This work was supported by the Deutsche Forschungsgemeinschaft by grant Z within Sonderforschungsbereich 944 &#8216;Physiology and Dynamics of Cellular Microcompartments&#8217; and HE1964/18 within priority program 1580.

1. Synthèse de 10 nm nanoparticules d&#39;or (Gold IP) 15 <ol><li> Préparer la solution A: ajouter 2 ml aqueuse à 1% de chlorure d&#39;or dans 160 ml Milli-Q, ou double distillée, eau. </li><li> Préparer la solution B: ajouter 8 ml 1% de tri-citrate de sodium x 2 H 2 O et l&#39;acide tannique 160 pi de 1% dans 32 ml Milli-Q, ou double distillée, eau. </li><li> Réchauffer la solution A et B à 60 ° C et les mélanger en remuant. Observez immédiatement une couleur bleu foncé. Observez la couleur rouge après environ 15 min. Ensuite, la chaleur jusqu&#39;à 95 ° C, 5 min et maintenir la solution refroidir à température ambiante. </li><li> Ajouter une goutte de la suspension sur une grille NP revêtu de carbone et laisser sécher à l&#39;air. Vérifier la taille et la morphologie des IP par microscopie électronique à transmission. </li></ol> 2. couche d&#39;or IP avec de la BSA et d&#39;étiquetage avec la rhodamine N-hydroxysuccinimidyle Ester (NHS) 16 <ol><li> Ajouter 900 pi IP or dans un tube Eppendorf de 1,5 ml, centrifuger à 15 000 g pendant 30 min. Opnellement, préparer tubes multiples peuvent à la fois. </li><li> Jeter le surnageant, remettre en suspension le culot dans 900 pi d&#39;eau stérilisée Milli-Q et ajouter 100 pi 2 mg / ml de BSA / PBS, mélanger au vortex à 800 tours par minute pendant 30 min. </li><li> Pour enlever l&#39;excès de BSA, centrifuger la préparation à 15 000 g pendant 60 min. </li><li> Jeter le surnageant et remettre en suspension les IP or BSA dans 125 pi de PBS, ajouter 12,5 pi de 1 M bicarbonate. </li><li> Immédiatement avant utilisation, préparer une solution à 10 mg / ml de rhodamine NHS / DMSO, ajouter 30 ul de solution rhodamine / DMSO NHS à 1 ml de la suspension or BSA IP. Incuber le mélange réactionnel pendant 2 heures (ou O / N) à la température ambiante pendant 800 tours par minute de mélange, en évitant l&#39;exposition à la lumière. </li><li> Purifier les IP d&#39;or-BSA-rhodamine par dialyse contre PBS à 4 ° C avec 5 changements de tampon sur la période de 36 à 48 h. </li><li> Pour stabiliser les IP, ajouter 10 ul de 200 mg / ml de BSA / PBS dans chacun 1 ml IP or BSA-rhodamine. Pour supprimer la centrifugeuse libre ou libéré BSA-rhodamine, à 15,000 xg pendant 60 min. Reprendre le culot dans 2 mg / ml de BSA / PBS, mesurer 520 OD, et conserver à 4 ° C, évitant l&#39;exposition à la lumière. </li><li> Diluer les IP 10 fois avec Milli-Q ou de l&#39;eau doublement distillée, ajouter une goutte sur une grille recouverte de carbone, et laisser sécher à l&#39;air. Vérifier la taille et la morphologie des IP par microscopie électronique à transmission (MET). NOTE: Tous les matériaux utilisés pour la préparation NP ont été stérilisés et l&#39;opération a été réalisée dans une hotte de culture cellulaire ou sur un banc à côté d&#39;une flamme. </li></ol> 3. Culture de cellules HeLa <ol><li> Des cellules HeLa exprimant de façon permanente LAMP1-GFP sont cultivées dans du DMEM avec 10% de sérum de veau foetal (FCS) et cultivées à 37 ° C dans une atmosphère contenant 5% de CO 2. </li><li> Ensemencer les cellules à une densité de 50 000 par puits dans une chambre diapositive 8 de puits (Ibidi) et incuber O / N. </li></ol> 4. culture de bactéries <ol><li> Utilisez Salmonella enterica sérotype Typhimurium NCTC 12023 wilD-Type (WT) souche. A titre de