我们感谢珍妮-李. Thomassin 在编辑手稿方面的帮助。这项工作得到了情报局赠款 MITOPATHO 和 PATHIMMUN 的支持, Labex Memolife 和人民党 IDEX Shigaforce 提供赠款。春晖孙是来自中国奖学金委员会的博士助学金的接受者。劳伦 Combettes 和家伙搬运车 Nhieu 是 WBI-法国交换 Tournesol 计划 N°31268YG (Wallonie 布鲁塞尔国际, 全宗研究所科学研究, 部法国, étrangères et européennes, 部 de 的接受者。l ' Enseignement 高级研究所 dans 乐干部 des Partenariats 休伯特克瑞恩)。

1. 筹备工作
<ol><li>
细菌制剂
	<ol>
<li>板细菌-志贺菌野生型菌株, 表达 AfaE 黏附 (M90T AfaE)-在胰酪胨大豆 (TCS) 琼脂板上含有0.01% 刚果红 (CR) 和孵化他们18小时37摄氏度。 注意: 为了提高其重现性, 在步骤1.1.1 中获得的志贺菌板块储存在4摄氏度, 应在一周内使用, 因为 CR 培养基上的所有菌落最终会随着时间而变红。</li>
		<li>接种 TCS 肉汤前培养, 从条纹板采摘3红殖民地。 注: 3 个菌落进行接种, 以限制选择单个克隆的可能性, 其毒性质粒经历了基因重组。</li>
		<li>在16小时在37摄氏度的振动孵化器中培养液体细菌培养 200 rpm。加入氨苄西林, 最后浓度为75毫克/毫升。抗生素浓度不应超过3x 的最小抑制浓度, 因为过量的抗生素可能导致大毒力质粒的损失。 注: 对志贺氏菌毒力质粒的维护应通过电镀铬板上的细菌培养物进行验证, 辅以适当的抗生素浓度。白色菌落的存在表明质粒损失。</li>
		<li>在1:100 稀释的情况下, 将 TCS 培养与细菌前培养接种。孵化它在37°c 在震动孵化器为2小时在200转每分钟。确保光学密度在 600 nm (OD600nm) 是 0.2-0.4。</li>
		<li>离心机细菌培养2分钟在 1.3万 x g 在21°c 和并用重悬颗粒在一个等效容量 EM 媒介 (120 毫米氯化钠, 7 毫米氯化钾, 1.8 毫米CaCl2, 0.8 毫米氯化镁2, 5 毫米葡萄糖和25毫米 HEPES, pH 值 = 7.3)。</li>
		<li>在0.1 的最终 OD600nm中稀释 EM 缓冲器中的细菌悬浮液, 并立即使用, 或将其存储在21摄氏度, 并在下一个60分钟内使用。</li>
	</ol>
</li>
	<li>
细胞制剂
	<ol>
<li>在 DMEM 中培养 HeLa 细胞, 1 克/升葡萄糖补充10% 胎小牛血清 (FCS), 并将其生长在37摄氏度, 10% CO2。生长 TC-7 生长在 DMEM 与4.5 克/升葡萄糖, 包含 10% FCS 和非必需氨基酸在37°c 孵化器包含 10% CO2。</li>
		<li>对于细胞的维护, 定期分裂细胞, 以避免与汇合状态相关的生长接触抑制;这对于高 Ca2 +探针加载/转染效率至关重要。</li>
		<li>在25毫米直径的6井板上, 以 3 x 105细胞的密度为单位, 在 hela 细胞实验前一天, 将 hela 细胞放到无菌的盖玻片上。</li>
		<li>对于 TC-7 细胞, 胰蛋白酶处理和计数细胞。种子他们在 4 x 105细胞/井和孵化他们 5-7 天在37°c 在 10% CO2-孵化器在实验之前允许极化。<ol>
<li>每天刷新细胞培养基, 直到实验当天。 注: 玻璃底部的35毫米直径的一次性房间也可以作为替代盖玻片含水井。如果使用后者, 温度控制通过加热的阶段或目标是不足的, 并且一个恒温器孵化室安装在显微镜阶段是必要的。</li>
