Wir danken H. Fleming für die kritische Durchsicht und Bearbeitung des Manuskripts. Diese Arbeit wurde durch ein Postdoc-Stipendium Misrock (TD) und NDSEG Graduiertenstipendium (AP) unterstützt. SNB ist ein HHMI Investigator.

1. Zellpräparation <ol><li> Passage Zelllinien unter Verwendung von Standard-Zellkultur-Techniken. In diesem Experiment verwendeten wir OVCAR-8-Zellen (NCI DCTD Tumor Repository, Frederick, MD). Wachsen Zellen zu einem Ziel Konfluenz von 80 - 100%. </li><li> Inkubieren Zellen mit 5 ml Trypsin für 5 min, dann 5 ml RPMI-Medium + FBS Trypsin zu inaktivieren. </li><li> Ernte und Anzahl Zellen auf einer Zählkammer. Resuspendieren in Phenolrot-freiem DMEM auf ein Ziel Konzentration von 5 x 10 7 Zellen / ml oder etwa 200 ul pro Kolben. </li><li> In 15% reduzierten Wachstumsfaktor Matrigel (BD Biosciences) und halten Sie die Zellsuspension auf Eis bis zur Implantation. </li></ol> 2. Tumorimplantation <ol><li> Anesthetize 4 Wochen alten weiblichen Ncr / Nu-Mäusen unter Verwendung von 3% Isofluran und warten etwa 5 min zu tiefe Narkose zu gewährleisten. Verwenden Tierarzt Salbe auf die Augen zu Trockenheit während der Narkose zu verhindern. </li><li> Legen Sie die Zellen (100 ul pro Tumor) in einer 1 ml Spritze eine 27 1/ 2-Gauge-Nadel. </li><li> Für jede der beiden bilateralen hinteren Flanke Injektionsstellen, heben Sie die Haut vorsichtig mit einer Pinzette, um ein Zelt zu machen und injizieren Zellen an der Basis. Achten Sie darauf, nicht zu durchdringen zu tief, wodurch Blut. Mit einer Pinzette vorsichtig entfernen Spritze ohne Spritzer. </li><li> Überwachen Tumorwachstum täglich für 10 - 20 Tage, bis ein Tumor Durchmesser von 2 - 4 mm erreicht ist. </li></ol> 3. Bakterien Vorbereitung <ol><li> Starten Sie eine Übernacht-Kultur von Bakterien in 3 ml LB-Medium + Ampicillin aus einer -80 C Gefrierschrank Lager oder gekühlten Teller. Bei diesem Experiment haben wir S. typhimurium Stamm ELH430 (SL1344 PhoPQ-aroA-). </li><li> In der Früh, verdünnen die Kultur 1:100 in LB gefiltert und wachsen, um OD600 0.4 - 0.6. </li><li> Abzentrifugieren und wäscht 3 mal in phosphatgepufferter physiologischer Kochsalzlösung (PBS) resuspendiert und in einer endgültigen OD 600 von 0,1 (etwa 1 x 10 7 Zellen / ml). </li></ol> 4. Bakterien Injection <ol><li> Anesthetize das Tier eind Position auf seiner Seite mit der Schwanz zeigte auf Ihrer dominanten Hand. Verwenden Tierarzt Salbe auf die Augen zu Trockenheit während der Narkose zu verhindern. </li><li> Dilate die Schwanzvene mit warmem Wasser oder eine Wärmelampe. </li><li> Legen Sie die Bakterien (100 ul pro Maus) in einer 1 ml Spritze und biegen Sie die Spitze etwas weniger als 90 Grad. </li><li> Richten Sie die Spitze der Spritze mit der Schwanzvene, in geringer Tiefe (sollte kein Widerstand zu spüren ist) eindringen, und injizieren Bakterien. Beachten bloodflow Verschiebung für eine erfolgreiche Injektion. </li></ol> 5. Mouse Imaging <ol><li> Anesthetize Tiere in der Induktion Kammer dann platzieren Sie jede Maus auf dem Imaging-Plattform. Verwenden Tierarzt Salbe auf die Augen zu Trockenheit während der Narkose zu verhindern. Pflegen präzise Positionierung auf quantitative Ergebnisse zwischen den Zeitpunkten zu gewährleisten. </li><li> Bild Tiere mit dem IVIS Spectrum Imaging System (Caliper Life Sciences). Wenn Bildgebung mehr als 1 Tier, Ort Lichtschranken zwischen ihnen zu verhindern Cross-illumung. Tiere zunächst auf der Bauchseite abzubildenden Lokalisierung von Bakterien zu bestätigen. </li><li> Bild Tieren dorsal mit IVIS alle 2 Stunden für die Dauer des Experiments (36 Stunden). Generieren Sie einen zeitlichen Verlauf für einen bestimmten ROI. </li></ol> 6. Quantifizierung Bakterielle Biodistribution <ol><li> Euthanize Tiere mit CO 2 und Platz auf der Rückseite auf einem saugfähigen Windel. </li><li> Schalten Sie das Tier, die beiden hinteren Flanke subkutanen Tumoren freizulegen. Schneiden Sie das umliegende Hautgewebe zu trennen und entfernen Sie die Tumoren. Halten Sie den Tumor mit einer Pinzette aus der Haut mit einer Schere umgekehrte Bewegung. Wenn die Mehrheit der Haut abgetrennt wurde, vollständig zu entfernen den Tumor mit einer Pinzette. </li><li> Legen Sie die Organe in vorgewogenen Mikrozentrifugenröhrchen. Da die Masse dieser Rohre erheblich variieren können, ist es wichtig, vor der gewogen für quantitative Ergebnisse. </li></ol> 7. Quantifizierung Bakterielle Biodistribution <ol><li> Für jeden Zeitpunkt, Verbrauchsteuern und wiegen die Tumoren. </li><li> In 500 ul PBS + 15% Glycerin und homogenisieren mit einem Tissue-Tearor (BioSpec). Spülen Sie die Homogenisator 3 mal mit Ethanol zwischen den Proben. </li><li> Generieren serielle Verdünnungen und Platte für bakterielle Koloniezahlbestimmung. Um Platte mehrere Verdünnungen auf einer einzigen Platte Verwendung pre-geschnittene Platten. </li></ol>

