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. Sintesi di 10 nm oro nanoparticelle (NP Oro) 15 <ol><li> Preparare la soluzione A: aggiungere 2 ml di 1% acquosa di cloruro di oro in 160 ml Milli-Q, o doppio distillata, acqua. </li><li> Preparare la soluzione B: aggiungere 8 ml 1% trisodico citrato x 2 H 2 O e 160 ml 1% di acido tannico in 32 ml Milli-Q, o doppio distillata, acqua. </li><li> Riscaldare soluzione A e B a 60 ° C e mescolare agitando. Osservare immediatamente un colore blu scuro. Osservare il colore rosso dopo circa 15 min. Poi riscaldare fino a 95 ° C, 5 min mantenere e raffreddare la soluzione a RT. </li><li> Aggiungere una goccia della sospensione NP su una griglia di carbonio rivestito e lasciare asciugare all&#39;aria. Controllare la dimensione e la morfologia delle NP mediante microscopia elettronica a trasmissione. </li></ol> 2. Rivestimento di oro NP con BSA e etichettatura con Rhodamine N-hydroxysuccinimidyl Ester (NHS) 16 <ol><li> Aggiungere 900 microlitri NP oro in una provetta Eppendorf da 1,5 ml, centrifugare a 15.000 xg per 30 min. Opzionale, preparare più tubi possono contemporaneamente. </li><li> Eliminare il surnatante, risospendere il pellet in 900 ml sterilizzato Milli-Q acqua e aggiungere 100 ml 2 mg / ml BSA / PBS, mescolare in un Vortex a 800 rpm per 30 min. </li><li> Per rimuovere l&#39;eccesso di BSA, centrifugare la preparazione a 15.000 xg per 60 min. </li><li> Eliminare il surnatante, e ri-sospendere i NP oro BSA in 125 microlitri PBS, aggiungere 12,5 ml di 1 M di bicarbonato. </li><li> Immediatamente prima dell&#39;uso, preparare una soluzione di 10 mg / ml di rodamina NHS / DMSO, aggiungere 30 ml di soluzione rhodamine / DMSO NHS a 1 ml della sospensione oro-BSA NP. Incubare la reazione per 2 ore (o O / N) a RT 800 rpm durante la miscelazione, evitando l&#39;esposizione alla luce. </li><li> Purificare le NP d&#39;oro-BSA-rhodamine tramite dialisi contro PBS a 4 ° C con 5 cambi tampone nel periodo di 36 - 48 ore. </li><li> Per stabilizzare NP, aggiungere 10 ml di 200 mg / ml BSA / PBS in ogni 1 ml NP gold-BSA-rodamina. Per rimuovere la centrifuga gratuito o rilasciato BSA-rodamina, a 15,000 xg per 60 min. Risospendere il pellet in 2 mg / ml BSA / PBS, misurare OD 520, e conservare a 4 ° C, evitando l&#39;esposizione alla luce. </li><li> Diluire le NP 10 volte con Milli-Q o acqua bidistillata, aggiungere una goccia su una griglia di carbonio rivestita, e lasciare asciugare all&#39;aria. Controllare la dimensione e morfologia delle NP mediante microscopia elettronica a trasmissione (TEM). NOTA: Tutti i materiali utilizzati per la preparazione NP sono stati sterilizzati e l&#39;operazione è stata condotta in una cappa di coltura cellulare o su una panchina accanto a una fiamma. </li></ol> 3. Cultura di cellule HeLa <ol><li> Cellule HeLa esprimenti stabilmente LAMP1-GFP sono coltivate in DMEM con 10% siero fetale di vitello (FCS) e coltivate a 37 ° C in atmosfera contenente il 5% di CO 2. </li><li> Seme le cellule ad una densità di 50.000 per bene in un 8-pozzo vetrino camera (Ibidi) e incubare O / N. </li></ol> 4. Cultura di batteri <ol><li> Usa Salmonella enterica sierotipo Typhimurium NCTC 12023 wild-type (WT) ceppo. Per confronto, utilizzare ceppi mutanti Δ SSAV difettosi nel SPI2-T3SS e Δ SIFA privi effettori chiave Sifa per SIF. Utilizzare ceppi ospitano plasmide pFPV25.1 per