocate from the phagolysosome to the cytosol in myeloid cells.</a> Cell. 129, 1287-1298 (2007).</li><li>Sansonetti, P. J., Ryter, A., Clerc, P., Maurelli, A. T., Mounier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplication+of+Shigella+flexneri+within+HeLa+cells:+lysis+of+the+phagocytic+vacuole+and+plasmid-mediated+contact+hemolysis.">Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis.</a> Infect Immun. 51, 461-469 (1986).</li><li>Bobard, A., Mellouk, N., Enninga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spotting+the+right+location-+imaging+approaches+to+resolve+the+intracellular+localization+of+invasive+pathogens.">Spotting the right location- imaging approaches to resolve the intracellular localization of invasive pathogens.</a> Biochim. Biophys. Acta. 1810, 297-307 (2011).</li><li>Beauregard, K. E., Lee, K. D., Collier, R. J., 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=pH-dependent+perforation+of+macrophage+phagosomes+by+listeriolysin+O+from+Listeria+monocytogenes.">pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes.</a> J. Exp. Med. 186, 1159-1163 (1997).</li><li>Shaughnessy, L. M., Hoppe, A. D., Christensen, K. A., 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=Membrane+perforations+inhibit+lysosome+fusion+by+altering+pH+and+calcium+in+Listeria+monocytogenes+vacuoles.">Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles.</a> Cell Microbiol. 8, 781-792 (2006).</li><li>Zlokarnik, G., 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=Quantitation+of+transcription+and+clonal+selection+of+single+living+cells+with+beta-lactamase+as+reporter.">Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter.</a> Science. 279, 84-88 (1998).</li><li>Charpentier, X., Oswald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+secretion+and+translocation+domain+of+the+enteropathogenic+and+enterohemorrhagic+Escherichia+coli+effector+Cif,+using+TEM-1+beta-lactamase+as+a+new+fluorescence-based+reporter.">Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter.</a> J. Bacteriol. 186, 5486-5495 (2004).</li><li>Mills, E., Baruch, K., Charpentier, X., Kobi, S., Rosenshine, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+effector+translocation+by+the+type+III+secretion+system+of+enteropathogenic+Escherichia+coli.">Real-time analysis of effector translocation by the type III secretion system of enteropathogenic Escherichia coli.</a> Cell Host Microbe. 3, 104-113 (2008).</li><li>Bish, S. E., Song, W., Stein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+bacterial+internalization+by+host+cells+using+a+beta-lactamase+reporter+strain:+Neisseria+gonorrhoeae+invasion+into+cervical+epithelial+cells+requires+bacterial+viability.">Quantification of bacterial internalization by host cells using a beta-lactamase reporter strain: Neisseria gonorrhoeae invasion into cervical epithelial cells requires bacterial viability.</a> Microbes Infect. 10, 1182-1191 (2008).</li><li>Ray, K., 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=Tracking+the+dynamic+interplay+between+bacterial+and+host+factors+during+pathogen-induced+vacuole+rupture+in+real+time.">Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time.</a> Cell Microbiol. 12, 545-556 (2010).</li><li>Blocker, A., 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+tripartite+type+III+secreton+of+Shigella+flexneri+inserts+IpaB+and+IpaC+into+host+membranes.">The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes.</a> J. Cell Biol. 147, 683-693 (1999).</li><li>Mounier, 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=The+IpaC+carboxyterminal+effector+domain+mediates+Src-dependent+actin+polymerization+during+Shigella+invasion+of+epithelial+cells.">The IpaC carboxyterminal effector domain mediates Src-dependent actin polymerization during Shigella invasion of epithelial cells.</a> PLoS Pathog. 5, e1000271 (2009).</li><li>Nowicki, B., Hart, A., Coyne, K. E., Lublin, D. M., Nowicki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+consensus+repeat-3+domain+of+recombinant+decay-accelerating+factor+is+recognized+by+Escherichia+coli+recombinant+Dr+adhesin+in+a+model+of+a+cell-cell+interaction.">Short consensus repeat-3 domain of recombinant decay-accelerating factor is recognized by Escherichia coli recombinant Dr adhesin in a model of a cell-cell interaction.</a> The Journal of Experimental Medicine. 178, 2115-2121 (1993).</li><li>Nothelfer, K., Dias Rodrigues, C., Bobard, A., Phalipon, A., Enninga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Shigella+flexneri+vacuolar+escape+by+flow+cytometry.">Monitoring Shigella flexneri vacuolar escape by flow cytometry.</a> Virulence. 2, 54-57 (2011).</li><li>Simeone, R., 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=Phagosomal+rupture+by+Mycobacterium+tuberculosis+results+in+toxicity+and+host+cell+death.">Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death.</a> PLoS Pathog. 8, e1002507 (2012).</li></ol>

