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. Synthesis of 10 nm Gold Nanoparticles (Gold NPs)15
<ol>
	<li>Prepare solution A: add 2 ml 1% aqueous gold chloride into 160 ml Milli-Q, or double distilled, water.</li>
	<li>Prepare solution B: add 8 ml 1% tri-sodium citrate x 2 H2O and 160 &#181;l 1% Tannic acid into 32 ml Milli-Q, or double distilled, water.</li>
	<li>Warm up solution A and B to 60 &#176;C and mix them while stirring. Observe a dark blue color immediately. Observe red color after about 15 min. Then heat up to 95&#176;C, keep ...

<ol>
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The endo-lysosomal system of mammalian cells controls important physiologic processes, including nutrient absorption, hormone-mediated signal transduction, immune surveillance, and antigen presentation17. Up to now, a variety of markers have been used for labeling of the endocytic pathway and tracking studies. For example, LysoTracker probes are fluorescent acidotropic probes developed by Molecular Probes (Life Technologies, USA) for lysosome labeling, which can selectively accumulate in cellular compartments ...

Fluorescent nanoparticles (NPs), including metal NPs, quantum dots, polymer NPs, silica NPs, carbon dots, etc., have attracted considerable attention during the past decades1,2. Compared to traditional organic dyes, fluorescent NPs show desirable chemical, optical and mechanical properties, such as strong signal strength, resistance to photobleaching and high biocompatibility3,4. These advantages make them the method of choice for intracellular sensing and live cell imaging. Furthermore, a variety of electron-dense NPs are visible by electron microscopy (EM), facilitating their use for correlated microscopic analysis, which allows combination of live cell tracking with light microscopy (LM) and higher resolution at ultrastructural level with EM5. For example, gold NPs have been long time efficiently used as biosensors in living cells for sensitive diagnosis as well as in the field of immuno-labeling6. Recent studies indicate that gold NPs with different size and shape can be readily uptake by a large variety of cell lines and routinely transport through the endosomal pathway, therefore have great potential being applied for intracellular vesicle transportation tracking and the endo-lysosomal system labeling7,8.
Microbial pathogens, such as Salmonella enterica, Shigella flexneri and Listeria monocytogenes, have developed different mechanisms to invade non-phagocytic host cells9. After being internalized, the pathogens, either localized in the cytosol or sequestered in membrane-bound compartments, interact extensively with their host environments and modulate these to favor their own survival10. For instance, Salmonella enterica resides and replicates within an intracellular phagosomal compartment termed the Salmonella-containing vacuole (SCV) upon infection11. The maturing SCV traffics towards the Golgi apparatus, undergoing continuous interactions with the endocytic pathway, and induces formation of extensive tubular structures, such as Salmonella-induced filaments (SIF), sorting nexin tubules, Salmonella-induced secretory carrier membrane protein 3 (SCAMP3) tubules, etc.12-14. Studying how these bacterial pathogens manipulate host-cell pathways is essential to understanding infectious disease.
Here, gold-BSA-rhodamine NPs were used as fluid tracers to label the host cellular endo-lysosomal system, and the human gastrointestinal pathogen Salmonella enterica serovar Typhimurium (Salmonella) was used as a model bacterium to study the interactions of the pathogen with the host endocytic pathway. Intracellular gold-BSA-rhodamine NPs in non-infected cells and cells infected with WT Salmonella or mutant strains were imaged by a confocal laser-scanning microscope (CLSM). Then Imaris software was used to quantify the distribution of NPs, indicating that Salmonella infection induced extreme rearrangement of the endosomes/lysosomes. Following the description of this method, analogous experiments can be designed to track long-term fate of the internalized NPs and to investigate the influence of various exogenous substances or endogenous factors on the endocytic pathway of eukaryotic cells.

<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

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.

Gold NPs were generated through a well-established method via reduction of chloroauric acid by citrate and tannic acid. As shown in Figure 2A, the synthesized gold NPs were quasi-spherical in shape with a size of approximately 10 nm. BSA-coating and rhodamine-labeling did not influence their morphology or size (Figure 2B).
It has been reported that gold NPs can be readily taken up by various mammalian cells and ended up in the endocytic systems7. In...

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

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.

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

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Research

JoVE Journal

Immunology and Infection

9.9K vues.  Universität Osnabrück.  Cet article décrit les méthodes pour la synthèse et le marquage fluorescent de nanoparticules (NP). Les IP ont été appliquées dans des expériences pulse-chase d'étiqueter le système endo-lysosomale des cellules eucaryotes. La manipulation du système d'endo-lysosomal par les activités de l'agent pathogène intracellulaire Salmonella enterica ont été suivis par imagerie des cellules vivantes et quantifiée. 