Questo progetto è stato sostenuto in parte da una sovvenzione per i National Institutes of Health NIAID (per AJB e LGA, R01 AI076246). La collezione mutante di Salmonella è stato in parte sostenuto dal National Institutes of Health sovvenzioni (per MM, U01 A152237-05, R01, R01 AI07397-01 AI039557-11 e R01 AI075093-01), in parte dal National Institutes of Health sovvenzioni (per HAP, R21 AI083964-01, 1R0 1AI083646-01, 1R56AI077645, R01 AI075093). Ringraziamo Steffen Prowollik per la placcatura replica e confermando i mutanti della collezione.

1 murino macrofagi RAW264.7 Colture Cellulari <ol><li> Crescere basso numero passaggio macrofagi murini, RAW264.7 (Il numero ATCC ®, TIB-71) in una coltura cellulare pallone ventilato cap T-75 filtro Dulbecco Modified Eagle Medium (DMEM) supplementato con 10% di siero fetale bovino (FBS) , 0,5% NaHCO 3 e 1% 100x non essenziali aminoacidi (NEAA) a 37 ° C, in un incubatore CO 2 5%. </li><li> Una volta che le cellule raggiungono una confluenza del 60-80% nel pallone, utilizzare un raschietto cella per raccogliere le cellule, e contare le cellule in un emocitometro e calcolare la concentrazione cellulare. </li><li> Risospendere le cellule e diluire la concentrazione cellulare in 2,5 x 10 5 / ml in mezzo di coltura cellulare fresca DMEM. Utilizzare pipette multicanale al piatto 200 ml di mezzo di coltura cellulare DMEM contenente 5 x 10 4 cellule in ciascun pozzetto di 96 pozzetti piastre di coltura cellulare. </li><li> Piatto quattro pozzi di ogni ceppo Salmonella. Impostare tre piatti separati per un setdi phagocytic dosaggio cellule invasione, e contrassegnarli con &quot;associazione&quot;, &quot;invasione&quot; e &quot;replica&quot;, rispettivamente. </li><li> Posizionare le piastre del 5% di CO 2 incubatore a 37 ° C ON per consentire alle cellule macrofagi per attaccare al fondo dei pozzetti. </li></ol> 2 Preparazione di Salmonella wild-type e mutanti <ol><li> Lo stesso giorno della preparazione delle cellule macrofagi RAW264.7, raccogliere singole colonie di wild-type (WT) Salmonella enterica sierotipo Typhimurium 14028s, DinvA mutanti, DphoP mutanti, e ceppi mutanti di prova desiderati, e la cultura li in Luria-Bertani ( LB) brodo integrato con antibiotici come appropriato. Usare ogni set dei fagociti saggi cellule invasione di testare fino a 10 ceppi mutanti (DinvA mutante e DphoP mutanti sono difettosi in invasione e replicazione intracellulare, rispettivamente). </li><li> Crescere i batteri per 14 ore a 37 ° C con sHaking a 220 rpm in 5 ml di brodo LB in vagamente 14 ml con tappo in polipropilene in una provetta a fondo tondo. LB brodo contiene antibiotici appropriati, nel nostro caso, la maggior parte dei ceppi mutanti sono resistenti alla kanamicina, 50 mg / ml </li><li> Il giorno successivo, Subculture ciascuna delle ON colture di ceppi di Salmonella in pianura 5 ml di brodo LB in un rapporto di 1:50 per un ulteriore 4 ore con agitazione a 220 rpm. </li><li> Leggi OD 600 valori di ogni coltura batterica su uno spettrofotometro, e ogni lettura dovrebbe variare 0,6-1,2 per ottimizzare Salmonella patogenicità isola-1 di tipo III, sistema di secrezione (SPI-1 TTSS) l&#39;espressione del gene per l&#39;invasione. </li><li> Utilizzare la formula di 1 OD 600 = 7,5 x 10 8 UFC / ml di valutare la concentrazione batterica, poi diluire batteri ad una concentrazione di 5 x 10 6 UFC / ml in mezzo di coltura cellulare fresca DMEM. </li></ol> 3 Invasion Assay in 96 pozzetti Cultura Piatto <ol><li> Rimuovere le tre cel 96 pozzettipiastre di l di coltura dalla CO 2 incubatore e lavare i pozzetti una volta con 200 ml di 1x tampone PBS. </li><li> Dopo il lavaggio e la rimozione completa di PBS, aggiungere 200 microlitri batteri in terreno DMEM (vedi sopra) in ogni pozzetto. Ciò si traduce in 1 x 10 6 cellule di Salmonella in ciascun molteplicità bene e di infezione (MOI) di 20: 1. </li><li> Centrifugare le piastre a 1000 xg in un vettore sigillata per 10 minuti, quindi sostituire piastre (tempo zero) a 37 ° C, 5% CO 2 incubatore per 30 min. Per ogni