我们什么都没有透露。

的CCF4-AM/β-lactamase检测是一个简单的方法来跟踪诱导细胞内痢疾杆菌和分枝杆菌在不同类型的细胞的液泡中断。它利用一内酰胺酶敏感的细胞质FRET记者活跃的表面上的细菌的酶裂解。 CCF4-AM基板的亏损是很容易避免加入丙磺舒所有的解决方案后，装载的基板。正如所演示的那样，该法可以适应多种类型的细胞（上皮细胞，吞噬细胞）和格式（96孔，35毫米的玻璃底菜，6/12/24孔板）。对于6/12/24孔板的使用，，无菌盖玻片分布在播种前的细胞的各孔的底部。在实验结束时，在载玻片上转移盖玻片安装介质，如延长黄金抗淬灭试剂（Invitrogen公司）。通过这种方式，信号是稳定的较长的时间周期后，测定相比，固定后的样品保存于PBS中的96孔格式。的CCF4-AM基板的成本高，应考虑之前确定的样本量。这就是为什么我们建议缩放下来。实验6/12/24以及格式更昂贵，但信号是稳定的天。与此相反，实验是在96孔格式的便宜，但在实验当天，将要分析的样品。值得注意的是，在96孔或384孔的格式也可以进行现场实验可以。这使得在同一时间（突变的细菌，内政部，质粒或转染，化学物质等）的使用条件下的几十个数字每口井的位置，进行现场实验。虽然适用于痢疾杆菌 ，我们强调的是"真正的"实时或延时实验分枝杆菌的研究是不可行的，因为感染周期超过CCF4可测量的浓度可保留在细胞质。为这个原因，CCF4-AM的细胞基板上涂敷后，才实现入侵。 MetaMorph软件用于采集和分析，我们建议使用该模块"屏幕收购"，允许获得一个完整的96/384孔板指定数目的每口井的照片。此外，该模块的"审查屏幕数据"允许（一）可视缝合每个图片马赛克以及任何通道的同时上了一个大的"大字报"及（ii）循环测量强度在535和一个专门的算法450纳米通道。虽然，我们已经由单一的细菌，使用时间推移显微镜能够测量空泡破裂，我们想提醒说，表面上单个细菌酶的活性变化使其难以精确关联胞内细菌的数量和有效性空泡破裂。 由于检测的鲁棒性，它是适合的hIGH吞吐量接近96或384孔的格式。我们还成功地适应这个协议流式细胞仪分析研究感染的细胞悬浮液中16。该试剂盒也可以用于为调查其他细菌或载体，在其表面上呈现内酰胺酶，导致广泛的应用。例如，这种方法可以用来研究空泡破裂引起的其他病原体，如β-内酰胺酶的表达嗜肺军团菌 ， 李斯特菌或鼠伤寒沙门氏菌 12。由于单核细胞增生李斯特氏菌具有与痢疾杆菌的感染周期短，时间推移实验是可能的。相比之下，因为嗜肺军团菌和鼠伤寒沙门氏菌显示感染周期长，我们建议进行终点实验。 的各种可能的应用使得CCF4-AM/β-lactamase的测定有趣的荧光法用于跟踪固定样品或实时细胞内的病原体引起的空泡破裂。