		</ol>
</li>
	</ol>
</li>
	<li>实验当天, 取出培养基, 用1毫升 EM 培养基在21摄氏度处冲洗细胞3x。</li>
	<li>将 3 mM Fluo-4-AM14的单元格加载到 EM 缓冲区中, 其值为21°c 30 分钟。 注: 虽然亚汇合 HeLa 细胞的加载没有引起重大问题, 但极化 TC-7 细胞的加载往往是异构的, 效率低下。对于这些细胞, 我们发现, 增加分散剂-pluronic 酸-在最终浓度为0.1% 和阴离子转运抑制剂 bromosulfophtalein, 增加了加载效率, 最终浓度为20µM 到Fluo-4-AM-containing 他们使用比例化学探针或基因编码的烦恼探头, 要求双采集不符合对本地 Ca2 +响应的映像所需的采集速度。</li>
	<li>用 em 缓冲器清洗样品 3x, 再在21摄氏度的1毫升 em 缓冲液中孵化, 使 AM 基团水解。立即或在下一个90分钟内使用 Fluo-4 加载的单元格 (保持在21°c)。</li>
</ol>2. 感染和图像获取本地 Ca2 +响应
<ol><li>将包含 Fluo-4 加载单元格的盖玻片放置在成像室或玻璃底部腔室中。用 EM 缓冲器将成像室或玻璃底部一次性腔内的样品洗净, 去除可能因细胞裂解而产生的化合物。添加1毫升的 EM 缓冲器。</li>
	<li>将房间放在33摄氏度加热的倒置荧光显微镜阶段。 注意: 此温度被选择为与志贺菌入侵过程兼容的最佳温度与 Ca2 +响应减慢以可视化本地响应之间的折衷。我们使用63X 浸入油目标 (NA = 1.25) 配有相衬环。</li>
	<li>选择一个显微领域, 并设置采集参数, 包括曝光时间和 binning, 如有必要, 以优化 Fluo-4 荧光信号。 注意: 本地 Ca2 +映像的主要挑战是响应的低振幅和较小的持续时间, 要求至少每30毫秒获取一次. 一些商用的背照明的 EM CCD 或 C MOS 相机提供了所需的使用 Fluo-4 检测本地 Ca2 +响应的灵敏度。检测灵敏度也是一个重要问题, 以限制光损伤或文物, 如自发反应链接到荧光激发的 Fluo-4 在高频率。在这里, LED 照明的470毫微米, 与 480, 40 nm 带通励磁过滤器, 505 nm 分色过滤器, 和 527 @ 30 nm 带通发射, 使用5% 的最大强度与1.0 中性密度过滤器, 以减少这些问题在有限工期的收购下。常规地执行120s 流批处理的本地 Ca2 +信号的分析。在这些低光照条件下, 荧光漂白在2分钟流收购期间没有被观察到。如果使用不同的字段, 则可以执行相同示例的连续收购。一般来说, 在没有细菌刺激的情况下, 第一个采集流是在控制场上进行的, 以确保没有自发反应。如果观察到自发反应, 样品将用 EM 缓冲器冲洗, 直到没有观察到反应。</li>
	<li>要将细菌添加到样品中, 请从该腔中取出500µL 的 EM 缓冲器, 并添加500µL 在步骤1.1.6 中制备的细菌悬浮液, 以获得最终 OD600nm 0.05, 并适当注意避免移动选定的显微领域;这些体积确保细菌的适当混合和细菌在细胞样品上的均匀分布。</li>
	<li>对细菌添加进行采集。另外, 在细菌添加后执行10分钟的采集, 这与细菌在使用条件下沉淀到细胞所需的时间相对应。在采集流的末尾, 获取选定字段的相对比图像, 以可视化与细菌侵入点相关的细胞和膜褶边的细菌。</li>
	<li>按照步骤2.5 中的规定, 重复采集过程, 时间间隔可满足图像获取的持续时间、文件的节省以及选择新字段以覆盖整个过程。 注: 对于志贺菌, 我们发现, 在10分钟细菌沉淀之后, 采集流每5分钟20分钟, 允许覆盖大多数入侵事件。</li>
