This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1.合成10nm的纳米金（金纳米粒）15 <ol><li>准备解决方案答:加2毫升1％水溶液氯化金成160毫升的Milli-Q，或双蒸水。 </li><li>制备溶液B:加入8毫升1％柠檬酸三钠×2的H 2 O和160微升1％的单宁酸成32毫升的Milli-Q，或双蒸水。 </li><li>热身方案A和B到60°C和混合他们同时进行搅拌。立即观察到深蓝色。约15分钟后，观察到红色。然后升温至95℃，保持5分钟，冷却溶液至室温。 </li><li>添加一滴的NP悬浮到碳包覆的网格并允许在空气中干燥。检查纳米粒的通过透射电子显微镜的大小和形态。 </li></ol> 2.涂料黄金纳米粒子与BSA和标签罗丹明N-羟基酯（NHS）16 <ol><li>添加900微升金纳米粒到1.5ml的Eppendorf管中，离心机以15000×g离心30分钟。欧普倚重，准备多个管可以一次。 </li><li>弃去上清液，重新悬浮沉淀在900微升灭菌的Milli-Q水，并添加100微升2毫克/毫升BSA / PBS中，混合在Vortex以800rpm进行30分钟。 </li><li>以除去过量的BSA，离心制备以15,000×g离心60分钟。 </li><li>弃去上清液，并重新暂停金纳米粒BSA在125微升PBS，加入12.5微升1M的碳酸氢盐。 </li><li>在即将使用前，制备10毫克/毫升的溶液罗丹明NHS / DMSO中，加入30微升罗丹明NHS / DMSO溶液至1ml金 - 牛血清白蛋白纳米粒混悬液。孵育2小时（或O / N）在室温时800转混合反应，避免暴露在光线下。 </li><li>通过净化透析对PBS的黄金BSA罗丹明纳米粒在4ºC超过36期5缓冲变化 - 48小时。 </li><li>为了稳定纳米颗粒，加10微升的200毫克/毫升BSA / PBS到每个1毫升金 - 牛血清白蛋白 - 若丹明纳米粒。以除去游离的或释放BSA的若丹明，离心机15，000 XG 60分钟。重悬沉淀在2毫克/毫升BSA / PBS，测量OD 520，并储存在4℃下，以避免暴露于光。 </li><li>稀释的纳米颗粒10次用Milli-Q或双蒸水，加一滴到碳包覆的网格，并允许在空气中干燥。检查纳米粒的大小和形态由透射电子显微镜（TEM）。 注意:用于NP制剂的所有材料进行灭菌和操作被在细胞培养罩或在火焰旁长凳进行。 </li></ol> 3.文化HeLa细胞<ol><li> HeLa细胞永久表达LAMP1-GFP在DMEM中培养，用10％胎牛血清（FCS），在37℃下在含有5％的CO 2的气氛中生长。 </li><li>种子细胞在50000每孔的密度在8孔室滑动（Ibidi）和孵化O / N。 </li></ol>细菌4.文化<ol><li>使用沙门伤寒NCTC 12023 WILd型（WT）菌株。为了便于比较，使用突变株ΔSSAV缺陷在SPI2-T3SS和Δ 西法缺乏关键效应西法为SIF。使用的菌株窝藏质粒pFPV25.1增强GFP的组成型表达。 注:细菌菌株是常规培养在LB肉汤（LB）并加入50微克/毫升羧苄青霉素（WT）和LB并加入50微克/毫升羧苄青霉素和/或50微克/毫升卡那霉素（ΔSSAV，ΔSIFA ）的要求保持质粒。 </li><li>接种细菌的单个菌落3毫升的LB用适当的抗生素和摇动的条件下进行曝气，在37ºC生长的O / N，则稀释1点31分在新鲜LB，并继续生长3.5小时。在这个时间点，将培养达到晚期对数期和细菌是高度侵入。 A&#39;辊筒&#39;方便孵化试管培养与通风。 </li></ol> 5.通过感染HeLa细胞沙门氏菌和金BSA-罗丹明纳米粒脉冲大通标签（见图1计划） <ol><li>测量子培养细菌的OD 600，并稀释至OD 600 = 0.2的1ml的PBS（〜3×10 8 CFU / ml）的，在8孔室玻片中添加细菌HeLa细胞的适当的量来实现的多个感染的100（MOI）。 </li><li>孵育在细胞培养箱30分钟，洗3次，用PBS以除去未内在化的细菌（该时间点被设定为0小时后的感染，或0小时圆周率）。加入300微升新鲜培养基含有100μg/ ml庆大霉素和维持1小时。然后用含10微克/毫升庆大霉素的温育时间，其余的新鲜培养基更换培养基。 </li><li>孵育与含有100微克/毫升庆大霉素1小时培养基后，将培养基替换为成像介质（鹰MEM不含FCS，L-谷氨酰胺，酚红和碳酸氢钠，用30毫米的HEPES，pH值7.4）共ntaining 10微克/毫升庆大霉素。金-牛血清白蛋白-若丹明纳米粒加入到HeLa细胞中，以获得外径520 = 0.1的终浓度。 注:金 - 牛血清白蛋白 - 若丹明纳米粒也可加入到细胞中感染之前，或在不同的时间点PI </li><li>经过1小时温育后，除去介质，洗3次，用PBS中，并加入300微升含有10％FCS和10μg/ ml的庆大霉素的温育时间的其余部分新鲜成像介质。 注:温育持续时间可以根据所用NP和细胞系的浓度而变化。对于RAW264.7巨噬细胞，我们观察到，30分钟孵化与纳米粒子在OD 520 = 0.05的允许浓度足够的内在。 </li></ol> 6.成像<ol><li>使用共焦成像系统，例如共焦激光扫描（CLSM）或旋转式圆盘（SD）的显微镜用的潮湿环境室，用于高分辨率成像在不同的时间点。 </li><li>开关上的温度下ONTROL系统和等待，直到它是稳定的。优化成像设置，例如放大，扫描速度，分辨率，Z-栈等使用适当的激发/发射设置GFP和金BSA-罗丹明纳米粒。对于这个协议，使用400 Hz的扫描速度，分辨率为512×512像素和0.25微米的Z-步长。激发GFP和使用Ar激光（488纳米）和氦氖激光器（543纳米），分别金 - 牛血清白蛋白 - 若丹明。包括一个亮场（BF）信道用于观察细胞的形状。对于其他显微镜系统和感染的条件下，设置有作相应的调整。 注:使用相同的Ar激光作为高炉信道和信号的光源与光电倍增（PMT）检测器进行检测。 </li><li>在指定的时间点PI，包含安装在显微镜舞台和影像记录受感染的细胞的8孔玻片。 </li></ol>图像7.分析<ol><li>采用显微图像分析软件（见<stron克&gt;材料和设备的数据表）通过细胞内沙门氏菌分析内溶酶体系统的重塑。通过点击"打开"，然后选择文件打开数据。 注:另外，开源软件如ImageJ的或FIJI可用于定量图像分析。 </li><li>在图标上超越视图中点击工具栏上的物体<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" />以添加新的表面项目。点击<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （下一个）。 </li><li>为了分析的感兴趣区域（ROI）的区域，选择一个细分的投资回报率。在一个可视面积，覆盖在图像矩形边框部分代表的投资回报率。在相应的X轴输入值，y-和z-域，或者直接点击箭头在预览矩形来修改大小和ROI的位置。然后点击<img alt="图标" fo:content-width=".1in" src<html> ="/文件/ ftp_upload / 52058 / 52058icon2.jpg"/&gt;（下一个）。 </li><li>作为源通道，选择频道3（信号金BSA-罗丹明NPS）。检查平滑选项来设置所得到的区域的平滑度。手动定义的值或接受自动生成的值。对于阈值，选择绝对强度的选项。 </li><li>对于阈值的调整，选择手动选项，并设置一个值。在可视面积，表面门槛预览显示为灰色。 </li><li>上的标签分类曲面所得表面可通过各种标准来过滤。默认过滤器是，和其他过滤器"体素&gt; 10号"也可以包括通过点击"添加"。如果一个过滤是没有必要的，然后通过点击删除按钮删除所有过滤器。点击<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> （下一个）。 </li><li>要完成表面创建，点击/files/ftp_upload/52058/52058icon3.jpg"/&gt;（完成）。在对象列表，目前未检查的项目量和新创建面框显示在可视面积。 </li><li>出口统计数据。现在，在超越视图根据其区域中物体的颜色变化从紫色到红色。和"情节数字区"表显示的统计信息（ 如身份证号码和对象的面积）。出口由点击Excel文件表面1所有统计<img alt="图标" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> （保存）。 </li></ol>