在感染过程中，众多的细菌性病原体的真核细胞中的膜封闭的室内部。发生单元格输入或者通过专门的宿主细胞的吞噬作用，是用于结核分枝杆菌的情况下，被巨噬细胞摄取，或病原体积极诱导其典型的非吞噬细胞摄取到。病原体引起的吸收的情况下，例如福氏志贺菌 ，在其他细胞功能紧密地调控内膜造成细菌定位在核内体的隔室1,2之内的分选机械劫持到宿主胞质溶胶注入效应蛋白。随后， 志贺氏菌破坏封闭的膜导 ​​致的空泡破裂和干扰主机的膜贩运和避免传递到溶酶体的病原体胞浆访问。最近，phagolysosomal破裂也被发现感染STRategy用于由结核杆菌 ，膜结合隔间3,17只局限在很长一段时间被认为病原体。 调查病原体侵入细胞内的膜贩运动态，已经取得了很大的改进，由于透射电子显微镜（TEM）的基础研究20世纪80年代后期4,5。例如，使用染料的荧光显微镜方法，对细菌表面成分，或与亚细胞定位标记的抗体接手6,7。然而，他们仍然没有得到精确的时空分辨率和鲁棒性由细菌病原体定量测量空泡破裂。 这一障碍已经解决与试验的基础上派生头孢CCF4-AM FRET记者，最初是用来研究基因表达8。然后，将其INF的上下文中使用调查挠度生物学效应的分泌，并按照奈瑟菌吸收到宿主细胞9,10,11。我们开发了一个检测研究空泡破裂引起的痢疾杆菌和结核杆菌 17本报记者趁着。我们的方法的原理是利用图1中描述痢疾杆菌 。首先，宿主细胞被加载与FRET CCF4-AM，被困住的细胞质内裂解后的AM酯部分的基板。然后，细胞与痢疾杆菌感染。的表面上存在的β-内酰胺酶的细菌是能够裂解尽快液泡破裂时，CCF4基板。这将导致切换从535 nm至450nm的发射峰在405nm处激发时探头的FRET信号的损失。 450/535 nm的强度的比例测量突出空泡完整性:低比率反映膜而高比例反映的封闭或胞菌的细菌和宿主细胞质之间的接触。我们也适应这种方法的研究由结核分枝杆菌引起的THP-1巨噬细胞的吞噬体破裂。该实验的原理是相同的，但顺序相反，只适用于细菌感染后CCF4-AM加载。 因此，在单细胞水平上通过的比例荧光定量测量， 痢疾杆菌空泡破裂可以实时跟踪和固定样本从不同类型的细胞12,13,14。此外，这种方法可适应所示，在本研究中使用牛分枝杆菌和结核分枝杆菌的一些其他病原体侵入。最后，我们的协议（96孔或384孔）格式的小型化，可在高吞吐量的许多条件的筛选。

<table><tbody><tr><td>LiveBlazer FRET-B/G Loading Kit (CCF4-AM) </td><td>Invitrogen</td><td>K1089</td><td>Protect from light, stock in - 80 &amp;deg; aliquots</td></tr><tr><td>Draq5</td><td>Biostatus</td><td>DR50050</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>&amp;mu;CLEAR-PLATE, BLACK, 96 well </td><td>Greiner Bio-One</td><td>655090</td><td></td></tr><tr><td>35 mm glass bottom dishes</td><td>MatTek corp.</td><td>P35G-1.5-10-C</td><td></td></tr><tr><td>Probenecid</td><td>Sigma</td><td>P8761</td><td></td></tr><tr><td>&amp;#946;-lactamase</td><td>Sigma</td><td>P0389</td><td></td></tr></tbody></table>

