Ringraziamo H. Fleming per la lettura critica e la modifica del manoscritto. Questo lavoro è stato sostenuto da una borsa di studio post-dottorato Misrock (TD) e NDSEG Graduate Fellowship (AP). BNS è un investigatore HHMI.

1. Preparazione delle cellule <ol><li> Linee cellulari passaggio utilizzando tecniche standard di coltura cellulare. In questo esperimento, abbiamo utilizzato OVCAR-8 (NCI DCTD Tumore Repository, Frederick, MD). Coltivare le cellule a un target confluenza di 80 - 100%. </li><li> Incubare le cellule con 5 ml di tripsina per 5 minuti, quindi aggiungere 5 ml di terreno RPMI + FBS per inattivare la tripsina. </li><li> Celle di raccolta e di contare su un emocitometro. Risospendere in rosso fenolo-DMEM privo bersaglio ad una concentrazione di 5 x 10 7 cellule / ml, o circa 200 pl per bombola. </li><li> Aggiungere 15% ridotta del fattore di crescita Matrigel (BD Biosciences) e mantenere la sospensione cellulare in ghiaccio fino all&#39;impianto. </li></ol> 2. Impianto del tumore <ol><li> Anestetizzare 4 settimane vecchia femmina Ncr / Nu topo, utilizzando 3% isoflurano e attendere circa 5 minuti per garantire l&#39;anestesia profonda. Utilizzare vet unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Caricare le cellule (100 microlitri per tumore) in una siringa da 1 ml e allegare un 27 1/ 2 ago calibro. </li><li> Per ciascuno dei due siti di iniezione fianchi posteriori bilaterali, sollevare delicatamente la pelle con una pinza per fare una tenda e iniettare le cellule alla base. Fare attenzione a non penetrare troppo in profondità, producendo sangue. Utilizzare le pinzette per rimuovere delicatamente la siringa senza spruzzi. </li><li> Monitorare la crescita del tumore al giorno per 10-20 giorni fino a un diametro del tumore di 2 - viene raggiunto da 4 mm. </li></ol> 3. Batteri Preparazione <ol><li> Avviare una cultura durante la notte di batteri in 3 ml di LB + ampicillina multimediale da un -80 C magazzino congelatore o piastra refrigerata. In questo esperimento, abbiamo utilizzato S. typhimurium ceppo ELH430 (SL1344 PhoPQ-Aroa-). </li><li> La mattina, diluire la cultura 1:100 in LB filtrata e crescere per OD600 0,4-0,6. </li><li> Spin down e lavare 3 volte in tampone fosfato salino (PBS) e risospendere ad una finale OD600 di 0,1 (circa 1 x 10 7 cellule / ml). </li></ol> 4. Batteri Iniezione <ol><li> Anestetizzare l&#39;animale unposizione d su un lato con la coda rivolta verso la mano dominante. Utilizzare vet unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Dilatare la vena caudale utilizzando acqua calda o una lampada di calore. </li><li> Caricare i batteri (100 microlitri per topo) in una siringa da 1 ml e piegare la punta poco meno di 90 gradi. </li><li> Allineare la punta della siringa con la vena della coda, penetrare a bassa profondità (dovrebbe sentire alcuna resistenza), e iniettare batteri. Osservare lo spostamento flusso sanguigno per una iniezione di successo. </li></ol> 5. Topo Imaging <ol><li> Anestetizzare gli animali nella camera di induzione quindi posizionare ogni mouse sulla piattaforma di imaging. Utilizzare vet unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. Mantenere il posizionamento preciso per garantire risultati quantitativi tra i punti di tempo. </li><li> Animali immagine utilizzando il sistema di imaging Spectrum IVIS (Caliper Life Sciences). Se l&#39;imaging più di 1 animale, luogo fotocellule tra di loro per evitare cross-illuminazione. Animali dovrebbero essere ripreso prima sul lato ventrale per confermare la localizzazione di batteri. </li><li> Animali Immagine dorsalmente con IVIS ogni 2 ore per la durata dell&#39;esperimento (36 ore). Generare un corso di tempo per un dato ROI. </li></ol> 6. Quantificare Biodistribuzione batterica <ol><li> Euthanize animali utilizzando CO 2 e posto sul retro su un pannolino assorbente. </li><li> Girare l&#39;animale per esporre i due tumori posteriori fianco sottocutanei. Tagliare il tessuto della pelle circostante per separare e rimuovere i tumori. Tenendo il tumore con una pinzetta, togliere la pelle con un movimento a forbice rovesciata. Quando la maggior parte della pelle è stata separata, rimuovere completamente il tumore usando pinzette. </li><li> Posizionare gli organi in provette da microcentrifuga pre-pesati. Poiché la massa di questi tubi può variare significativamente è importante per loro pre-pesare per risultati quantitativi. </li></ol> 7. Quantificare Biodistribuzione batterica <ol><li> Per ogni punto, accise e pesare i tumori. </li><li> Aggiungere 500 microlitri di PBS + 15% glicerolo e omogeneizzare con un tessuto-Tearor (BioSpec). Sciacquare l&#39;omogeneizzatore 3 volte con etanolo tra i campioni. </li><li> Generare diluizioni seriali e piastra per il conteggio delle colonie batteriche. Per piastra diluizioni multiple su un&#39;unica piastra Usare piatti pre-sezionati. </li></ol>