l&#39;espressione costitutiva di una maggiore GFP. NOTA: ceppi batterici sono regolarmente coltivate in Luria-Bertani brodo (LB) con l&#39;aggiunta di 50 mg / ml carbenicillina (WT) e LB con l&#39;aggiunta di 50 mg / ml carbenicillina e / o 50 mg / ml kanamicina (Δ SSAV, Δ SIFA ) di richiesta per mantenere plasmidi. </li><li> Inoculare una singola colonia di batteri in 3 ml LB con antibiotici appropriati e crescere O / N a 37 ° C, in condizioni che agitano per l&#39;aerazione, quindi diluire 1:31 in fresco LB e continuare la crescita per 3,5 ore. A questo punto-punto, le culture raggiungono la fase log tardi e batteri sono altamente invasiva. A &#39;tamburo roller&#39; è conveniente per incubare culture provetta con aerazione. </li></ol> 5. L&#39;infezione di cellule HeLa daSalmonella e oro-BSA-Rodamina NP Pulse Chase-etichettatura (vedi Figura 1 per programma) <ol><li> Misurare OD 600 dei batteri sub-coltura e diluire a OD 600 = 0.2 in 1 ml di PBS (~ 3 × 10 8 cfu / ml), aggiungere quantità appropriate di batteri alle cellule HeLa in vetrini camera 8 pozzetti per ottenere una molteplicità di infezione (MOI) di 100. </li><li> Incubare per 30 minuti in incubatrice cellulare, lavare 3 volte con PBS per rimuovere i batteri non internalizzati (questo punto il tempo è stato impostato come 0 ore dopo l&#39;infezione, o 0 hr pi). Aggiungere 300 ml di terreno di coltura fresco contenente 100 mg / ml di gentamicina e mantenere per 1 ora. Quindi sostituire il mezzo con mezzo fresco contenente 10 ug / ml gentamicina per il resto del tempo di incubazione. </li><li> Dopo l&#39;incubazione con mezzo di coltura contenente 100 ug / ml gentamicina per 1 ora, il mezzo viene sostituito con mezzo di imaging (Eagles MEM senza FCS, L-glutammina, rosso fenolo e bicarbonato di sodio, con HEPES 30 mM, pH 7,4) containing 10 mg / ml di gentamicina. NP oro-BSA-rodamina vengono aggiunti alle cellule HeLa per ottenere una concentrazione finale di OD 520 = 0.1. NOTA: NP Gold-BSA-rodamina può anche essere aggiunto alle cellule prima dell&#39;infezione, o in vari punti temporali pi </li><li> Dopo 1 ora di incubazione, rimuovere il terreno, lavare 3 volte con PBS e aggiungere 300 ml di mezzo fresco contenente immagini 10% FCS e 10 ug / ml gentamicina per il resto del tempo di incubazione. NOTA: Durata di incubazione può variare a seconda della concentrazione di NP e linee cellulari utilizzate. Per macrofagi RAW264.7, abbiamo osservato che 30 min incubazione con NP ad una concentrazione di OD 520 = 0,05 consentito internalizzazione sufficiente. </li></ol> 6. Imaging <ol><li> Utilizzare un sistema di imaging confocale come un confocale a scansione laser (CLSM) o disco rotante (SD) microscopio con una camera umidificata ambiente per immagini ad alta risoluzione in diversi punti temporali. </li><li> Accendere la temperatura control sistema e attendere che sia stabile. Ottimizzare le impostazioni di imaging come l&#39;ingrandimento, velocità di scansione, la risoluzione, Z-stack ecc Utilizzare adeguate impostazioni di eccitazione / emissione per GFP e NP gold-BSA-rodamina. Per questo protocollo, utilizzare una velocità di scansione di 400 Hz, risoluzione di 512 x 512 pixel e formato Z-passo di 0,25 micron. Excite GFP e oro-BSA-rodamina utilizzando un laser Ar (488 nm) e un laser HeNe (543 nm), rispettivamente. Include un campo chiaro (BF) canale per osservare la forma delle cellule. Per