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No conflicts of interest declared.

哺乳动物细胞内溶酶体系统控制重要的生理过程，包括营养吸收，激素介导的信号转导，免疫监视和抗原呈递17。到现在为止，各种标记已经用于内吞途径和跟踪研究的标记。例如，LysoTracker探针是由Molecular Probes公司（Life Technologies公司，美国）为溶酶体标记开发荧光acidotropic探针，其可以选择性地积聚在细胞区室与低内pH和有效地在纳摩尔浓度18标记的活细胞。然而，LysoTracker探针...

荧光纳米粒子（纳米粒子），包括金属纳米粒子，量子点，聚合物纳米颗粒，二氧化硅纳米颗粒，碳圆点， 等等 ，都在过去几十年1,2-引起了相当的重视。相比传统的有机染料，荧光纳米颗粒显示出理想的化学，光学和机械性质，如强的信号强度，耐漂白和高生物相容性3,4。这些优势使他们的首选细胞内检测和活细胞成像的方法。此外，各种电子致密纳米粒子是通过电子显微镜（EM）可见，促进它们的使用为相关显微分析，这使得活细胞在光学显微镜（LM）和更高的分辨率跟踪在超微结构水平使用EM 5的组合。例如金纳米粒已经有效地用作生物传感器在活细胞中对敏感的诊断，以及在免疫标记6的字段长的时间。近期s案例研究表明，金纳米粒具有不同的尺寸和形状可容易地通过大量的各种细胞系摄取并通过内体途径常规输送，因此有很大的潜力被施加于细胞内囊泡运输跟踪和内溶酶体系统贴标7,8- 。 微生物病原体，如沙门氏菌 ， 志贺氏菌和李斯特菌 ，开发不同的机制侵入非吞噬宿主细胞9。被内化后，病原体，无论是定位于细胞质或螯合在膜结合隔间，广泛的互动与他们的主机环境和调节这些有利于自己的生存10。例如， 沙门氏菌驻留并复制感染11后，在细胞内的吞噬体舱称为含沙门氏菌的空泡（SCV）。成熟SCV贩卖朝向高尔基体，进行与内吞途径连续的相互作用，并诱导形成广泛的管状结构，例如沙门氏菌诱导长丝（SIF），分拣连接蛋白肾小管， 沙门氏菌诱导分泌载体膜蛋白3（SCAMP3）小管等12-14。研究这些细菌病原体如何操纵宿主细胞途径是必不可少的理解传染病。 这里，金-牛血清白蛋白-若丹明纳米粒被用作流体示踪剂标记的宿主细胞内溶酶体系统，以及人体胃肠病原体沙门鼠伤寒（ 沙门氏菌 ）用作模型细菌来研究所述病原体与相互作用主机内吞途径。在非感染细胞和感染的细胞与WT 沙门氏菌或突变株细胞内的金-牛血清白蛋白-若丹明纳米粒通过共焦激光扫描显微镜（CLSM）成像。然后了Imaris软件用于定量纳米粒的分布，表明沙门氏菌感染引起的内涵体/溶酶体极端重排。下面该方法的描述中，类似的实验，可以设计成跟踪内在化纳米颗粒的长期命运，并调查各外源物质或内源性因子对真核细胞的内吞途径的影响。

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

荧光纳米粒子（纳米粒子）具有理想的化学，光学和机械性能是有希望的工具来标记胞内细胞器。在这里，我们介绍利用金-牛血清白蛋白-若丹明纳米粒标记的真核细胞的内溶酶体系统以及由细胞内病原体沙门氏菌监测的宿主细胞途径的操纵的方法。该纳米粒子易于被HeLa细胞内化和本地化下旬内涵体/溶酶体。 沙门氏菌感染引起的沙门氏菌中引起膜结构的囊泡和纳米颗粒的积累重排。我们部署的共聚焦显微镜图像定量分析的了Imaris软件包。对象的数目和在非感染细胞的大小分布是从那些在沙门氏菌 -感染细胞明显，指示由WT 沙门氏菌极其重塑内溶酶体系统。

通过完善的通过由柠檬酸和鞣酸减少氯金酸的方法生成的黄金纳米粒子。 如图2A所示 ，合成的金纳米粒是准球形的形状具有大约10nm的尺寸。 BSA的涂层和罗丹明-标记不影响它们的形态或尺寸（ 图2B）。 已经报道了金纳米粒可容易地采取了由各种哺乳动物细胞和内吞系统7结束了。按照以前的作品，明亮的红色荧光信号，观察在HeLa细胞后1小时?...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

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

本文介绍了合成和纳米粒子的荧光标记（NPS）的方法。所述纳米颗粒是在脉冲追踪实验施加标记的真核细胞的内溶酶体系统。内溶酶体系统的细胞内病原体沙门氏菌的活动操纵其次是活细胞成像和量化。

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 ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  Universität Osnabrück. 本文介绍了合成和纳米粒子的荧光标记（NPS）的方法。所述纳米颗粒是在脉冲追踪实验施加标记的真核细胞的内溶酶体系统。内溶酶体系统的细胞内病原体沙门氏菌的活动操纵其次是活细胞成像和量化。 

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

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

科研

免疫与感染

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

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 ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

生物膜启动对病毒感染的细胞测定细菌和真菌

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

通过"Acufection"和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

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

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