comparaison, utiliser des souches mutantes Δ ssaV défectueux dans le SPI2-SST3 et Δ SIFA manquant effecteur clé SIFA pour SIF. Utilisez les souches portant le plasmide pFPV25.1 pour l&#39;expression constitutive de la GFP améliorée. REMARQUE: Les souches bactériennes sont cultivées en routine dans du bouillon de Luria-Bertani (LB) avec addition de 50 ug / ml de carbénicilline (WT) et LB avec addition de 50 ug / ml de carbénicilline et / ou 50 pg / ml de kanamycine (ssa V Δ, Δ SIFA ) nécessaire au maintien de plasmides. </li><li> Inoculer une seule colonie de bactéries dans 3 ml de LB avec les antibiotiques appropriés et grandir O / N à 37 ° C dans des conditions secousse pour l&#39;aération, puis diluer 01h31 dans LB frais et continuer la croissance pendant 3,5 heures. A ce moment-là, les cultures atteignent la phase logarithmique tardive et les bactéries sont très envahissantes. A &#39;rouleau tambour »est pratique pour incuber des cultures de tube à essai avec l&#39;aération. </li></ol> 5. L&#39;infection des cellules HeLa parSalmonella et Gold-BSA-rhodamine IP Pulse Chase-étiquetage (voir la figure 1 pour le régime) <ol><li> Mesurer la DO 600 de la bactérie de la sous-culture et diluer à DO600 = 0,2 dans 1 ml de PBS (~ 3 x 10 8 ufc / ml), ajouter des quantités appropriées de bactéries à des cellules HeLa dans des lames à chambres 8 puits pour parvenir à une multiplicité de infection (MOI) de 100. </li><li> Incuber pendant 30 min dans l&#39;incubateur de cellules, laver 3 fois avec du PBS pour éliminer les bactéries non-internalisés (ce point de temps a été défini comme post-infection 0 h, ou 0 h pi). Ajouter 300 ul de milieu de culture frais contenant 100 ug / ml de gentamicine et maintenir pendant 1 heure. Remplacer ensuite le milieu par du milieu frais contenant 10 ug / ml de gentamicine pendant le reste du temps d&#39;incubation. </li><li> Après incubation avec le milieu de culture contenant 100 ug / ml de gentamicine pendant 1 h, le milieu est remplacé par imagerie milieu (Eagles MEM sans FCS, de L-glutamine, le phénol rouge et bicarbonate de sodium, avec 30 mM de HEPES, pH 7,4) containing 10 ug / ml de gentamicine. NP or-rhodamine-BSA sont ajoutés à des cellules HeLa pour obtenir une concentration finale de OD 520 = 0,1. REMARQUE: NP-BSA-or rhodamine peut également être ajouté aux cellules avant l&#39;infection, ou à divers points de temps pi </li><li> Après 1 h d&#39;incubation, éliminer le milieu, laver trois fois avec du PBS, et ajouter 300 ul de milieu de formation d&#39;image fraîche contenant 10% de FCS et 10 ug / ml de gentamicine pendant le reste du temps d&#39;incubation. NOTE: Durée d&#39;incubation peut varier en fonction de la concentration des IP et des lignées cellulaires utilisées. Pour les macrophages RAW264.7, nous avons observé que 30 minutes d&#39;incubation avec des IP à une concentration de OD 520 = 0,05 permis internalisation suffisante. </li></ol> 6. Imaging <ol><li> Utilisation d&#39;un système d&#39;imagerie confocale, tel qu&#39;un laser à balayage confocal (CLSM) ou disque de filage (SD) microscope avec une chambre à environnement humidifié pour imagerie à haute résolution à différents points de temps. </li><li> Allumez la température control système et attendez jusqu&#39;à ce qu&#39;il soit stable. Optimiser les paramètres d&#39;imagerie telles que l&#39;agrandissement, la vitesse de numérisation, résolution, Z-pile, etc. Utiliser les paramètres d&#39;excitation / émission appropriées pour la GFP et IP or BSA-rhodamine. Pour ce protocole, utiliser un 