ceppo, istituito almeno quattro pozzetti in duplicato. </li><li> Dopo 30 minuti, togliere le piastre dal termostato e lavare tre volte con 200 ml di PBS 1x per rimuovere i batteri non associati. </li><li> Dopo il lavaggio, salvare la piastra etichettato con &quot;associazione&quot; per un ulteriore trattamento. Aggiungere 200 ml di terreno DMEM contenente 100 mg / ml di gentamicina in ogni pozzetto delle piastre contrassegnati con &quot;invasione&quot; e &quot;replica&quot; per to uccidere la salmonella extracellulare. Riportare le piastre a 37 ° C, 5% CO 2 incubatore per un ora supplementare. </li><li> Salvare un bene da ogni ceppo infetto per registrare la conta delle cellule macrofagi, trattare il resto dei pozzi sul piatto &quot;associazione&quot; con 200 ml di 1x PBS contenente 1% Triton X-100 per 10 min. La soluzione Triton lisare cellule macrofagi e rilasciare Salmonella associata. </li><li> Harvest Salmonella da ogni pozzetto e metterli in 1,5 ml provette Eppendorf. Eseguire tre 10 diluizioni seriali di piegatura con 900μl 1x PBS sui campioni raccolti da ogni pozzetto, vortice viene eseguita tra ogni diluizione. </li><li> Piatto terza diluizione, 10 -3, sia su LB (WT) o LB Kan (mutanti) piatti. Etichettare le piastre con un&#39;adeguata nome Salmonella ceppo, numero di diluizione, il punto ora e la data. </li><li> Dopo vortex la terza provetta di diluizione, dispensare campione di 10 microlitri sulla superficie del agar e ripetere fnostri tempi per un totale di 5 gocce. Assicurarsi di spazio cinque 10 microlitri scende in modo uniforme sulle piastre di Petri piatto 7. </li><li> Dopo le gocce penetrare nel agar, girare i piatti più e incubare una notte a 37 ° C, 5% CO 2 incubatore. </li><li> Trattare pozzi salvati per il conteggio dei macrofagi con 150 1XPBS microlitri contenenti 0,25% tripsina-EDTA per 10 min. </li><li> Dopo tripsinizzazione, posto macrofagi in 1,5 ml provette Eppendorf e li trattano con 50 ml di FBS per neutralizzare soluzione di tripsina / EDTA, colorano le cellule con 0,4% Trypan blu e punteggio attraverso l&#39;utilizzo emocitometro. </li><li> Una volta trascorso il incubazione di un hr, rimuovere il &quot;invasione&quot; e piatti &quot;replica&quot; dal CO 2 incubatore, lavare i pozzetti come &quot;Step 3.4&quot;. </li><li> Salvare la piastra &quot;invasione&quot; per un ulteriore trattamento come &quot;Step 3,6-3,12&quot;. </li><li> Aggiungere 200 ml di terreno DMEM contenente 10 mg / ml di gentamicina perla piastra &quot;replica&quot; per mantenere la clearance della Salmonella extracellulare nel mezzo. Riportare la piastra a 37 ° C, 5% CO 2 incubatore per un ulteriore 22,5 ore. </li><li> Il giorno seguente, rimuovere la piastra &quot;replica&quot; di 37 ° C, 5% CO 2 incubatore, lavare i pozzetti come &quot;Step 3.4&quot; e trattarli come &quot;Step 3,6-3,12&quot;. </li><li> Infine, rimuovere le piastre di agar dopo incubazione overnight a 37 ° C incubatore e segnare il numero di colonie batteriche. </li><li> Una buona distribuzione delle colonie deve essere compresa tra 10 e 100 colonie, ogni punto contiene circa 2 a 20 colonie, sarebbe difficile segnare se ci fossero più di 20 colonie su un punto. Se il conteggio è troppo bassa o troppo alta, replate il campione con 10-diluizioni maggiori o minori a seconda delle necessità. </li></ol> Analisi 4. Dati <ol><li> Calcolare i (unità di formazione di colonie per ml) CFU di ogni piatto in base alla placcaturavolume e fattore di diluizione. </li><li> Accertare il numero di Salmonella per macrofagi attraverso la conta delle cellule macrofagi registrato da ogni ceppo. </li><li> Calcolare le medie geometriche della CFU per macrofagi di almeno tre esperimenti indipendenti per associazione cellulare, invasione o la replica rispettivamente, e normalizzare ulteriormente la WT per il valore relativo. </li><li> Analizzare i dati per l&#39;associazione, l&#39;invasione, e la replica da parte di Student t-test, rispettivamente, confrontando ogni ceppo di WT, e determinare la significatività statistica del p-value. </li></ol>

