Ce travail a été soutenu par des subventions du National Institutes of Health et la Fondation de la fibrose kystique.

1. Préparation de bactéries pour l&#39;extraction LPS <ol><li> Démarrer une culture de nuit dans 5 ml de milieu Luria Broth (LB), complété avec des antibiotiques si nécessaire. Cultiver la culture pendant la nuit (12-18 heures) dans un incubateur agitateur à 37 ° C et 200 rpm. </li><li> Diluer la culture avec 1:10 LB et de prendre une DO 600 de lecture dans un spectrophotomètre. Sur la base de la DO 600 lecture, faire une suspension de 1,5 mL de vos bactéries à une DO 600 de 0,5. </li><li> Pellet les bactéries dans une microcentrifugeuse à 10.600 x g pendant 10 minutes. Retirer et jeter le surnageant. Le culot peut être conservé à -20 ° C, si LPS ne va pas être extrait immédiatement. </li></ol> 2. Extraction de LPS <ol><li> Tout d&#39;abord, préparer 2x tampon SDS. Faire une solution à 50 ml de 4% β-mercaptoéthanol (BME), 4% de SDS et 20% de glycérol dans 0,1 M de Tris-HCl, pH 6,8. Ajouter une pincée de bleu de bromophénol pour teindre la solution. Faire un 1x SDS-stock tampon en diluant 2X stac 1:1 dans distillée stérile H 2 O. Cela peut être conservé à température ambiante. </li><li> Faites trois jumeaux 10 mg / ml des solutions de DNase I, RNase, et la protéinase K dans distillée stérile H 2 O. </li><li> Remettre en suspension les bactéries enrobées de l&#39;étape 1.3 dans 200 ul de SDS-1x tampon. Veiller à ce que le culot est complètement remis en suspension par pipetage la solution de haut en bas lentement. Ne pas vortex. </li><li> Faire bouillir les bactéries en suspension dans un bain d&#39;eau pendant 15 minutes. Laisser la solution refroidir à température ambiante pendant 15 minutes. </li><li> Ajouter 5 pi de deux DNase I et des solutions RNase préparé en 2.2. Incuber les échantillons à 37 ° C pendant 30 minutes. * Cette étape est facultative, il ya une différence minime de la qualité de LPS entre les échantillons nucléase traités et non traités. </li><li> Ajouter 10 ul de la solution K protéinase préparé en 2.2. Incuber les échantillons à 59 ° C pendant 3 heures. Cette étape peut être réalisée du jour au lendemain, si il ya des contraintes de temps. </li><li> Pour chaque échantillon, annonced 200 pl de glacée Tris-phénol saturé (eau-phénol saturé peut être utilisée comme substitut si Tris-phénol saturé n&#39;est pas disponible). S&#39;assurer que les bouchons sur les tubes sont fermés hermétiquement et vortex chaque échantillon pendant environ 5 à 10 secondes. </li><li> Incuber les échantillons à 65 ° C pendant 15 minutes, vortex occasionnellement. Après une incubation refroidir à température ambiante, puis ajouter 1 mL de la température ambiante diéthyléther à chaque échantillon et vortex pendant 