single cell measurements of vacuolar rupture caused by intracellular pathogens

痢疾杆菌是致病菌侵入宿主细胞的进入内吞液泡的。随后，该膜封闭隔室的破裂使细菌胞质溶胶内移动，增殖和，进一步侵入相邻小区。 结核分枝杆菌的免疫细胞吞噬，最近已被证明在巨噬细胞的吞噬体膜破裂。我们开发出一个强大的痢疾杆菌或结核杆菌进入宿主细胞的吞噬体膜破坏后的跟踪检测。方法利用CCF4，荧光共振能量转移，在宿主细胞的胞质溶胶中达到平衡的β-内酰胺酶敏感的记者。入侵的细菌病原体的宿主细胞后，探头保持不变，只要细菌驻留在膜封闭车厢。后的表面上的细胞内病原体劈开CCF4瞬间中断的液泡，β-内酰胺酶的活性，导致ING FRET信号的损失和开关其发射光谱。这一功能强大的比例测定产生细菌的侵入引起的液泡破裂的定时的准确信息，并且它可以被耦合到由专门的算法，用于检测发射信号的自动显微镜和图像处理FRET的供体和受体。此外，它允许调查液泡中断引起的细胞内细菌在单细胞实时动态。最后，它是非常适合高通量分析与时空分辨率，超过以前的方法。在这里，我们提供了实验示范协议的细节CCF4对HeLa细胞和THP-1巨噬细胞的液泡破裂试验时间推移实验或终点实验中使用的痢疾杆菌以及多分枝杆菌菌株，如分枝杆菌 ， 牛分枝杆菌，结核杆菌 。

