Diese Arbeit wurde durch Mittel der National Institutes of Health und der Mukoviszidose-Stiftung unterstützt.

1. Vorbereitung der Bakterien für die LPS-Extraktion <ol><li> Starten Sie eine Übernachtkultur in 5 ml Luria Broth (LB), die mit Antibiotika bei Bedarf ergänzt. Kultur wachsen über Nacht (12-18 Stunden) in einem Schüttelinkubator bei 37 ° C und 200 Umdrehungen pro Minute. </li><li> Verdünnen Sie die Kultur im Verhältnis 1:10 mit LB und nehmen eine OD 600 Lektüre in einem Spektralphotometer. Basierend auf der OD 600 Lektüre, machen Sie eine 1,5 ml Suspension aus Ihrem Bakterien auf eine OD 600 von 0,5. </li><li> Pellet die Bakterien in einer Mikrozentrifuge bei 10.600 x g für 10 Minuten. Entfernen und den Überstand verwerfen. Das Pellet kann bei -20 ° C gelagert werden, wenn LPS nicht geht ist, sofort extrahiert werden. </li></ol> 2. Extraktion von LPS <ol><li> Zuerst bereiten 2x SDS-Puffer. Einen 50 ml Lösung von 4% β-Mercaptoethanol (BME), 4% SDS und 20% Glycerin in 0,1 M Tris-HCl, pH 6,8. Fügen Sie eine Prise Bromphenolblau, um die Lösung zu färben. Machen Sie einen 1x SDS-Puffer Lager durch Verdünnung von 2x Stock 1:1 in sterilem destilliertem H 2 O. Dies kann bei Raumtemperatur gelagert werden. </li><li> Machen Sie drei separate 10 mg / ml Lösung von DNase I, RNase-und Proteinase K in sterilem destilliertem H 2 O. </li><li> Resuspendieren pelletierten Bakterien aus Schritt 1.3 in 200 ul 1x SDS-Puffer. Sie sicher, dass das Pellet vollständig durch Pipettieren der Lösung langsam auf und ab resuspendiert. Nicht vortexen. </li><li> Kochen suspendierter Bakterien in einem Wasserbad für 15 Minuten. Lassen Sie die Lösung bei Raumtemperatur für 15 Minuten abkühlen. </li><li> 5 l der beiden DNase I und RNase-Lösungen in 2.2 vorbereitet. Inkubieren der Proben bei 37 ° C für 30 Minuten. * Dieser Schritt ist optional, es gibt minimale Unterschied in der Qualität von LPS zwischen Nuklease-behandelten und unbehandelten Proben. </li><li> In 10 ul der Proteinase K-Lösung in 2.2 vorbereitet. Inkubieren der Proben bei 59 ° C für 3 Stunden. Dieser Schritt kann über Nacht durchgeführt werden, wenn es Zeitdruck sind. </li><li> Zu jeder Probe add 200 pl eiskaltem Tris-gesättigtem Phenol (Wasser gesättigtem Phenol kann als Ersatz verwendet werden, wenn Tris-gesättigtem Phenol steht nicht zur Verfügung). Stellen Sie sicher, die Kappen an die Rohre dicht verschlossen sind, und Vortex jede Probe für etwa 5 bis 10 Sekunden. </li><li> Inkubieren der Proben bei 65 ° C für 15 Minuten, gelegentlich Vortexen. Nach Inkubation bei Raumtemperatur abkühlen lassen, dann fügen Sie 1 mL bei Raumtemperatur Diethylether zu jeder Probe und