5 à 10 secondes. Veillez à effectuer l&#39;éther poignée dans une hotte, car il est volatil. </li><li> Centrifuger les échantillons à 20600 x g pendant 10 minutes. Retirez délicatement les échantillons provenant de la centrifugeuse et extraire la couche fond bleu. Soyez sûr d&#39;éviter la couche supérieure claire. Laissant une petite quantité de la couche bleue est préférable à la contamination de la couche supérieure. </li><li> Ré-extraire les échantillons en répétant les étapes 2.7 à 2.9. Deux extractions sont généralement suffisantes. Si les échantillons apparaissent nuageux, plusieurs extractions peuvent être performed. Ajouter 200 ul de 2x SDS-tampon pour chacun des échantillons prélevés avant de se séparer par SDS-PAGE. Les échantillons peuvent être exécutés sur 8% -15% de SDS-polyacrylamide gels. Cinq à quinze ul de LPS préparés en utilisant cette méthode est généralement suffisante pour la visualisation. </li></ol> 3. Les résultats représentatifs Échantillons LPS préparés comme ci-dessus peuvent être visualisées par coloration directe après la séparation par SDS-PAGE en utilisant un étalon-argent-coloration protocole ou d&#39;un kit de coloration disponibles dans le commerce LPS. Alternativement, LPS séparés sur un gel de polyacrylamide peut être transféré à une membrane de nitrocellulose et soumis à un Western immunoblot en utilisant le LPS spécifique anti-sérums. Pour ce protocole, nous avons utilisé le Pro-Q Emerald 300 Kit Gel Teinté Lipopolysaccharide (Molecular Probes), et suivi les instructions du fabricant. Illustré à la figure 1 est une Pro-Q Emerald 300 teinté 12% SDS-gel de polyacrylamide. Chaque voie contient 15 pl de LPS préparés à partir de difsouches différentes de Burkholderia dolosa isolé à partir d&#39;échantillons de crachats de patients atteints de mucoviscidose. LPS Différents profils de bandes réfléchissantes échelle, des nombres différents de l&#39;antigène O répéter les unités rattachées au noyau oligosaccharide, sont manifestes en utilisant cette méthode, par exemple, des échantillons de la piste 1 et 6 ont les mêmes profils de bandes à l&#39;autre (encadré en rouge). <img alt="Figure 1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Figure 1. Pro-Q Emerald 300 LPS colorées à partir d&#39;isolats cliniques. Dolosa Burkholderia LPS de sept B. souches dolosa d&#39;une éclosion chez les patients atteints de fibrose kystique ont été isolés comme décrit dans le présent protocole. Quinze ul ont été séparés sur un 12% SDS-gel de polyacrylamide, et colorées avec Pro-Q Emerald 300, selon les instructions du fabricant. LPS noyau est indiquée d&#39;une flèche, et le LPS montrant des profils de bandes similaires de O-antigène répète sont encadrés en rouge. M = marqueur de poids moléculaire.