<ol>
	<li>Haraga, A., Ohlson, M. B., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonellae+interplay+with+host+cells.">Salmonellae interplay with host cells.</a> Nat Rev Microbiol. 6, 53-66 (2008).</li><li>Mandell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+intraleukocytic+bacteria+and+antibiotics.">Interaction of intraleukocytic bacteria and antibiotics.</a> The Journal of clinical investigation. 52, 1673-1679 (1973).</li><li>Elsinghorst, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+invasion+by+gentamicin+resistance.">Measurement of invasion by gentamicin resistance.</a> Methods Enzymol. 236, 405-420 (1994).</li><li>Elsinghorst, E. A., Weitz, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+invasion+and+adherence+directed+by+the+enterotoxigenic+Escherichia+coli+tib+locus+is+associated+with+a+104-kilodalton+outer+membrane+protein.">Epithelial cell invasion and adherence directed by the enterotoxigenic Escherichia coli tib locus is associated with a 104-kilodalton outer membrane protein.</a> Infect Immun. 62, 3463-3471 (1994).</li><li>Behlau, I., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PhoP-repressed+gene+promotes+Salmonella+typhimurium+invasion+of+epithelial+cells.">A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells.</a> J Bacteriol. 175, 4475-4484 (1993).</li><li>Hueck, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+typhimurium+secreted+invasion+determinants+are+homologous+to+Shigella+Ipa+proteins.">Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.</a> Mol Microbiol. 18, 479-490 (1995).</li><li>Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+epithelial+cells+with+Salmonella+enterica.">Infection of epithelial cells with Salmonella enterica.</a> Methods Mol Biol. 431, 201-211 (2008).</li><li>Foster, J. W., Spector, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Salmonella+survive+against+the+odds.">How Salmonella survive against the odds.</a> Annu Rev Microbiol. 49, 145-174 (1995).</li><li>Edwards, A. M., Massey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+human+cells+by+a+bacterial+pathogen.">Invasion of human cells by a bacterial pathogen.</a> Journal of visualized experiments JoVE. 10, (2011).</li><li>Molinari, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+played+by+the+group+A+streptococcal+negative+regulator+Nra+on+bacterial+interactions+with+epithelial+cells.">The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells.</a> Mol Microbiol. 40, 99-114 (2001).</li><li>Van der Velden, A. W., Lindgren, S. W., Worley, M. J., Heffron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+pathogenicity+island+1-independent+induction+of+apoptosis+in+infected+macrophages+by+Salmonella+enterica+serotype+typhimurium.">Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium.</a> Infect Immun. 68, 5702-5709 (2000).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet Pathol. 39, 200-215 (2002).</li><li>Lawhon, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+SPI-1+secreted+effectors+in+acute+bovine+response+to+Salmonella+enterica+Serovar+Typhimurium+a+systems+biology+analysis+approach.">Role of SPI-1 secreted effectors in acute bovine response to Salmonella enterica Serovar Typhimurium a systems biology analysis approach.</a> PLoS One. 6, e26869 (2011).</li><li>Drecktrah, D., Knodler, L. A., Galbraith, K., Steele-Mortimer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Salmonella+SPI1+effector+SopB+stimulates+nitric+oxide+production+long+after+invasion.">The Salmonella SPI1 effector SopB stimulates nitric oxide production long after invasion.</a> Cell Microbiol. 7, 105-113 (2005).</li></ol>