CCF4-AM/β-lactamase方法是一个强大和敏感的方法跟踪空泡破裂，细胞内的病原体，如痢疾杆菌 （ 图1）。本研究中所使用的志贺氏菌菌株被称为M90T AFAI和BS176 AFAI。 M90T是一个AFAI 福氏志贺氏菌的菌株，表达黏附AfaE，这是能够有效地结合CD55的在表面上的上皮细胞15。因此的表达AfaE菌株显示M90T在上皮细胞株与野生型相比，更高的侵袭能力。 BS176 AFAI是表达突变型痢疾杆菌菌株没有志贺氏菌毒力质粒AfaE。该菌株是无法侵入HeLa细胞。然而，该菌株能够进入吸收在该细胞系的THP-1细胞，因为只依赖于古典的吞噬作用，并且不需要的功能3型分泌系统。这两个菌株表达β-内酰胺酶，以及显示在其表面上由于的的AfaE编码质粒的存在下β-内酰胺酶的活性。 HeLa细胞感染的非侵入性的BS176 AFAI菌株1小时后，CCF4 FRET探针保持完好，如在图2A中所示的绿色信号（535 nm）的。与此相反，毒性M90T AFAI应变1小时的感染导致的开关信号向蓝色（450nm处）突出在胞液中的探针的裂解。为了量化这种情况，我们开发了一个脚本的的MetaMorph阿卡佩拉的软件，可以让细胞自动检测，在535和450纳米通道的决心比例信号强度测量。正如在图2B中所示，细胞的细胞核和细胞质中的分割使用DRAQ5通道。然后，该算法能够为每个单独的电池，它是用于计算两个强度之间的比率检测450nm和535nm处的阳性细胞种群（以直方图形式表示在图2C）。低的比例，得到了与高比例的突变株毒株。虽然这代表性的感染实验使用共聚焦显微镜获得的，荧光显微镜也适合这项任务， 图3和图4示出了使用2个不同的人类细胞类型:HeLa细胞的上皮细胞和THP-1巨噬细胞样细胞的例子。毒性M90T AFAI 志贺氏菌菌株1小时和90分钟，HeLa细胞和THP-1细胞，绿色（535纳米），蓝色信号的开关感染后（450 nm）的观察相比，BS176 AFAI感染的细胞（ 图3A 4A）。的MetaMorph软件的脚本中，我们开发了直接检测CCF4阳性的细胞群体，并计算在450和535 nm的通道为每个单独的单元格的强度之间的比率。然后，它们的比值作为一个函数使用一个宏开发的细胞分类PED在Excel中，从而产生细胞分布的直方图显示。因为它已经开发的Acapella其他脚本的情况下，BS176感染AFAI的细胞比例低的特点，而M90T感染AFAI的细胞显示高比例的HeLa细胞和THP-1巨噬细胞（ 图3B和4B使用的MetaMorph算法） 。最后，在图5示出了该方法的适应分枝杆菌的研究是牛分枝杆菌 BCG的吞噬体驻留在整个实验过程中，在整个实验过程中检测到的强535 nm信号所反映的。相比之下， 结核分枝杆菌引发的THP-1巨噬细胞的吞噬体膜破裂后超过3天感染所强调的450 nm信号，在7天的感染（ 图5A和5B）。使用相同的算法由志贺氏菌研究空泡破裂</em&gt;的，我们发现结核分枝杆菌感染的细胞显示更高的450/535纳米比卡介苗后7天的感染（ 图5C，5D）。 <img alt="图1" fo:content-width="4in" fo:src="/files/ftp_upload/50116/50116fig1highres.jpg" src="/files/ftp_upload/50116/50116fig1.jpg" /> 图1。计划的原则的CCF4-AM/β-lactamase测定用于跟踪福氏志贺氏菌液泡破裂。CCF4-AM可自由扩散通过质膜进入细胞质CCF4阴离子通过细胞内酯酶的酯部分裂解掉。这种反应可以防止CCF4，进入任何膜嵌入式车厢。在这一步，CCF4引起在405nm处激发后，在535 nm处的荧光共振能量转移。探头保持不变，直到β-内酰胺酶的细菌表达rupturE为内吞泡。在此步骤中，FRET信号会丢失，因为CCF4裂解β-内酰胺酶在发射触发开关从535 nm至450nm，在405nm处激发时。 <img alt="图2" fo:content-width="2.25in" fo:src="/files/ftp_upload/50116/50116fig2highres.jpg" src="/files/ftp_upload/50116/50116fig2.jpg" /> 图2。跟踪福氏痢疾杆菌空泡破裂，利用共聚焦显微镜（A）CCF4-AM负载2小时30分钟后，HeLa细胞与β-内酰胺酶的表达痢疾杆菌 BS176 AFAI突变株或1小时，固定M90T AFAI毒株感染用4％多聚甲醛固定10分钟。然后，核染色与DRAQ5和细胞成像利用共聚焦显微镜用10倍的目标。具有代表性的图片，选择下面合并的渠道:完整CCF4探头出现在535纳米（格力n）时，切割的CCF4探头出现在450nm处（蓝色）（B）的实施例的图片的突出我们的自动算法上阿卡佩拉软件的检测系统。分割获得的细胞（细胞核+细胞质）使用DRAQ5通道。 CCF4阳性细胞是使用450和535 nm的渠道汇集起来的（C）直方图显示的结果，我们的自动分析阿卡贝拉痢疾杆菌 BS176 AFAI突变株M90T AFAI毒株。平均比率指之间的强度在450和535 nm的通道。 <a href="https://www.jove.com/files/ftp_upload/50116/50116fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="4.5in" fo:src="/files/ftp_upload/50116/50116fig3highres.jpg" src="/files/ftp_upload/50116/50116fig3.jpg" /> 图3。跟踪痢疾FLECCF4-AM负载2小时30分钟后xneri空泡破裂在HeLa细胞中使用荧光显微镜（A），HeLa细胞与β-内酰胺酶的表达痢疾杆菌 BS176 AFAI突变株或1小时，M90T AFAI毒株感染用4％多聚甲醛固定10分钟。然后，细胞使用的落射荧光显微镜用20倍的目标成像。图片被选为代表（B）有以下途径:CCF4完整的探头535 nm（绿色）和CCF4裂解探头450纳米（蓝色）。MetaMorph软件自动分析结果的直方图。的各个细胞的分布的函数，它们的比值的强度在450和535 nm的通道。 <img alt="图4" fo:content-width="4.5in" fo:src="/files/ftp_upload/50116/50116fig4highres.jpg" src="/files/ftp_upload/50116/50116fig4.jpg" /> &gt; 如图4所示。 THP-1细胞用荧光显微镜跟踪痢疾杆菌空泡破裂（A）经过2小时30分钟的CCF4-AM装载，THP-1细胞被感染，β-内酰胺酶的表达痢疾杆菌 BS176 AFAI突变株或M90T AFAI的毒株为1小时30分钟，并用4％多聚甲醛固定10分钟。然后，细胞使用的落射荧光显微镜用20倍的目标成像。代表图片以下渠道:CCF4完整的探头535 nm（绿色）和CCF4裂解探头450纳米（蓝色）选择（B）的MetaMorph软件自动分析结果的直方图。的各个细胞的分布的函数，它们的比值的强度在450和535 nm的通道。 ghres.jpg"SRC ="/ files/ftp_upload/50116/50116fig5.jpg"/&gt; 图5。跟踪牛分枝杆菌卡介苗和结核分枝杆菌的吞噬体破裂THP-1巨噬细胞用荧光显微镜（A，B）THP-1细胞感染与β-内酰胺酶的表达结核分枝杆菌或结核杆菌 2小时，并培养3至7天。洗涤后，细胞被装入的CCF-4-AM 2小时后，用4％多聚甲醛固定30分钟，在成像之前使用的落射荧光显微镜用40倍物镜。被选为代表图片以下渠道:完整CCF4探头，535纳米（绿色）; CCF4裂，探头450纳米（蓝色）（C，D）呈现使用MetaMorph软件自动分析的结果直方图。各自的细胞的分布为在450和535 nm的通道的强度之间的比率的功能。