Vortex für 5 bis 10 Sekunden. Achten Sie darauf, Griff Äther in einem Abzug durchführen, da es flüchtig ist. </li><li> Zentrifugation der Proben bei 20.600 × g für 10 Minuten. Entfernen Sie vorsichtig die Proben aus der Zentrifuge und extrahieren Sie die untere blaue Schicht. Achten Sie darauf, die obere, klare Schicht zu vermeiden. Zurücklassung einer kleinen Menge des blauen Schicht ist bevorzugt, um eine Kontamination mit der oberen Schicht. </li><li> Extrahieren Sie erneut die Proben durch Wiederholen der Schritte von 2,7 bis 2,9. Zwei Extraktionen sind in der Regel ausreichend. Wenn die Proben bedeckt erscheinen, kann mehr sein Extraktionen performed. Zugabe von 200 ul 2x SDS-Puffer zu jedem der extrahierten Proben vor der Trennung durch SDS-PAGE. Die Proben können bei 8% -15% SDS-Polyacrylamid-Gelen laufen gelassen werden. Fünf bis fünfzehn ul von LPS unter Verwendung dieser Methode ist in der Regel ausreichend für die Visualisierung. </li></ol> 3. Repräsentative Ergebnisse LPS Proben wie oben hergestellt visualisiert werden kann durch direkte Färbung nach der Trennung auf SDS-PAGE unter Verwendung eines Standard-Silber-Färbung Protokoll oder ein im Handel erhältliches LPS-Färbekit. Alternativ kann LPS auf einem Polyacrylamidgel getrennt auf eine Nitrocellulosemembran übertragen werden und Western-Immunoblotting unter Verwendung LPS-spezifischen Anti-Seren. Aus diesem Protokoll verwendeten wir die Pro-Q Smaragd 300 Lipopolysaccharid Gel Stain Kit (Molecular Probes), und folgte den Anweisungen des Herstellers. Dargestellt in Abbildung 1 ist ein Pro-Q 300 Smaragd gefärbten 12% SDS-Polyacrylamidgel. Jede Spur enthält 15 ul LPS aus unterschiedlichen vorbereitetschiedenen Stämme von Burkholderia dolosa isoliert aus Sputum-Proben von Patienten mit zystischer Fibrose. Verschiedene Streifen LPS Leitermuster, reflektierende unterschiedlicher Zahlen O-Antigen sich wiederholenden Einheiten, die an Kernoligosaccharid ergeben sich unter Verwendung dieses Verfahrens, zum Beispiel, müssen die Proben in Spur 1 und 6 ähnliche Bandenmuster miteinander (Kasten in rot). <img alt="1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Abbildung 1. Pro-Q Smaragd 300 gebeizt LPS von Burkholderia dolosa klinischen Isolaten. LPS aus sieben B. dolosa Stämme aus einem Ausbruch bei Mukoviszidose-Patienten isoliert wurden, wie in diesem Protokoll beschrieben. Fünfzehn ul wurden auf einem 12% SDS-Polyacrylamid-Gel, und gefärbt mit Pro-Q Smaragd 300 getrennt, pro Anweisungen des Herstellers. LPS-Kern einen Pfeil gekennzeichnet, und LPS zu ähnlichen Bandenmuster von O-Antigen Wiederholungen sind in rot eingerahmt. M = Molekulargewichtsmarker.

