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。

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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 border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>LiveBlazer FRET-B/G Loading Kit (CCF4-AM) </td>
			<td>Invitrogen</td>
			<td>K1089</td>
			<td>Protect from light, stock in - 80 &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>&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>&beta;-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

13.4K 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

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

胞内相互作用的可视化和量化脑膜炎奈瑟菌和人类α-辅肌动蛋白共聚焦成像

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

科研

免疫与感染

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

斑马鱼的胚胎细胞内细菌病原体感染

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

新的细胞内病原体由Amoebal共培养和Amoebal富集途径探索

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

荧光标记基因的基因枪转化成机会性真菌病原<em&gt;新型隐球菌</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

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 ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

荧光寿命成像显微镜观察细胞内圈套的贩运

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

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 ...