We have nothing to disclose.

Il test di protezione gentamicina è ampiamente usato per studiare l&#39;invasione e la replica di batteri patogeni intracellulari all&#39;interno della cellula ospite, ed è soprattutto un importante strumento biologico per lo studio di agenti patogeni, come la Salmonella, la cui invasione è il passo presupposto indispensabile per l&#39;infezione 1. La protezione dosaggio standard di gentamicina nella comunità di ricerca di Salmonella è implementato in 24-ben piatto cultura 5....

Salmonella non tifoidi sono importanti cause di malattie enteriche in tutti i vertebrati. Salmonellosi nell&#39;uomo è tra le prime malattie di origine alimentare batterica 1. Caratterizzazione dei meccanismi molecolari che sono alla base delle interazioni di Salmonella con i loro host di origine animale è ottenuta principalmente attraverso lo studio di Salmonella enterica sierotipo Typhimurium (STM) in colture di tessuti e modelli animali di infezione. Guadagnando intuizioni nelle interazioni STM-ospite ci aiuterà a capire come Salmonella sopravvivere e crescere all&#39;interno di cellule ospiti. La prima sfida nello studio di queste interazioni è quello di identificare il maggior numero possibile di fattori che partecipano sia da ospite e patogeno, ma questi sforzi sono in gran parte ostacolato dalle notevoli difficoltà di trattare con due sistemi biologici complessi indipendenti simultaneamente, vale a dire, di accoglienza e di Salmonella, in condizioni fisiologiche. Inoltre, la grande repertoOire di Salmonella e geni ospitanti potenzialmente codificanti fattori coinvolti in interazioni ospite necessitano di high-throughput piattaforma biologico per affrontare questa sfida. Un modificato, test standardizzato per fenotipo associazione Salmonella, l&#39;invasione e la replica nei macrofagi con capacità high-throughput è stato sviluppato per esaminare un ampio set di geni probabilmente impegnati in interazioni Salmonella -host. Il saggio di protezione gentamicina è stato sviluppato nel 1973 2, ma è stato accuratamente descritto da Elsinghorst nel 1994 3,4. Ora è diventato uno strumento standard per lo studio di molti batteri patogeni intracellulari ex vivo, tra cui Salmonella 5,6. Batteri internalizzati evitare di essere ucciso da alcuni antibiotici, come la gentamicina, che non possono penetrare le cellule eucariotiche 3. Sfruttando questo fenomeno, il saggio protezione Gentamicina misura la sopravvivenza e la crescita di ba intracellularepatogeni cterial. Tre eventi durante l&#39;infezione, cioè, l&#39;associazione con le cellule eucarioti, l&#39;invasione e la replica, possono essere valutati per batteri patogeni intracellulari in base l&#39;intervallo di tempo tra l&#39;infezione, il trattamento Gentamicina, e in seguito di incubazione (Figura 1). Linee di cellule eucariotiche forniscono un ambiente fisiologico che è meno complessa di modelli animali per studi di interazione ospite-patogeno. Il test di protezione gentamicina è una piattaforma appropriata per studiare le interazioni STM-ospite, ma il test standard in un piatto di coltura 24 pozzetti num bassa velocità. Analisi computazionale dei set di dati in vivo identificato 149 prodotti genici Salmonella che si prevede di interagire con circa 300 prodotti genici ospite (dati non pubblicati). Il test di protezione gentamicina standard non ha la capacità di fenotipo questo numero di mutanti in modo efficiente. In Additione, il saggio di protezione Gentamicina può teoricamente rilevare l&#39;invasione di anche un singolo batterio. A causa di questa sensibilità intrinseca, i dati grezzi sono suscettibili di variazioni tecniche quando ripetuto in tempi diversi. I controlli interni e la presentazione dei dati relativi dopo la