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <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+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

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Wir haben ein Verfahren zur Reinigung von LPS von anderen zellulären Komponenten, einschließlich Nukleinsäuren und Proteinen beschrieben. Diese Methode bietet eine hohe Qualität LPS, die in einer Reihe von verschiedenen Visualisierungsmethoden, einschließlich des Kohlenhydrat-Färbung der SDS-PAGE-Gelen, wie in 1 gezeigt ist, verwendet werden kann. Dieses Verfahren kann verwendet werden, um LPS aus einer Vielzahl von Stämmen Serotyp, unter Verwendung spezifischer Antiseren, oder um eine Verwandtschaft zwischen den...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> DNase I rekombinanten, RNase-frei </td><td> Roche </td><td> 04716728001 </td></tr><tr><td> RNase A </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> Proteinase K </td><td> Fischer </td><td> BP1700 </td></tr><tr><td> Tris-gesättigtem Phenol </td><td> Fischer </td><td> BP1750-100 </td></tr><tr><td> Diethylether </td><td> Thomas Scientific </td><td> C313K31 </td></tr><tr><td> Pro-Q Smaragd 300 Lipopolysaccharid Gel Stain Kit </td><td> Molecular Probes </td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

Lipopolysaccharid (LPS) ist ein wichtiger Bestandteil von Gram-negativen bakteriellen äußeren Membranen. Es ist eine dreigliedrige Molekül, das aus Lipid A, das in der äußeren Membran eingebettet ist, ein Kern-Oligosaccharid und Wiederholen der O-Antigen-Einheiten, die nach außen erstrecken sich von der Oberfläche der Zelle 1, 2. LPS ist ein Molekül, das immundominante wichtig für die Virulenz und Pathogenese vieler Bakterienarten, darunter Pseudomonas aeruginosa, Salmonella-Arten, und Escherichia coli 3-5, und Unterschiede in der LPS O-Antigen-Zusammensetzung bilden die Grundlage für die Serotypisierung der Stämme. LPS wird in Anlage beteiligten Zellen zu Beginn der Infektion Host und bietet Schutz vor Komplement-vermittelte Tötung; Stämme, die LPS fehlt, gedämpft werden kann für die Virulenz 6-8. Aus diesen Gründen ist es wichtig, LPS visualisieren, insbesondere aus klinischen Isolaten. Visualisierung von LPS Bandenmuster und Erkennung durch spezifische einntibodies können nützliche Werkzeuge sein, um Stamm-Linien zu identifizieren und verschiedenen Mutanten zu charakterisieren. In diesem Bericht beschreiben wir eine heiße wässrige Phenol-Verfahren zur Isolierung und Reinigung von LPS aus Gram-negativen bakteriellen Zellen. Dieses Protokoll ermöglicht die Extraktion von LPS von Nukleinsäuren und Proteinen, die mit Visualisierung von LPS, die mit kürzeren, weniger intensiv Extraktionsverfahren 9 erfolgt stören können. LPS diese Weise hergestellten durch Natriumdodecylsulfat (SDS)-Polyacrylamid-Gelelektrophorese (PAGE) und gefärbt direkt unter Verwendung von Kohlenhydrat / Glykoprotein Flecken oder Standard-Silberfärbung Verfahren getrennt werden können. Viele Anti-Seren gegen LPS Antikörper enthalten, dass mit der äußeren Membran-Proteine ​​oder andere antigene Ziele, kann ein Hindernis für kreuzreagieren Reaktivität nach Western-Immunoblot-SDS-PAGE getrennt Zelllysat beobachtet. Protease-Behandlung von Zelllysat allein ist nicht immer ein wirksames Mittel zur Beseitigung dieserHintergrund der Nutzung dieser oder andere Visualisierungs-Methoden. Ferner kann umfangreiche Proteasebehandlung in einem Versuch, dieses Objekt zu entfernen, schlechter Qualität LPS, die nicht gut von einem der vorgenannten Verfahren gelöst führen. Aus diesen Gründen glauben wir, dass das folgende Protokoll, aus Westpahl Jann und 10 angepasst, ideal für die LPS-Extraktion ist.

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Wir beschreiben ein modifiziertes heißen wässrigen-Phenolextraktion Verfahren zum Reinigen von Lipopolysaccharid (LPS) von Gram-negativen Bakterien. Sobald extrahiert wurden, kann die LPS anschließend durch SDS-PAGE analysiert und visualisiert werden durch direkte Färbung oder Western-Immunoblot-.

Reinigung und Visualisierung von Lipopolysaccharid aus Gram-negativen Bakterien durch heiße wäßrige-Phenol-Extraktion

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

purification-visualization-lipopolysaccharide-from-gram-negative

Research

JoVE Journal

Immunology and Infection

34.7K Aufrufe.  University of Virginia Health System. Wir beschreiben ein modifiziertes heißen wässrigen-Phenolextraktion Verfahren zum Reinigen von Lipopolysaccharid (LPS) von Gram-negativen Bakterien. Sobald extrahiert wurden, kann die LPS anschließend durch SDS-PAGE analysiert und visualisiert werden durch direkte Färbung oder Western-Immunoblot-.

Serie: Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Reinigung von spezifischen Zellpopulation durch Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Forschung

Immunologie und Infektion

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY-Technologie: Extraction of Chicken Antikörper aus Eidotter durch Polyethylenglykol (PEG) Niederschlag

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Die Reinigung von Pathogen Vakuolen aus<em&gt; Legionellen</em&gt;-Infizierten Phagozyten

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Verfahren zur Erzeugung von Lungen Neutrophilie Mit aerosolisiertes Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

Schnelle Identifizierung von Gram-negativen Bakterien aus Blutkulturbrühe unter Verwendung von MALDI-TOF-Massenspektrometrie

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Die Anwendung der Fluoreszenz-Nanopartikel zur Umgestaltung des Endo-lysosomalen System-Studie durch intrazelluläre Bakterien

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

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T-Zellen erfassen Bakterien durch Transinfection von dendritischen Zellen

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Bestimmung von Biofilm Initiation auf Virus-infizierte Zellen durch Bakterien und Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Isolierung, Charakterisierung und Reinigung der Makrophagen aus den Geweben von Fettleibigkeit bedingte Entzündung betroffen

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Sezieren Signalisierung Multi-Protein-komplexe durch eine Bimolekulare Ergänzung Affinitätsreinigung (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...