Questo lavoro è stato supportato da sovvenzioni da parte del National Institutes of Health e dalla Fondazione Fibrosi Cistica.

1. Preparazione di batteri per l&#39;estrazione LPS <ol><li> Avviare una cultura pernottamento in 5 ml di Luria Broth (LB), integrato con antibiotici se necessario. Grow coltura durante la notte (12-18 ore) in un incubatore agitazione a 37 ° C e 200 rpm. </li><li> Diluire il 01:10 cultura con LB e prendere un 600 lettura OD in uno spettrofotometro. Sulla base della lettura 600 OD, una sospensione 1,5 mL dei batteri ad una OD 600 di 0,5. </li><li> Pellet i batteri in una microcentrifuga a 10.600 xg per 10 minuti. Rimuovere e scartare il surnatante. Il pellet può essere conservato a -20 ° C, se LPS non sta per essere estratto immediatamente. </li></ol> 2. Estrazione di LPS <ol><li> In primo luogo, preparare 2x tampone SDS. Effettuare una soluzione di 50 mL del 4% β-mercaptoetanolo (BME), 4% SDS e 20% glicerolo in 0,1 M Tris-HCl, pH 6,8. Aggiungere un pizzico di blu di bromofenolo per tingere la soluzione. Fai una SDS-buffer 1x magazzino diluendo 2X stock 1:1 in sterile distillata H 2 O. Questo può essere conservato a temperatura ambiente. </li><li> Fare tre distinti a 10 mg / mL soluzioni di DNasi, RNasi e proteinasi K in sterile distillata H 2 O. </li><li> Risospendere i batteri pellet a partire dal punto 1,3 in 200 microlitri di 1x SDS-buffer. Assicurarsi che il pellet è completamente risospeso pipettando la soluzione su e giù lentamente. Non vortice. </li><li> Lessare i batteri in sospensione in un bagnomaria per 15 minuti. Lasciare raffreddare la soluzione a temperatura ambiente per 15 minuti. </li><li> Aggiungere 5 microlitri di entrambi I DNasi RNasi e soluzioni preparate in 2.2. Incubare i campioni a 37 ° C per 30 minuti. * Questo passaggio è facoltativo, c&#39;è minima differenza nella qualità di LPS tra campioni nucleasi trattati e non trattati. </li><li> Aggiungere 10 pl di soluzione di proteinasi K preparata in 2,2. Incubare i campioni a 59 ° C per 3 ore. Questa fase può essere eseguita durante la notte, se ci sono vincoli di tempo. </li><li> Per ogni annuncio campione,d 200 pl di ghiacciato Tris-fenolo saturo (acqua-fenolo saturo può essere utilizzato come sostituto se Tris-fenolo saturo non è disponibile). Assicurarsi che i tappi sui tubi sono chiuse ermeticamente, e vortex ogni campione per circa 5 a 10 secondi. </li><li> Incubare i campioni a 65 ° C per 15 minuti, vortex occasionalmente. Dopo incubazione raffreddare a temperatura ambiente, poi aggiungere 1 ml di temperatura ambiente etere dietilico per ciascun campione e vortex per 5 a 10 secondi. Assicurarsi di eseguire etere manico in una cappa aspirante, in quanto è volatile. </li><li> Centrifugare i campioni a 20600 x g per 10 minuti. Rimuovere con cautela i campioni dalla centrifuga ed estrarre lo strato di fondo blu. Essere sicuri di evitare la parte superiore, strato trasparente. Lasciando una piccola quantità dello strato blu è preferibile contaminazione con lo strato superiore. </li><li> Ri-estrarre i campioni ripetendo i punti da 2,7-2,9. Due estrazioni sono di solito sufficienti. Se i campioni appaiono nuvoloso, più estrazioni può essere Effettuared. Aggiungere 200 pl di 2x SDS-buffer per ciascuno dei campioni estratti prima separazione mediante SDS-PAGE. I campioni possono essere eseguiti su 8% -15% SDS-poliacrilammide gel. Cinque a quindici microlitri di LPS preparate utilizzando questo metodo è di solito sufficiente per la visualizzazione. </li></ol> 3. Risultati rappresentativi LPS campioni preparati come sopra può essere visualizzato mediante colorazione diretta dopo la separazione su SDS-PAGE usando uno standard di argento o di un protocollo di colorazione disponibile in commercio kit di colorazione LPS. In alternativa, LPS separati su un gel di poliacrilammide può essere trasferita ad una membrana di nitrocellulosa e sottoposto a Western immunoblotting usando LPS-specifici anti-sieri. Per questo protocollo, abbiamo usato il Pro-Q Emerald 300 Kit Gel Stain Lipopolisaccaride (Molecular Probes), e seguito le istruzioni del produttore. In figura 1 è una Pro-Q Emerald 300 tinto 12% SDS-gel di poliacrilammide. Ciascuna corsia contiene 15 pl di LPS preparati da DIFdifferenti ceppi di Burkholderia dolosa isolati da campioni di espettorato di pazienti affetti da fibrosi cistica. Differenti modelli di LPS banding scala, riflettenti di numeri diversi O-antigene unità ripetitive attaccato al centro oligosaccaride, sono evidenti con questo metodo, ad esempio, campioni in corsia 1 e 6 sono simili schemi bande tra loro (scatolato in rosso). <img alt="Figura 1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Figura 1. Pro-Q Emerald 300 LPS macchiate da Burkholderia dolosa isolati clinici. LPS di sette B. ceppi dolosa di un focolaio in pazienti affetti da fibrosi cistica sono stati isolati come descritto in questo protocollo. Quindici pl sono stati separati su un 12% SDS-gel di poliacrilammide, e colorate con Pro-Q Emerald 300, secondo le istruzioni del produttore. LPS nucleo è indicata una freccia, e LPS che mostrano modelli simili bande di O-antigene ripetizioni sono boxed in rosso. M = marcatore di peso molecolare.