normalizzazione sono essenziali per l&#39;interpretazione significativa dei risultati. Alla luce di queste considerazioni, una versione modificata, standardizzato test di protezione gentamicina è stato sviluppato per migliorare la capacità di test e aumentare la precisione. Il protocollo seguito è dettagliata e illustrata per eseguire il test di protezione Gentamicina modificato utilizzando 96 pozzetti piatti della cultura e la linea cellulare di macrofagi RAW264.7 murino. Rispetto al protocollo standard a 24 pozzetti piastre di coltura, il protocollo modificato presenta i seguenti vantaggi: 1) Uso di 96 pozzetti piatti della cultura permette fino a 10 diversi ceppi mutanti per essere phenotyped compresi i controlli interni positivi e negativi con potenza statistica sufficiente;2) La varianza dei risultati è notevolmente ridotta, in quanto i ceppi mutanti vengono testate nello stesso piatto cultura e allo stesso tempo; 3) L&#39;uso di pipette multicanale aumenta il throughput riducendo la fatica dell&#39;operatore. Infine, il confronto a 24 pozzetti piastre di coltura, le preoccupazioni di meno cellule ospiti per pozzetto a 96-ben piatto cultura sono state affrontate attraverso l&#39;ottimizzazione e la standardizzazione del protocollo. In sintesi, la modifica, test standardizzato per vitro fenotipo Salmonella o altra associazione batterica, l&#39;invasione e la replica in fagociti aumenta la precisione e raggiunge la capacità high-throughput riducendo la fatica dell&#39;operatore.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:173px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11965</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Fetal bovine serum</td>
			<td style="width:173px;">HyClone</td>
			<td style="width:173px;">SH30910.03</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">T-75 cell culture flask vented filter cap</td>
			<td style="width:173px;">Nest Biotechnology</td>
			<td style="width:173px;">708003</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">100X Non-Essential Amino Acids</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">11140</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Cell scraper</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">353086</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">96-well cell culture plate</td>
			<td style="width:173px;">Corning Incorporated</td>
			<td style="width:173px;">3595</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">Luria-Bertani (LB) broth</td>
			<td style="width:173px;">MP Biomedicals</td>
			<td style="width:173px;">3002-075</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:173px;">14 mL Polypropylene Round-Bottom Tube</td>
			<td style="width:173px;">BD Falcon</td>
			<td style="width:173px;">352059</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">PBS pH7.4 (1x)</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">10010</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Triton X-100</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T-8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Kanamycin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">K0254</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Gentamicin solution</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">G1272</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">0.25% Trypsin-EDTA</td>
			<td style="width:173px;">Life Technologies</td>
			<td style="width:173px;">25200</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:173px;">Trypan blue</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:173px;">T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput assay to phenotype salmonella enterica typhimurium association, invasion, and replication in macrophages