	<li>在获取过程的末尾, 添加 ca2 +载体 ionomycin 示例的2µM 的最终浓度, 以确定 ca2 +信号的最大振幅。每 3-5 秒获取图像直到信号稳定, 通常不到10分钟。</li>
	<li>通过添加 ca2 +离子螯合剂 EGTA 在最终浓度为 10 mM 的情况下, 确定在缺少Ca2 + 的情况下的荧光信号。每 3-5 秒获取图像直到信号稳定, 通常不到10分钟。 注意: 由于全局 Ca2 +变体的动态相对缓慢, 每 3-5 秒 (不在流模式下) 执行这些获取, 在后续处理过程中允许 Ca2 +成像在相同的微观领域。</li>
</ol>3. 分析
<ol><li>快速胞浆 Ca2 +随时间变化的ΔF/F0的百分比, 其中ΔF 表示感兴趣区域的平均荧光强度 (roi) 和 F0在相同 ROI 中的基线荧光的变化, 其中一个减去单元格的字段的非相关区域。<ol>
<li>去除不同领域中每个细胞的基底荧光水平, 对应基线水平;由于给定流捕获中本地 Ca2 +响应的低频和相对短的持续时间, 可以明确确定这些级别。 注: 量化与所询问的问题有关的反应的显著特征和研究的致病模型。经典情况下, 考虑了各种参数来分析 Ca2 +信号, 包括显示本地或全局 Ca2 +响应的单元格的频率, 以及这些响应的持续时间、振幅和频率。在志贺菌和局部反应的情况下, 分析的另一个重要方面是与细菌入侵部位的反应的空间关联。</li>
	</ol>
</li>
	<li>为了量化显示全球或局部反应的应答细胞的百分比, 在细胞中绘制 ROIs, 时间序列对应 Fluo-4 强度的变化。<ol>
<li>设置严格的参数以标识本地 Ca2 +响应, 例如, 在平均荧光强度单元基线的背景变化的三倍以上的振幅, 或至少200毫秒的持续时间, 以避免得分为假阳性。 注意: 作为一般规则, 即使小的本地 Ca2 +响应也可以与其配置文件中的任何背景噪音区分开来。ROIs 应该足够大, 能够集成足够的荧光信号, 以便检测局部变化。根据 Fluo-4 检测的强度, 这些 ROIs 可能对应于直径从 2-5 毫米不等的圆。</li>
		<li>测量同一单元格不同区域中2个区域中平均 Fluo-4 荧光强度的变化, 以确定响应是局部的还是全局的。 注: 通过使用成像软件, 允许在线屏幕显示所选区域的平均荧光强度变化, 从而促进了对大量细胞的分析。商业上可用的成像软件有这样一个选项, 但也可以使用ROI 强度演化插件在冰冷的软件中执行此操作。</li>
		<li>确定显示本地响应的单元格的百分比。 注意: 这个百分比是从显微镜领域分析的细胞总数计算出来的, 应该有足够的数字来支持使用非参数测试的统计学意义。</li>
		<li>确定本地响应的持续时间。 注意: 本地 Ca2 +响应可根据其持续时间范围 (即小于500毫秒, 介于5和十年代之间, 或在十年代以上) 进一步区分, 并与志贺氏菌入侵站点关联。</li>
	</ol>
</li>
	<li>量化响应的振幅。首先, 校准最大 Ca2 +和确定为步骤2.1.7 的单元格背景级别。由于每个单元格的 Fluo-4 负载可能会有所不同, 请对该字段中的单个单元格执行这些决定。<ol>
<li>将响应的振幅表示为最大响应的相对百分比。</li>
		<li>使用正常测试进行统计测试, 然后进行参数化测试, 分析样本间响应幅度的潜在差异。</li>
		<li>根据相应的相衬图像中的细菌诱导膜褶边, 确定这些反应相对于细菌侵入点的相关性。</li>
	</ol>
</li>
</ol>