<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>

Pas de conflits d&#39;intérêt déclarés.

Nous avons décrit un procédé de purification LPS l&#39;écart des autres composants cellulaires, y compris les acides nucléiques et des protéines. Cette méthode offre une haute qualité LPS qui peuvent être utilisés dans un certain nombre de méthodes de visualisation différentes, y compris la coloration des glucides gels SDS-PAGE, comme le montre la figure 1. Cette méthode peut être utilisée au sérotype LPS à partir d&#39;une variété de souches, à l&#39;aide spécifique anti-sérums, ou pour montrer la...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> DNase I recombinante, sans RNase </td><td> Roche </td><td> 04716728001 </td></tr><tr><td> RNase A </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> Protéinase K </td><td> Pêcheur </td><td> BP1700 </td></tr><tr><td> Tris-phénol saturé </td><td> Pêcheur </td><td> BP1750-100 </td></tr><tr><td> Ether diéthylique </td><td> Thomas scientifique </td><td> C313K31 </td></tr><tr><td> Pro-Q Emerald 300 Kit Gel Teinté Lipopolysaccharide </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

Lipopolysaccharide (LPS) est une composante majeure de bactéries Gram-négatives membranes externes bactériennes. Il est une molécule tripartite composé de lipide A, qui est incorporé dans la membrane externe, un noyau oligosaccharides et répéter O-antigène unités qui s&#39;étendent vers l&#39;extérieur depuis la surface de la cellule 1, 2. LPS est une molécule immunodominant qui est important pour la virulence et la pathogenèse de nombreuses espèces bactériennes, y compris Pseudomonas aeruginosa, Salmonella et Escherichia coli 3-5, et les différences dans la forme LPS composition de l&#39;antigène O la base pour le sérotypage des souches. LPS est impliqué dans l&#39;attachement à des cellules hôtes lors de l&#39;initiation de l&#39;infection et offre une protection contre médiée par le complément meurtre; souches qui n&#39;ont pas LPS peuvent être atténuées pour la virulence 6-8. Pour ces raisons, il est important de visualiser LPS, en particulier à partir d&#39;isolats cliniques. LPS Visualisation profils de bandes et de la reconnaissance spécifique d&#39;un parntibodies peuvent être des outils utiles pour identifier les lignées de contrainte et de caractériser différents mutants. Dans ce rapport, nous décrivons une chaude aqueuse-phénol méthode pour l&#39;isolement et la purification de LPS de bactéries Gram-négatives des cellules bactériennes. Ce protocole permet l&#39;extraction de LPS l&#39;écart des acides nucléiques et des protéines qui peuvent interférer avec la visualisation de LPS qui se produit avec plus courts, des méthodes d&#39;extraction moins intensives 9. LPS préparés de cette façon peuvent être séparés par le dodécylsulfate de sodium (SDS)-électrophorèse sur gel de polyacrylamide (PAGE) et directement teinté à l&#39;aide de glucides / glycoprotéine taches ou les méthodes standard de coloration à l&#39;argent. Beaucoup anti-sérums contiennent des anticorps au LPS que des réactions croisées avec les protéines de membrane externe ou des cibles antigéniques d&#39;autres qui peuvent nuire à la réactivité observés à la suite de l&#39;Ouest de la immunoblot SDS-PAGE-séparés lysats cellulaires bruts. Le traitement aux protéases de lysats cellulaires bruts seuls n&#39;est pas toujours un moyen efficace d&#39;éliminer cettede fond en utilisant tel ou autres méthodes de visualisation. En outre, le traitement protéase vaste dans une tentative de supprimer ce fond peut conduire à LPS de mauvaise qualité qui n&#39;est pas bien résolus par l&#39;une des méthodes ci-dessus. Pour ces raisons, nous croyons que le protocole suivant, adapté de Westpahl et Jann 10, est idéal pour l&#39;extraction de LPS.

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

Nous décrivons une méthode modifiée de chaud extraction aqueuse de phénol pour purifier le lipopolysaccharide (LPS) de bactéries Gram-négatives. Une fois extraite, les LPS peuvent ensuite être analysées par SDS-PAGE et visualisés par coloration directe ou immunoblot de l&#39;Ouest.

Purification et visualisation de lipopolysaccharide de bactéries à Gram négatif par Hot-phénol aqueux d&#39;extraction

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 vues.  University of Virginia Health System. Nous décrivons une méthode modifiée de chaud extraction aqueuse de phénol pour purifier le lipopolysaccharide (LPS) de bactéries Gram-négatives. Une fois extraite, les LPS peuvent ensuite être analysées par SDS-PAGE et visualisés par coloration directe ou immunoblot de l'Ouest.

Vidéo: 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.

Purification de population cellulaire spécifique par le tri cellulaire activé par fluorescence (FACS)

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

Recherche

Immunologie et infection

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.

Technologie IgY: Extraction d&#39;anticorps de jaune d&#39;œuf de poulet par polyéthylène glycol (PEG) Précipitations

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.

Purification de vacuoles agent pathogène à partir<em&gt; Legionella</em&gt; Infectées par les phagocytes

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.

Une méthode pour générer pulmonaire Neutrophilie Utilisation Aerosolized 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.

Identification rapide des bactéries à Gram négatif de sang Bouillon de culture à l&#39;aide MALDI-TOF spectrométrie de masse

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.

Application de nanoparticules fluorescentes pour étudier Rénovation du système Endo-lysosomale par des bactéries intracellulaires

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.

Cellules T Capturez bactéries par Transinfection de cellules dendritiques

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.

Détermination de biofilm Initiation sur les cellules infectées par le virus par les bactéries et les champignons

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.

Isolement, caractérisation et Purification des Macrophages dans les tissus touchés par l’Inflammation associées à l’obésité

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.

Dissection multi-signalisation Complexes protéiques par Purification d’affinité de complémentation bimoléculaire (BiCAP)

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