gli altri sistemi di microscopia e condizioni di infezione, l&#39;impostazione deve essere adeguato di conseguenza. NOTA: utilizzare lo stesso laser Ar come la sorgente luminosa del canale BF e il segnale è stato rilevato con un rilevatore fotomoltiplicatore (PMT). </li><li> A indicata punti temporali pi, montare le diapositive camera 8 pozzetti contenenti cellule infettate sul palco microscopio e registrare le immagini. </li></ol> 7. Analisi di Immagini <ol><li> Utilizzare microscopia software di analisi dell&#39;immagine (vedi <strong&gt; Materiali e Table di Equipment) per analizzare il rimodellamento del sistema endo-lisosomiale by intracellulare Salmonella. Aprire i dati cliccando su &#39;Open&#39; e scegliendo il file. NOTA: In alternativa, i pacchetti di software open source, come ImageJ o FIJI possono essere utilizzati per l&#39;immagine analisi quantitative. </li><li> Nella barra degli strumenti oggetti del click Surpass vista sull&#39;icona <img alt="Icona" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> aggiungere un nuovo elemento di superficie. Clicca su <img alt="Icona" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Il Prossimo). </li><li> Per analizzare una regione di interesse (ROI), selezionare un segmento ROI. In un area di visualizzazione, una sezione rettangolare, delimitata sovrapposto sull&#39;immagine sta rappresentando il ROI. Immettere i valori nelle direzioni x corrispondente, Y e campi Z-, o cliccare direttamente sulle frecce nel rettangolo di anteprima per modificare le dimensioni e la posizione della ROI. Quindi fare clic su <img alt="Icona" fo:content-width=".1in" src<html> = &quot;/ Files / ftp_upload / 52058 / 52058icon2.jpg&quot; /&gt; (Avanti). </li><li> Come canale sorgente, selezionare Canale 3 (segnale per Gold-BSA-Rhodamine NP). Selezionare l&#39;opzione liscio per impostare la morbidezza della superficie risultante. Definire un valore manualmente o accettare il valore generato automaticamente. Per Soglia, selezionare l&#39;opzione Intensità Assoluto. </li><li> Per la regolazione della soglia, selezionare l&#39;opzione manuale e impostare un valore. Nell&#39;area di visualizzazione, un&#39;anteprima soglia superficie viene visualizzato in grigio. </li><li> Sulla scheda Classificare Superfici superficie risultante può essere filtrato da vari criteri. Un filtro predefinito è &#39;il numero di voxel&gt; 10&#39;, e altri filtri può essere inclusa, cliccando su &#39;Aggiungi&#39;. Se un filtraggio non è necessario, quindi eliminare tutti i filtri facendo clic sul pulsante Elimina. Fare clic <img alt="Icona" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Il Prossimo). </li><li> Per completare la creazione di superficie, cliccate su/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt; (Fine). Nell&#39;elenco Oggetto, ora deselezionare la casella per il volume prodotto e nuova superficie creata viene visualizzata nell&#39;area di visualizzazione. </li><li> Esportare le statistiche. Ora, nella vista superare i colori dei soggetti variano da porpora a rosso in base alla loro superficie. E il tavolo &#39;Numbers Superficie Totale&#39; mostra le informazioni statistiche (ad esempio, il numero ID e l&#39;area degli oggetti). Esportare tutte le statistiche di superficie 1 in un file Excel per click <img alt="Icona" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Save). </li></ol>