Watch this Scientific Journal Video about Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens at JoVE.com

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

我们描述了一种用于跟踪内膜破裂引起的细胞内的细菌<em&gt;痢疾杆菌<em&gt;</em</em&gt;和<em<em&gt;<em&gt;结核杆菌</em&gt;</em</em&gt;侵入宿主细胞后。我们的检测利用CCF4，宿主细胞质FRET探针在活的或固定的细胞。记者降解的酶活性存在于细菌表面。

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

single-cell-measurements-vacuolar-rupture-caused-intracellular

Research

JoVE Journal

Immunology and Infection

13.5K 次观看.  Institut Pasteur, Paris, France. 我们描述了一种用于跟踪内膜破裂引起的细胞内的细菌痢疾杆菌

视频: Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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

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

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

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

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

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

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

生物学

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液泡和胞质pH值的测量<em&gt;在体内</em在酵母细胞悬浮液

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly understood. Here assays are described to monitor the ability and kinetics of healing of cultured cells following localized injury. The first protocol describes an end point based approach to simultaneously assess cell membrane repair ability of hundreds of cells. The second protocol describes a real time imaging approach to monitor the kinetics of cell membrane repair in individual cells following localized injury with a pulsed laser. As healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of injury, the third protocol describes the use of above end point based approach to assess one such trafficking event (lysosomal exocytosis) in hundreds of cells injured simultaneously and the last protocol describes the use of pulsed laser injury together with TIRF microscopy to monitor the dynamics of individual subcellular compartments in injured cells at high spatial and temporal resolution. While the protocols here describe the use of these approaches to study the link between cell membrane repair and lysosomal exocytosis in cultured muscle cells, they can be applied as such for any other adherent cultured cell and subcellular compartment of choice.

The process of healing injured cells involves trafficking of specific proteins and subcellular compartments to the site of cell membrane injury. This protocol describes assays to monitor these processes.

