Name: application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria
Uploaded: 2015-01-02T14:00:00
Description: Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

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?...

<ol>
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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 ...

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