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No conflicts of interest declared.

Il sistema endo-lisosomiale di cellule di mammiferi controlla importanti processi fisiologici, tra cui l&#39;assorbimento dei nutrienti, la trasduzione del segnale ormonale mediata, sorveglianza immunitaria, e l&#39;antigene presentazione 17. Fino ad ora, una varietà di marcatori sono stati utilizzati per l&#39;etichettatura dei pathway studi e monitoraggio endocitici. Ad esempio, le sonde LysoTracker sono sonde fluorescenti acidotropic sviluppati da Molecular Probes (Life Technologies, USA) per l&#39;etiche...

Nanoparticelle fluorescenti (NP), comprese le nanoparticelle di metallo, i punti quantici, nanoparticelle polimeriche, NP di silice, punti di carbonio, ecc, hanno attirato una notevole attenzione nel corso degli ultimi decenni 1,2. Rispetto ai tradizionali coloranti organici, NP fluorescenti mostrano chimiche, proprietà ottiche e meccaniche desiderabili, come forte intensità del segnale, resistenza photobleaching ed alta 3,4 biocompatibilità. Questi vantaggi loro il metodo di scelta per il rilevamento intracellulare e imaging cellulare dal vivo fanno. Inoltre, una varietà di elettroni NP-dense sono visibili mediante microscopia elettronica (EM), facilitando il loro utilizzo per l&#39;analisi microscopica correlata, che consente combinazione di cellule vive di inseguimento con microscopio ottico (LM) e la risoluzione superiore a livello ultrastrutturale con EM 5. Ad esempio, NP oro sono stati da tempo utilizzati in modo efficiente come biosensori nelle cellule sensibili per la diagnosi vita così come nel campo della immuno-etichettatura 6. S recentiTUDI indicano che NP oro con forme e dimensioni diverse possono essere facilmente assorbimento da una grande varietà di linee cellulari e trasportare regolarmente attraverso la via endosomal, hanno quindi grande essere potenziale applicato per il monitoraggio del trasporto intracellulare di vescicole e l&#39;etichettatura sistema endo-lisosomiale 7,8 . Agenti patogeni microbici, come la Salmonella enterica, Shigella flexneri e Listeria monocytogenes, hanno sviluppato diversi meccanismi di invadere le cellule ospiti non fagocitiche 9. Dopo essere stato interiorizzato, gli agenti patogeni, sia localizzate nel citoplasma o sequestrati in compartimenti di membrana, interagiscono molto con i loro ambienti host e modulano questi per favorire la propria sopravvivenza 10. Ad esempio, Salmonella enterica risiede e replica all&#39;interno di un vano phagosomal intracellulare definito il -contenenti vacuolo Salmonella (SCV) dopo l&#39;infezione 11. Il SCV stagionaturatraffici verso l&#39;apparato di Golgi, subendo continue interazioni con il percorso endocitica, e induce la formazione di estese strutture tubolari, come Salmonella filamenti -indotta (SIF), ordinamento tubuli nexin, Salmonella carrier secretoria indotta proteine ​​di membrana (3) SCAMP3 tubuli, etc . 12-14. Studiare come questi batteri patogeni manipolano percorsi host-cellule è fondamentale per la comprensione di malattie infettive. Qui, NP gold-BSA-rodamina sono stati utilizzati come traccianti fluido per etichettare il cellulare sistema endo-lisosomiale di accoglienza, e l&#39;umano patogeno gastrointestinale Salmonella enterica sierotipo Typhimurium (Salmonella) è stato utilizzato come modello batterio per studiare le interazioni del patogeno con il ospiterà pathway endocitico. Intracellulari NP gold-BSA-rodamina nelle cellule e le cellule non infette infettate con WT Salmonella o ceppi mutanti sono stati ripresi da un microscopio confocale a scansione laser (CLSM).Poi software Imaris è stato utilizzato per quantificare la distribuzione di NP, che indica che l&#39;infezione da Salmonella indotto estrema riassetto endosomi / lisosomi. Dopo la descrizione di questo metodo, gli esperimenti analoghi possono essere progettati per tenere traccia destino a lungo termine delle NP interiorizzato e per indagare l&#39;influenza di varie sostanze esogene o fattori endogeni sulla via endocitica delle cellule eucariotiche.