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Nessun conflitto di interessi dichiarati.

Utilizzando questa procedura, siamo in grado di generare corsi di tempo per la crescita e la dinamica di espressione genica dei batteri colonizzano i tumori. Mentre queste misurazioni sono eseguite di routine in vitro in coltura batch o dispositivi microfluidici, sono molto più difficili da eseguire in vivo. Ci sono diverse modifiche che possono essere applicate a questi metodi. Mentre abbiamo utilizzato la linea cellulare OVCAR-8 per generare nostri modelli murini, un cer...

La biologia sintetica è progredita rapidamente nel corso degli ultimi dieci anni ed è ora posizionato a impatto dei problemi importanti nella energia e salute. Tuttavia, l&#39;espansione in ambito clinico è stato rallentato da problemi di sicurezza e l&#39;assenza di sviluppo di criteri di progettazione per circuiti genetici in vivo. Accelerare le applicazioni mediche di grande impatto richiede metodi che si interfacciano direttamente con infrastrutture mediche, circuiti genetici che funzionano al di fuori del setting di laboratorio controllato, e padroni di casa microbici sicuri e clinicamente accettato che utilizza. Un numero di ceppi sono state studiate per la terapia del cancro a causa della loro capacità di crescere preferenzialmente nei tumori. Questi hanno incluso C. novyi, E. coli, V. cholorae, B. longum, e S. typhimurium 3-8. S. typhimurium ha generato particolare interesse come hanno esibito la sicurezza e la tolleranza in un certo numero di prove cliniche umane 9-12. Questi batteriologiche<html> un stati inizialmente mostrati per creare effetti antitumorali attraverso la stimolazione del sistema immunitario dell&#39;ospite e dalla deplezione di sostanze nutritive necessarie per il metabolismo delle cellule del cancro. Produzione di merci terapeutico è stato poi aggiunto attraverso modificazioni genetiche. Mentre questi studi rappresentano importanti progressi nell&#39;uso di batteri per le terapie tumorali, la maggior parte degli sforzi esistenti hanno fatto affidamento su un&#39;espressione di alto livello, che in genere si traduce nella fornitura di alti dosaggi, effetti off-target, e lo sviluppo di resistenza dell&#39;ospite 13-16 . Ora, la biologia sintetica può aggiungere la produzione del carico programmabile utilizzando circuiti genetici computazionalmente-progettati in grado di eseguire il rilevamento avanzate e di consegna 17-20. Questi circuiti possono essere progettati per agire come sistema di consegna che rilevano gli stimoli tumore-specifici e di auto-regolare la produzione di merci, se necessario. Tuttavia, studiando la funzione di questi circuiti in vivo è stato quindi finora impegnativo. ve_content &quot;&gt; Dal plasmidi sono il quadro di riferimento comune per i circuiti sintetici, si descrive un metodo per caratterizzare la dinamica di espressione genica plasmide-based in vivo, utilizzando un modello di topo. Questi metodi utilizzano time-lapse luminescenza imaging e misurazione quantitativa di biodistribuzione. Insieme, questi approcci forniscono un quadro per studiare le reti basate plasmide in vivo per applicazioni cliniche.