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本手稿描述了在志贺菌入侵的相对较短的动力学过程中, 我们设计为遵循本地 ca2 +信号的协议, 以及志贺菌扩展动力学过程中的全局 ca2 +响应。下面, 需要解决的关键问题是优化 Ca2 +信号的检测, 同时尽量减少对生物过程的任何干扰。
化学vs基因编码的 Ca2 +探头:
为了图像本地 Ca2 +的变化, 我们使用?...

Ca2 +调节所有已知的单元进程, 包括骨架重组、炎症反应和与宿主-病原体相互作用有关的细胞死亡通路1,2,3。在生理条件下, 基底胞浆 Ca2 +浓度低, 在数以百计的 nM 范围内, 但可能会受到瞬变增加后激动剂刺激。这些变化经常表现出振荡行为, 通过泵和渠道在血浆和内质网膜的作用。这些振荡的模式以 ca2 +的周期、持续时间和振幅为特征, 并由单元格进行解密, 然后在被称为 Ca2 +代码4,5 中触发特定响应..在病理条件下, 胞浆 Ca2 +浓度的持续增加可能导致与线粒体膜通透相关的细胞死亡, 并释放亲凋亡或坏死因子6, 7。
志贺菌是细菌性痢疾的致病剂, 通过使用 III. 型分泌系统 (T3SS)8、9, 将效应器注入宿主细胞, 侵入上皮细胞。志贺菌入侵宿主单元格与 T3SS 引发的本地和全局 Ca2 +信号相关联。至于成孔毒素, T3SS translocon 插入宿主细胞膜, 并需要注入 T3SS 效应剂可能负责激活 PLC 和肌醇 (1, 4, 5) trisphosphate (InsP3) 依赖 Ca2 +释放。局部 PLC 刺激和聚合肌动蛋白在志贺菌入侵部位的聚集, 导致超持久的 InsP3-dependent Ca2 +发行10。III. 型效应器 IpgD, 磷脂 45 bisphosphate (PIP2)-4-磷酸酶, 限制了局部 PIP2 量, 从而控制了可编程序控制器生成 InsP3 的可用基板的数量, 这有助于限制本地 Ca2 +细菌入侵站点的响应11,12。这些本地 Ca2 +响应可能会导致在志贺菌入侵站点10的肌动蛋白聚合。但是, 由志贺氏引起的全局 Ca2 +响应是细菌入侵过程不可缺少的, 但会触发连接蛋白 hemichannels 在等离子膜上的打开和胞外 ATP 的释放。车厢。以分泌方式释放的 ATP 将反过来刺激受感染细胞旁边的单元格中的 Ca2 +振荡响应。IpgD 还负责将全局 Ca2 +响应塑造为不稳定的孤立响应, 并具有缓慢的动态。最后, 在长时间的细菌感染后, IpgD 导致 InsP3-mediated Ca2 +信号的抑制。通过对 ca2 +信号的干扰, IpgD 延迟了与 ca2 +相关的钙蛋白酶激活, 导致了焦点黏附结构的拆卸和感染细胞的过早脱离 13.
虽然 Ca2 +信号涉及病机的关键方面, 但微生物的使用引发了一些在经典激动剂研究中没有遇到的技术挑战。此处描述的协议使用常用的荧光 Ca2 +化学指标 Fluo-4, 我们在志贺病毒感染期间设计了用于表征本地 Ca2 +信号的特性。讨论了对这些信号进行检测的关键步骤, 以及为进行定量分析所需的程序, 以确定细菌效应在 Ca2 +信号中的作用。