Le specie Salmonella sono agenti patogeni zoonotici e principali cause di malattie di origine alimentare negli esseri umani e bestiame 1. La comprensione dei meccanismi alla base delle interazioni Salmonella -host sono importanti per chiarire la patogenesi molecolare di infezione da Salmonella. Il dosaggio di protezione gentamicina a fenotipo associazione Salmonella, l&#39;invasione e la replica in fagociti è stato adattato per consentire lo screening high-throughput per definire i ruoli dei mutanti di delezione di Salmonella enterica sierotipo Typhimurium nelle interazioni host utilizzando RAW 264.7 macrofagi murini. Nell&#39;ambito di questo protocollo, la varianza nelle misurazioni è significativamente ridotto rispetto al protocollo standard, perché wild-type e più ceppi mutanti possono essere testati nello stesso piatto di cultura e, allo stesso tempo. L&#39;uso di pipette multicanale aumenta la produttività e migliora la precisione. Inoltre, le preoccupazioni relative a usare meno host cellule per pozzetto in 96-ben piatto cultura sono stati affrontati. Qui, il protocollo della vitro Salmonella invasione test modificati utilizzando fagociti è stato impiegato con successo per fenotipo 38 singoli mutanti di delezione Salmonella per l&#39;associazione, l&#39;invasione e la replica intracellulare. I fenotipi in vitro sono presentati, alcuni dei quali sono stati successivamente confermato di avere in fenotipi vivo in un modello animale. Pertanto, la modifica, test standardizzato per fenotipo Salmonella associazione, l&#39;invasione e la replica nei macrofagi con capacità high-throughput potrebbe essere utilizzato più ampiamente per studiare le interazioni batteri-ospite.

Vedere i risultati rappresentativi (Figura 2) dopo che i dati sono tracciate in base alla fagocitosi test invasione delle cellule modificate. I dati comprendono cinque diversi ceppi, WT, ΔinvA, ΔphoP, mutante A e B. mutante Δ Inva, noto per essere difettoso per l&#39;invasione, e ΔphoP noti per essere difettoso per la replica 8, sono utilizzati come controlli positivi per valutare la validità sperimentale . Infatti, nel saggio di invasione modificato, un mutant...

Watch this Scientific Journal Video about High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages at JoVE.com

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Un test high-throughput per fenotipo Salmonella in vitro o di altra associazione batterica, l&#39;invasione, e la replica in fagociti con capacità high-throughput è stato sviluppato. Il metodo è stato impiegato per valutare Salmonella gene knockout ceppi mutanti per il loro coinvolgimento in interazioni ospite-patogeno.

High-throughput Assay per Fenotipo<em&gt; Salmonella enterica</em&gt; Typhimurium Associazione, Invasion, e replica nei macrofagi

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

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Research

JoVE Journal

Immunology and Infection

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

12.5K Views.  Texas A&M University.  Un test high-throughput per fenotipo Salmonella in vitro o di altra associazione batterica, l'invasione, e la replica in fagociti con capacità high-throughput è stato sviluppato. Il metodo è stato impiegato per valutare Salmonella gene knockout ceppi mutanti per il loro coinvolgimento in interazioni ospite-patogeno. 

Video: High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Un Allelotyping PCR per l&#39;identificazione Salmonella enterica Sierotipi Enteritidis, Hadar, Heidelberg, e Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Ricerca

Immunologia e infezioni

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitativa High-throughput cella singola test di citotossicità per le cellule T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Un protocollo Infettare<em&gt; Caenorhabditis elegans</em&gt; Con<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Quantificazione dei citosolico vs. vacuolare<em&gt; Salmonella</em&gt; In primarie macrofagi di Permeabilization differenziale

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput di Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

Una fluorescenza a base di linfociti Assay Adatto per high-throughput screening di piccole molecole

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolamento di Salmonella typhimurium-contenente fagosomi dai macrofagi

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Una piattaforma di High-throughput per lo Screening della Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

Un'analisi di High-throughput per valutare e quantificare la trappola extracellulari del neutrofilo formazione

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...