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. Syntese af 10 nm Gold Nanopartikler (Gold NP&#39;er) 15 <ol><li> Forbered løsning A: tilsættes 2 ml 1% vandig guld chlorid i 160 ml Milli-Q, eller dobbelt destilleret vand. </li><li> Forbered opløsning B: tilsæt 8 ml 1% tri-natriumcitrat x 2 H2O og 160 pi 1% garvesyre i 32 ml Milli-Q eller dobbelt destilleret vand. </li><li> Varm op opløsning A og B til 60 ° C og blande dem under omrøring. Overhold en mørk blå farve med det samme. Observer rød farve efter ca. 15 minutter. Derefter op...

<ol>
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Den endo-lysosomal system pattedyrceller styrer vigtige fysiologiske processer, herunder absorption af næringsstoffer, hormon-formidlet signaltransduktion, immunovervågning, og antigenpræsentation 17. Indtil nu har en række markører er blevet anvendt til mærkning af de endocytiske pathway og sporing studier. For eksempel lysoTracker sonder er fluorescerende acidotropic sonder udviklet af Molecular Probes (Life Technologies, USA) for lysosom mærkning, som selektivt kan ophobes i cellulære rum med lav i...

Fluorescerende nanopartikler (NPS), herunder metal NP&#39;er, kvantepunkter, polymer NP&#39;er, silica NP&#39;er, carbon prikker osv har stor opmærksomhed i de seneste årtier 1,2. Sammenlignet med traditionelle organiske farvestoffer, fluorescerende NP&#39;er viser ønskelige kemiske, optiske og mekaniske egenskaber, såsom stærkt signal styrke, modstandsdygtighed over for fotoblegning og høj biokompatibilitet 3,4. Disse fordele gør dem til foretrukne metode til intracellulær detektering og levende celler billeddannelse. Endvidere en række elektron-tætte NP&#39;er er synlige ved elektronmikroskopi (EM), hvilket letter deres anvendelse til korreleret mikroskopisk analyse, som tillader kombination af levende celler sporing med lysmikroskopi (LM) og højere opløsning på ultrastrukturelle niveau med EM 5. For eksempel har guld NP&#39;er været lang tid effektivt anvendes som biosensorer i levende celler til følsom diagnose samt inden for immuno-mærkning 6. Seneste studies indikerer, at guld NP&#39;er med forskellig størrelse og form kan være let optagelse af en lang række cellelinier og rutinemæssigt transport gennem den endosomale pathway, har derfor stort potentiale nu anvendes til intracellulær vesikel transport sporing og endo-lysosomale mærkningsordning 7,8 . Mikrobielle patogener, såsom Salmonella enterica, Shigella flexneri og Listeria monocytogenes, har udviklet forskellige mekanismer til at invadere ikke-fagocytiske værtsceller 9. Efter at være blevet internaliseret, patogener, enten lokaliseret i cytosolen eller afsondret i membranbundne rum, samspil med deres værtsmiljøer og modulerer disse til at favorisere deres egen overlevelse 10. For eksempel Salmonella enterica bosat og replikerer i en intracellulær phagosomal rum kaldes Salmonella holdige vacuole (SCV) efter infektion 11. Den modning SCVtrafikker mod Golgi-apparatet, undergår kontinuerlige interaktioner med endocytiske proces, og inducerer dannelse af omfattende rørformede strukturer, såsom Salmonella -inducerede filamenter (SIF), sortering nexin tubuli, Salmonella induceret sekretorisk carrier membran protein 3 (SCAMP3) tubuli, etc . 12-14. Studer hvordan disse bakterielle patogener manipulere vært-celle veje er afgørende for forståelsen infektionssygdom. Her blev guld-BSA-rhodamin NP&#39;er anvendes som flydende sporstoffer til mærkning værten cellulære endo-lysosomal system og den humane mave patogen Salmonella enterica serovar Typhimurium (Salmonella) blev anvendt som en model bakterie at studere vekselvirkninger patogenet med vært endocytiske pathway. Intracellulære guld-BSA-rhodamin nationale parlamenter i ikke-inficerede celler og celler inficeret med WT Salmonella eller mutantstammer blev afbildet med en konfokal laser-scanning mikroskop (CLSM).Derefter Imaris software blev anvendt til at kvantificere fordelingen af NP&#39;er, hvilket indikerer, at Salmonella infektion induceret ekstrem omlejring af endosomer / lysosomer. Efter beskrivelse af denne metode, kan analoge eksperimenter designet til at spore langsigtet skæbne internaliserede NPs og til at undersøge indflydelsen af ​​forskellige eksogene stoffer eller endogene faktorer på endocytiske pathway af eukaryote celler.

<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

Fluorescerende nanopartikler (NPS) med ønskelig kemiske, optiske og mekaniske egenskaber er lovende redskaber at mærke intracellulære organeller. Her indfører vi en fremgangsmåde ved hjælp af guld-BSA-rhodamin NP&#39;er at mærke endo-lysosomale system eukaryote celler og overvåge manipulationer af værten cellulære veje af intracellulært patogen Salmonella enterica. NPS blev let internaliseret af HeLa-celler og lokaliseret i sene endosomer / lysosomer. Salmonella infektion fremkaldt omlægning af vesiklerne og akkumulering af nationale parlamenter i Salmonella- induceret membranstrukturer. Vi implementerede Imaris software pakke til kvantitative analyser af konfokal mikroskopi billeder. Antallet af objekter og deres størrelsesfordeling i ikke-inficerede celler var forskellige fra dem i Salmonella -inficerede celler, hvilket indikerer meget ombygning af endo-lysosomale system ved WT Salmonella.

Guld NP&#39;er blev genereret gennem et veletableret metode via reduktion af chlorguldsyre ved citrat og garvesyre. Som vist i figur 2A, de syntetiserede guld NP var kvasi-sfærisk form med en størrelse på ca. 10 nm. BSA-belægning og rhodamin-mærkning påvirkede ikke deres morfologi eller størrelse (figur 2B). Det er blevet rapporteret, at guld NP&#39;er kan let tages op af forskellige pattedyrceller og endte i de endocytiske systemer 7. I ov...

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

I denne artikel beskrives metoder til syntese og fluorescerende mærkning af nanopartikler (NP&#39;er). NPS blev anvendt i puls-chase forsøg at mærke endo-lysosomale system eukaryote celler. Manipulation af endo-lysosomale system ved aktiviteter af den intracellulære patogen Salmonella enterica blev fulgt af levende celler og kvantificeret.

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

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