<table><tbody><tr><td>Fluo-4 AM</td><td>Invitrogen</td><td>F14201</td><td></td></tr><tr><td>Metamorph version 7.7</td><td>Universal Imaging</td><td></td><td></td></tr><tr><td>CoolLED illumination system pE-2</td><td>Roper Scientific</td><td></td><td></td></tr><tr><td>micro-dish 35 mm, high&amp;nbsp;</td><td>IBIDI</td><td>81156</td><td></td></tr><tr><td>Trypticase Soy (TCS) broth</td><td>Thermofisher</td><td></td><td>B11768</td></tr><tr><td>TCS agar</td><td>Thermofisher</td><td></td><td>B11043</td></tr><tr><td>Congo red</td><td>Sigma-Aldrich</td><td></td><td>75768</td></tr><tr><td>M90T-AfaE</td><td>Sun et al. 2017</td><td>&lt;em&gt;Shigella flexneri&lt;/em&gt; serotype V. expressing the AfaE adhesin</td><td></td></tr><tr><td>&lt;em&gt;ipgD&lt;/em&gt;-AfaE</td><td>Sun et al. 2017</td><td>isogenic ipgD mutant strain expressing the AfaE adhesin</td><td></td></tr></tbody></table>

imaging ca2+ responses during shigella infection of epithelial cells

Ca2 +是所有已知的蜂窝进程中普遍存在的一个离子。尽管全局 Ca2 +响应可能会影响细胞的命运, 但在自由 Ca2 +胞浆浓度的局部变化中, 与内部存储区的释放有关, 或者通过等离子膜通道流入, 调节皮层细胞的过程。附着或侵入宿主细胞的病原体触发了宿主血浆膜肌动蛋白细胞骨架的重组, 这可能会影响全局和局部 Ca2 +信号。由于这些事件可能以伪随机方式出现在扩展动力学上, 因此, 分析由病原体引起的 Ca2 +信号会引发需要解决的重大技术挑战。
在这里, 我们报告的协议, 以检测全球和本地 Ca2 +信号后,志贺菌感染上皮细胞。在这些协议中, 与 Ca2 +荧光探针的激发相关的长时间暴露和光损伤关联的手工制品诊断在志贺菌入侵。在使用化学探针 Fluo-4 对扩展感染动力学过程中的全局胞浆 Ca2 +信号的振幅和频率进行严格分析的过程被实施。

志贺菌入侵与非典型的持久性本地 Ca2 +响应关联:
根据上述协议, Fluo-4-loaded HeLa 细胞受到了志贺菌的挑战, 并进行了流采集以分析 Ca2 +信号。一个具有代表性的实验显示在图 1中, 其中的时间推移图像系列的荧光强度的 Fluo-4 探针平均在一个区域感兴趣的一个单元, 和相应的相位?...

Watch this Scientific Journal Video about Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells at JoVE.com

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

在这里, 我们提出的协议, 可视化钙 (Ca2 +) 的反应引起的 HeLa 细胞感染, 由志贺菌。通过对 ca2 +荧光探针的细菌感染和成像参数进行优化, 提出了由细菌引起的非典型全局和局部 Ca2 +信号在大范围的感染动力学中的特征。

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

imaging-ca2-responses-during-shigella-infection-of-epithelial-cells

Research

JoVE Journal

Immunology and Infection

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

5.5K 次观看.  Collège de France. 在这里, 我们提出的协议, 可视化钙 (Ca2 +) 的反应引起的 HeLa 细胞感染, 由志贺菌。通过对 ca2 +荧光探针的细菌感染和成像参数进行优化, 提出了由细菌引起的非典型全局和局部 Ca2 +信号在大范围的感染动力学中的特征。

视频: Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

科研

免疫与感染

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

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 ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

从人类胸腺髓样树突状细胞和上皮细胞的分离

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

病毒感染的人肠道类器官单层的钙成像

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

作者聚焦：研究肠道上皮信号传导的病毒破坏 – 研究见解和未来方向 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

分析结肠上皮细胞中 志贺氏菌 感染的方案

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

作者聚焦:了解和对抗 志贺氏菌 感染的进展

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

志贺氏菌上皮细胞感染分析

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

成像 Ca^{2 +}在志贺菌感染上皮细胞期间的响应

摘要

探索更多视频

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

单细胞测量液泡破裂引起细胞内病原体

肺炎链球菌在鼻内殖民期间炎症反应的特征

从人类胸腺髓样树突状细胞和上皮细胞的分离

使用

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

使用基因编码的钙指示剂对病毒感染的人肠道类器官单层进行活体钙成像

志贺氏菌上皮细胞感染分析