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Keine Interessenskonflikte erklärt.

Mit diesem Verfahren sind wir in der Lage, Zeit-Verläufe für das Wachstum und die Genexpression Dynamik von Bakterien besiedeln Tumoren zu erzeugen. Während diese Messungen routinemäßig in vitro in Batch-Kultur oder Mikrofluidik-Geräte ausgeführt, sind sie viel schwieriger in vivo durchzuführen. Es gibt verschiedene Modifikationen zu diesen Methoden angewendet werden können. Während wir die OVCAR-8-Zelllinie verwendet, um unsere Mausmodellen zu erzeugen, kann eine...

Synthetische Biologie hat sich in den letzten zehn Jahren Fortschritte gemacht und ist nun positioniert, um wichtige Probleme in den Bereichen Energie und Gesundheit auswirken. Allerdings hat die Expansion in den klinischen Bereich von Sicherheitsbedenken und einer Abwesenheit von der Entwicklung Design-Kriterien für in vivo genetische Schaltkreise verlangsamt worden. Beschleunigung hohe Schlagzähigkeit medizinische Anwendungen erfordern Methoden verwenden, die eine direkte Schnittstelle mit medizinischen Einrichtung Genetische Schaltkreise, die außerhalb des gesteuerten Testumgebung funktionieren, zu regeln und klinisch akzeptierten mikrobiellen Wirten. Eine Reihe von Stämmen sind zur Krebstherapie aufgrund ihrer Fähigkeit, vorzugsweise wachsen in Tumoren untersucht. Diese haben C enthalten novyi, E. coli, V. cholorae, B. longum, und S. typhimurium 3-8. S. typhimurium ist besonderem Interesse erzeugt, wie sie Sicherheit und Verträglichkeit in einer Reihe von klinischen Studien am Menschen haben gezeigt 9-12. Diese bacteri<html> A wurden gezeigt, Anti-Tumor-Wirkung durch Stimulation des Immunsystems des Wirts und von Nährstoffmangel für Krebs Zellstoffwechsel erforderlich erstellen. Produktion von therapeutischen Ladung wurde später durch genetische Modifikationen aufgenommen. Während diese Studien wichtige Fortschritte bei der Verwendung von Bakterien für die Tumor-Therapien dar, die Mehrheit der bestehenden Bemühungen auf hoher Ebene Ausdruck, führt in der Regel die Lieferung von hohen Dosierungen, Off-Target-Effekte, und die Entwicklung von Host-Widerstand 13-16 berufen . Jetzt können synthetische Biologie programmierbare Ladung Produktion durch den Einsatz rechnerisch ausgelegt genetischer Schaltkreise, die erweiterte Erfassung und Lieferung 17-20 durchführen hinzufügen können. Diese Schaltungen können entworfen, um als Delivery-Systeme, die Tumor-spezifische Stimuli und selbst regulieren Fracht Produktion notwendigen Sinn zu handeln. Allerdings hat die Untersuchung der Funktion dieser Schaltungen in vivo bisher schwierig gewesen. ve_content &quot;&gt; Da Plasmide der gemeinsame Rahmen für synthetische Schaltungen sind, beschreiben wir eine Methode, um die Dynamik von Plasmid-basierte Genexpression in vivo mit einem Maus-Modell zu charakterisieren. Diese Methoden nutzen Zeitraffer Lumineszenz Bildgebung und quantitative Messung der Biodistribution. Gemeinsam Diese Ansätze bieten einen Rahmen für die Untersuchung Plasmid-basierte Netze in vivo für klinische Anwendungen.