<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

Nanoparticelle fluorescenti (NP) con proprietà ottiche e meccaniche chimiche desiderabile, sono strumenti promettenti per etichettare organelli intracellulari. Qui, si introduce un metodo che utilizza oro-BSA-rodamina PN per etichettare il sistema endo-lisosomiale di cellule eucariotiche e monitorare manipolazioni di accoglienza vie cellulari dal patogeno intracellulare Salmonella enterica. Le NP sono stati prontamente internalizzati dalle cellule HeLa e localizzate in tardo endosomi / lisosomi. Infezione da Salmonella indotto riassetto delle vescicole e accumulo di NP in strutture a membrana Salmonella- indotte. Abbiamo implementato il pacchetto software Imaris per analisi quantitative di immagini di microscopia confocale. Il numero di oggetti e la loro distribuzione dimensionale in cellule non infette erano distinte da quelle in cellule -infected Salmonella, indicando estremamente rimodellamento del sistema endo-lisosomiale by WT Salmonella.

NP d&#39;oro sono stati generati attraverso un metodo consolidato attraverso la riduzione dell&#39;acido cloroaurico per citrato e acido tannico. Come mostrato in Figura 2A, PN oro sintetizzati erano quasi-forma sferica con una dimensione di circa 10 nm. BSA-rivestimento e rodamina-etichettatura non hanno influenzato la loro morfologia e dimensione (Figura 2B). E &#39;stato riportato che le NP d&#39;oro possono essere facilmente assorbiti da varie cellule di...

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

Questo articolo descrive i metodi per la sintesi e l&#39;etichettatura fluorescente di nanoparticelle (NP). Le NP sono stati applicati in esperimenti di pulse-chase di etichettare il sistema endo-lisosomiale di cellule eucariotiche. Manipolazione del sistema endo-lisosomiale dalle attività del patogeno intracellulare Salmonella enterica sono state seguite da imaging cellulare dal vivo e quantificato.

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

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 Views.  Universität Osnabrück.  Questo articolo descrive i metodi per la sintesi e l'etichettatura fluorescente di nanoparticelle (NP). Le NP sono stati applicati in esperimenti di pulse-chase di etichettare il sistema endo-lisosomiale di cellule eucariotiche. Manipolazione del sistema endo-lisosomiale dalle attività del patogeno intracellulare Salmonella enterica sono state seguite da imaging cellulare dal vivo e quantificato. 

Video: 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.

Rilevazione multiplex di batteri in campioni clinici complessi ed Ambientali con oligonucleotidi accoppiati microsfere fluorescenti

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

Ricerca

Immunologia e infezioni

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.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

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.

Misure singole cellule di Rottura Vacuolar causate da patogeni intracellulari

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.

Istituzione di un<em&gt; In vitro</em&gt; Sistema per studiare intracellulare Comportamento<em&gt; Candida glabrata</em&gt; In umani THP-1 macrofagi

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.

L&#39;applicazione di un sistema di espressione inducibile per studiare interferenza di fattori batterici di virulenza con segnalazione intracellulare

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 ...

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.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

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.

Sviluppo di un sistema di consegna economico DNA da &quot;Acufection&quot; e la sua applicazione alla ricerca della pelle

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.

Sistema per l&#39;efficacia e la citotossicità Proiezione di inibitori di targeting intracellulare<em&gt; Mycobacterium tuberculosis</em

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

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.

Un nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini 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 ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

La macchiatura di Immunohistochemical fluorescente multiplex, Imaging e analisi in campioni istologici di linfoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Modelli di colonizzazione vaginale murina da parte di batteri cresciuti anaerobicamente

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...