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free in the cytosol (cytosolic hyper-replication). These different phenotypes of intracellular replication drive to different pathways of pathogenesis, i.e., cytosolic hyper-replication induces inflammatory cell death and extrusion into the gut lumen, while vacuolar replication leads to trans-epithelium penetration and systemic spread. Significant effort was made to create microscopy tools to study the behavior of Salmonella inside invaded cells, such as the pCHAR-Duo fluorescence reporter plasmid that allows discrimination between vacuolar and cytosolic bacteria by differential expression of mCherry and GFP. However, intracellular phenotypes are often manually scored, a time-consuming procedure that limits analysis to a small number of samples and cells. To overcome these limitations, two complementary and automated image analyses were developed using ImageJ, a freely available image analysis software. In the high-throughput protocol, epithelial cells were infected with Salmonella carrying pCHAR-Duo using 96-well plates. Imaging was performed using an automated fluorescence microscope. Then, two image analysis methods were applied to measure the intracellular behavior of Salmonella at different detail levels. The first method measures the overall intracellular bacterial load and the extent of cytosolic hyper-replication. It is fast and allows the scoring of a high number of cells and samples, making it suitable for high-throughput assays such as screening experiments. The second method performs single-cell analysis to determine the percentage of infected cells, the mean vacuolar load of Salmonella, and the cytosolic hyper-replication rate giving greater details about Salmonella behavior inside epithelial cells. The protocols can be performed by specifically designed ImageJ scripts to automatically run batch analyses of the major steps of Salmonella-enterocyte interaction.

Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ

Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.

使用 ImageJ 自动分析 沙门氏菌 的细胞内表型

Salmonella is an enteric pathogen able to invade the intestinal epithelium and replicate in enterocytes, both inside Salmonella-specific vacuoles and free ...

A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways through permeabilization of IV membranes and the formation of endosomes and lysosomes. A method named chromophore-assisted laser inactivation (CALI) is applied to study the formation of IVs and the materials in controlling IV regulation. CALI is an imaging-based photodynamic methodology to study the signaling pathway induced by membrane permeabilization. The method allows spatiotemporal manipulation of the selected organelle to be permeabilized in a cell. The CALI method has been applied to observe and monitor specific molecules through the permeabilization of endosomes and lysosomes. The membrane rupture of IVs is known to selectively recruit glycan-binding proteins, such as galectin-3. Here, the protocol describes the induction of IV rupture by AlPcS2a and the use of galectin-3 as a marker to label impaired lysosomes, which is useful in studying the downstream effects of IV membrane rupture and their downstream effects under various situations.

AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.

研究细胞内囊泡破裂功能的光动力方法

Intracellular vesicles (IVs) are formed through endocytosis of vesicles into cytoplasm. IV formation is involved in activating various signal pathways ...

单细胞测量液泡破裂引起细胞内病原体

摘要

探索更多视频

此视频中的章节

病原液泡的分离纯化

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

胞浆的定量与液泡

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

单细胞荧光显微镜定量 Efferocytosis 的研究

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

液泡和胞质pH值的测量

成像生物膜损伤和参与亚细胞修复过程

使用 ImageJ 自动分析沙门氏菌的细胞内表型

研究细胞内囊泡破裂功能的光动力方法

单细胞测量液泡破裂引起细胞内病原体

摘要

探索更多视频

此视频中的章节

病原液泡的分离纯化

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

胞浆的定量与液泡

高通量，实时，细胞内的抗菌活性的双读出测试和真核细胞的细胞毒性

单细胞荧光显微镜定量 Efferocytosis 的研究

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

液泡和胞质pH值的测量

成像生物膜损伤和参与亚细胞修复过程

使用 ImageJ 自动分析 沙门氏菌 的细胞内表型

研究细胞内囊泡破裂功能的光动力方法

使用 ImageJ 自动分析沙门氏菌的细胞内表型