<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

Das Ziel dieser Versuche ist es, quantitative zeitliche Verlauf Daten über das Wachstum und die Genexpression Dynamik abgeschwächt S. generieren typhimurium wachsenden Bakterienkolonien in den Tumoren. Wir generierte Modell Xenograft Tumoren in Mäusen durch subkutane Injektion eines menschlichen Ovarialkarzinom-Zelllinie, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). Wir verwandelt abgeschwächte Stämme von S. typhimurium Bakterien (ELH430: SL1344 phoPQ-1) mit einem konstitutiv exprimierten Luciferase (luxCDABE) Plasmid zur Visualisierung 2. Diese Stämme besiedeln speziell Tumoren während verbleibenden wesentlichen nicht-virulenten der Maus 1. Sobald messbare Tumore wurden hergestellt wurden Bakterien intravenös über die Schwanzvene mit unterschiedlicher Dosierung injiziert. Tumor-lokalisiert wurde bakterielle Genexpression in Echtzeit im Laufe von 60 Stunden unter Verwendung eines überwachtenin vivo Imaging System (IVIS). Zu jedem Zeitpunkt wurden Tumore herausgeschnitten, homogenisiert und ausplattiert, um Bakterienkolonien Korrelation mit Genexpressionsdaten quantifizieren. Zusammen ergibt sich daraus Daten ein quantitatives Maß für die in vivo-Wachstum und die Genexpression Dynamik der wachsenden Bakterien in den Tumoren.

Mit diesem Protokoll sind wir in der Lage, Daten über die in vivo-Wachstum und Genexpression Dynamik der Tumor gezielt Bakterien erzeugen. Der gesamte Workflow wird in Abbildung 1 zusammengefasst. In der ersten Stufe injizieren wir Bakterien (grün) und Bild des Tieres mit IVIS die Genexpression mit Lumineszenz-(Blau) als Reporter zu messen. Dann, in der zweiten Stufe, wir Verbrauchssteuern, zu homogenisieren und die Platte für Tumoren...

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

Das Ziel dieser Versuche ist es, quantitative zeitliche Verlauf Daten über das Wachstum und die Genexpression Dynamik abgeschwächt erzeugen<em&gt; S. typhimurium</em&gt; Wachsenden Bakterienkolonien in den Tumoren. In diesem Video werden Tumorzelle Vorbereitung und Implantation Bakterien Vorbereitung und Injektion, Ganzkörper-Tier Lumineszenz Imaging, Tumorentfernung und bakterielle Koloniezahlbestimmung.

Mess-Wachstum und Genexpression von Tumor-Dynamics Targeted<em&gt; S. Typhimurium</em&gt; Bakterien

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

measuring-growth-gene-expression-dynamics-tumor-targeted-s

Research

JoVE Journal

Immunology and Infection

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

12.1K Aufrufe.  Massachusetts Institute of Technology. Das Ziel dieser Versuche ist es, quantitative zeitliche Verlauf Daten über das Wachstum und die Genexpression Dynamik abgeschwächt erzeugen S. typhimurium Wachsenden Bakterienkolonien in den Tumoren. In diesem Video werden Tumorzelle Vorbereitung und Implantation Bakterien Vorbereitung und Injektion, Ganzkörper-Tier Lumineszenz Imaging, Tumorentfernung und bakterielle Koloniezahlbestimmung.

Serie: 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.

Verwendung Fluoreszierende Proteine, zu visualisieren und zu quantifizieren<em&gt; Chlamydia</em&gt; Vakuole Wachstumsdynamik in lebenden Zellen

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

Forschung

Immunologie und Infektion

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-Viren Reporter zur Quantifizierung von Viral-Funktion auf allen Stufen der Genexpression

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.

Ein Protokoll zum Infect<em&gt; Caenorhabditis elegans</em&gt; Mit<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.

Hochdurchsatz-Assay zum Phänotyp<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion und Replikation in Makrophagen

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.

Isolierung von Murine Peritonealmakrophagen zur Durchführung Genexpressionsanalyse Nach Toll-like Rezeptoren Stimulation

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.

Gene Expression Profiling von Infecting Mikroben Mit einem Digital-Strichcode-Plattform

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.

Eine Methode zur Bewertung von Fc-vermittelten Effektor-Funktionen durch Influenza Hämagglutinin spezifische Antikörper induziert

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.

Isolierung von Salmonella Typhimurium-mit Phagosom von Makrophagen

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.

Live-Cell Fluoreszenzmikroskopie zur Untersuchung subzellulärer Proteinlokalisierung und Zellmorphologie Veränderungen in Bakterien

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.

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.

NF-B-abhängige Luziferase-Aktivierung und Quantifizierung der Genexpression in Salmonellen-infizierten Gewebekulturzellen

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