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1 ml syringe</td>
 <td>BD Biosciences</td>
 <td>309602</td>
 <td></td>
 </tr>
 <tr>
 <td>3/10 cc Insulin Syringe</td>
 <td>BD Biosciences</td>
 <td>309301</td>
 <td></td>
 </tr>
 <tr>
 <td>PrecisionGlide Needle </td>
 <td>BD Biosciences </td>
 <td>301629</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 (1X), 
 liquid, with L-glutamine</td>
 <td>Invitrogen</td>
 <td>11875-119 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sigma Aldrich</td>
 <td>A0166-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>LB Agar Broth</td>
 <td>Sigma Aldrich</td>
 <td>L2897</td>
 <td></td>
 </tr>
 <tr>
 <td>Luria-Bertani broth</td>
 <td>BD Biosciences</td>
 <td>244610</td>
 <td></td>
 </tr>
 </tbody>
</table>

measuring growth and gene expression dynamics of tumor-targeted s. typhimurium bacteria

L&#39;obiettivo di questi esperimenti è generare dati quantitativi di andamento sulla crescita e la dinamica di espressione genica di S. attenuato typhimurium colonie batteriche che crescono all&#39;interno dei tumori. Abbiamo generato tumori trapiantati in topi modello per iniezione sottocutanea di una linea cellulare di carcinoma ovarico umano, OVCAR-8 (NCI DCTD Tumore Repository, Frederick, MD). Abbiamo trasformato ceppi attenuati di S. batteri typhimurium (ELH430: SL1344 phoPQ-1) con una luciferasi costitutivamente espresso (luxCDABE) plasmide per la visualizzazione 2. Questi ceppi specificamente colonizzano tumori rimanendo essenzialmente non virulento al mouse 1. Una volta tumori misurabili sono stati stabiliti, i batteri sono stati iniettati per via endovenosa attraverso la vena della coda con diversi dosaggi. Tumore localizzato, espressione del gene batterico è stata monitorata in tempo reale nel corso di 60 ore utilizzando unnel sistema di imaging in vivo (IVIS). Ad ogni tempo, i tumori sono stati asportati, omogeneizzato, e piastrate per quantificare colonie batteriche per la correlazione con i dati di espressione genica. Insieme, questi dati produce una misura quantitativa della crescita in vivo e la dinamica di espressione genica dei batteri in crescita all&#39;interno dei tumori.

Usando questo protocollo, siamo in grado di generare dati sulla crescita in vivo e la dinamica di espressione genica dei batteri mirati tumorali. Il flusso di lavoro generale è riassunto in Figura 1. Nella prima fase, abbiamo iniettare batteri (verde) e l&#39;immagine dell&#39;animale utilizzando IVIS per misurare l&#39;espressione genica con luminescenza (blu) come reporter. Poi, nella seconda fase, abbiamo accise, omogeneizzare, e tum...

Watch this Scientific Journal Video about Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria at JoVE.com

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

L&#39;obiettivo di questi esperimenti è generare dati quantitativi di andamento sulla crescita e la dinamica di espressione genica di attenuato<em&gt; S. typhimurium</em&gt; Colonie batteriche che crescono all&#39;interno dei tumori. Questo video illustra la preparazione delle cellule tumorali e l&#39;impianto, la preparazione e l&#39;iniezione di batteri, imaging intero animale luminescenza, l&#39;asportazione del tumore, e la colonia batterica conteggio.

Crescita di misura e di espressione genica Dinamica di tumore-mirata<em&gt; S. Typhimurium</em&gt; Batteri

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

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Immunology and Infection

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

12.0K Views.  Massachusetts Institute of Technology. L'obiettivo di questi esperimenti è generare dati quantitativi di andamento sulla crescita e la dinamica di espressione genica di attenuato S. typhimurium Colonie batteriche che crescono all'interno dei tumori. Questo video illustra la preparazione delle cellule tumorali e l'impianto, la preparazione e l'iniezione di batteri, imaging intero animale luminescenza, l'asportazione del tumore, e la colonia batterica conteggio.

Video: Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

Utilizzo di proteine ​​fluorescenti per visualizzare e Quantificazione<em&gt; Chlamydia</em&gt; Vacuolo crescita dinamica in cellule viventi

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

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Immunologia e infezioni

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Vaccinia Reporter virus per quantificare Funzione virale in tutte le fasi di espressione genica

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

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 ...

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

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

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

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

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 ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento di murini peritoneale macrofagi di effettuare analisi di espressione genica Al Toll-like recettori stimolazione

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

Profili di espressione genica di microbi che infettano utilizzare una piattaforma digitale di codificazione

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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. ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Microscopia a fluorescenza a cellule vive per studiare la localizzazione delle proteine subcellulari e i cambiamenti della morfologia cellulare nei batteri

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

NF-&amp;#954;B-dependent Luciferase Activation and Quantification of Gene Expression in &lt;em&gt;Salmonella&lt;/em&gt; Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Attivazione e quantificazione dell'espressione genica infette da NF-B nelle cellule di coltura dei tessuti infetti

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...