<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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Abbiamo descritto un metodo di purificazione LPS lontano dagli altri componenti cellulari, tra cui gli acidi nucleici e proteine. Questo metodo consente di alta qualità LPS che possono essere utilizzati in un certo numero di metodi di visualizzazione differenti, compresa la colorazione carboidrati di SDS-PAGE gel, come mostrato nella Figura 1. Questo metodo può essere utilizzato per sierotipo LPS da una varietà di ceppi, utilizzando specifici anti-sieri, o per mostrare connessione tra isolati tramite visualizzazione ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> DNasi ricombinante, RNasi-free </td><td> Roche </td><td> 04716728001 </td></tr><tr><td> RNasi A </td><td> Roche </td><td> 10109169001 </td></tr><tr><td> Proteinasi K </td><td> Pescatore </td><td> BP1700 </td></tr><tr><td> Tris-saturi Fenolo </td><td> Pescatore </td><td> BP1750-100 </td></tr><tr><td> Etere etilico </td><td> Thomas Scientific </td><td> C313K31 </td></tr><tr><td> Pro-Q Emerald 300 Gel Kit Lipopolisaccaride Stain </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

Lipopolisaccaride (LPS) è un componente principale delle membrane batteriche Gram-negativi esterni. Si tratta di una molecola composta dal lipide A, che è incorporato nella membrana esterna, un oligosaccaride e ripetibile nucleo O-antigene unità che si estendono verso l&#39;esterno dalla superficie della cella 1, 2. LPS è una molecola immunodominante che è importante per la virulenza e patogenesi di molte specie batteriche, tra Pseudomonas aeruginosa, Salmonella spp, Escherichia coli e 3-5, e le differenze di LPS O-antigene composizione forma la base per la sierotipizzazione di ceppi. LPS è coinvolto in attaccamento alle cellule ospiti all&#39;inizio della infezione e fornisce protezione da complemento-mediata uccisione; ceppi privi LPS può essere attenuato per virulenza 6-8. Per queste ragioni, è importante visualizzare LPS, in particolare da isolati clinici. LPS visualizzando bande modelli e il riconoscimento da uno specificontibodies possono essere strumenti utili per identificare i lignaggi di deformazione e caratterizzare i vari mutanti. In questa relazione, si descrive un hot-fenolo acquoso metodo per l&#39;isolamento e la purificazione di LPS da cellule batteriche gram-negativi. Questo protocollo consente l&#39;estrazione di LPS lontano da acidi nucleici e proteine ​​che possono interferire con visualizzazione di LPS che si verifica con brevi e metodi di estrazione meno intensi 9. LPS preparato in questo modo possono essere separati da sodio dodecil solfato (SDS)-poliacrilammide gel elettroforesi (PAGE) e direttamente colorati usando carboidrati / glicoproteina macchie o normali metodi di colorazione d&#39;argento. Molti anti-sieri di LPS contenere anticorpi che cross-reagiscono con le proteine ​​della membrana esterna o altri bersagli antigenici che possono ostacolare la reattività osservati dopo immunoblot occidentale SDS-PAGE separate da lisati cellulari grezzi. Proteasi trattamento di lisati cellulari grezzi da sola non è sempre un modo efficace per rimuovere questosfondo di utilizzare questo o altri metodi di visualizzazione. Inoltre, il trattamento della proteasi esteso nel tentativo di eliminare questo sfondo può portare a LPS scarsa qualità che non è ben risolto da uno qualsiasi dei metodi summenzionati. Per queste ragioni, riteniamo che il seguente protocollo, adattato da Westpahl Jann e 10, è l&#39;ideale per l&#39;estrazione 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

Descriviamo una modificato caldo acquosa-fenolo metodo di estrazione per purificare lipopolisaccaride (LPS) da batteri Gram-negativi. Una volta estratto, le LPS può essere successivamente analizzati mediante SDS-PAGE e visualizzati tramite colorazione diretta o immunoblot occidentale.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

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 Views.  University of Virginia Health System. Descriviamo una modificato caldo acquosa-fenolo metodo di estrazione per purificare lipopolisaccaride (LPS) da batteri Gram-negativi. Una volta estratto, le LPS può essere successivamente analizzati mediante SDS-PAGE e visualizzati tramite colorazione diretta o immunoblot occidentale.

Video: 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.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (FACS)

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

Ricerca

Immunologia e infezioni

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.

Tecnologia IgY: Estrazione di anticorpi pollo rosso d&#39;uovo con polietilenglicole (PEG) Precipitazioni

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.

Purificazione di Vacuoli patogeno da<em&gt; Legionella</em&gt;-Infetti fagociti

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.

Un metodo per generare polmonare Neutrofilia Uso inalato Lipopolisaccaride

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.

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

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.

Applicazione di nanoparticelle fluorescenti per studiare rimodellamento del sistema Endo-lisosomiale da batteri intracellulari

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.

Cellule T Capture batteri da Transinfection da cellule dendritiche

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.

Determinazione del biofilm iniziazione su cellule infettate da virus da batteri e funghi

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.

Isolamento, caratterizzazione e purificazione dei macrofagi dai tessuti colpiti da infiammazione obesità

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.

Dissezione multiproteici segnalazione di purificazione di affinità di complementazione bimolecolare (BiCAP)

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