400 Hz vitesse de numérisation, résolution de 512 x 512 pixels et la taille Z-étape de 0,25 um. Exciter et GFP-BSA-or rhodamine utilisant un laser Ar (488 nm) et un laser HeNe (543 nm), respectivement. Inclure un (BF) canal lumineux sur le terrain pour observer la forme des cellules. Pour les autres systèmes de microscopie et des conditions d&#39;infection, le réglage doivent être ajustées en conséquence. REMARQUE: Utilisez le même laser Ar comme la source de canal de BF et signal lumineux a été détectée avec un détecteur photomultiplicateur (PMT). </li><li> Au temps indiqué points pi, monter les diapositives de la chambre 8 puits contenant les cellules infectées sur la platine du microscope et enregistrer des images. </li></ol> 7. Analyse des Images <ol><li> Utilisez la microscopie logiciel d&#39;analyse d&#39;image (voir <strong&gt; Matériaux et table d&#39;équipement) pour analyser le remodelage du système endo-lysosomale par Salmonella intracellulaire. Ouvrez les données en cliquant sur &#39;Ouvrir&#39; et en choisissant le fichier. NOTE: Vous pouvez également ouvrir les paquets de logiciels libres tels que ImageJ ou FIDJI peuvent être utilisés pour l&#39;image des analyses quantitatives. </li><li> Dans la barre d&#39;outils des objets du clic Surpass vue sur l&#39;icône <img alt="Icône" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon1.jpg" /> pour ajouter un nouvel élément de surface. Cliquez sur <img alt="Icône" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Suivant). </li><li> Pour analyser une région d&#39;intérêt (ROI), sélectionnez le segment un retour sur investissement. Dans une zone de visualisation, une section de rectangle bordé superposée sur l&#39;image représente le retour sur investissement. Entrez les valeurs dans la direction x correspondant, y et champs Z-, ou cliquez directement sur les flèches dans le rectangle d&#39;aperçu de modifier la taille et la position de la ROI. Puis cliquez sur <img alt="Icône" fo:content-width=".1in" src<html> = &quot;/ Files / ftp_upload / 52058 / 52058icon2.jpg&quot; /&gt; (Suivant). </li><li> Comme un canal source, sélectionnez le canal 3 (signal pour l&#39;or-BSA-rhodamine IP). Cochez l&#39;option lisse de mettre en place la douceur de la zone résultant. Définir une valeur manuellement ou accepter la valeur générée automatiquement. Pour Seuil, sélectionnez l&#39;option absolue de l&#39;intensité. </li><li> Pour le réglage de seuil, sélectionnez l&#39;option manuelle et définir une valeur. Dans la zone de visualisation, un aperçu de seuil de surface se affiche en gris. </li><li> Dans l&#39;onglet Classer Surfaces la surface résultante peut être filtrée par divers critères. Un filtre par défaut est «nombre de voxels&gt; 10 &#39;, et d&#39;autres filtres peuvent également être inclus en cliquant sur&#39; Ajouter &#39;. Si un filtrage ne est pas nécessaire, puis supprimez tous les filtres en cliquant sur le bouton Supprimer. Cliquez <img alt="Icône" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon2.jpg" /> (Suivant). </li><li> Pour terminer la création de surface, cliquez sur/files/ftp_upload/52058/52058icon3.jpg &quot;/&gt; (Terminer). Dans la liste d&#39;objets, maintenant décochez la case pour le volume et la nouvelle surface créée élément se affiche dans la zone de visualisation. </li><li> Exporter les statistiques. Maintenant, dans la vue Surpass la couleur des sujets varient du violet au rouge en fonction de leur région. Et la table &#39;Numéros de courbe de surface&#39; affiche les informations (par exemple, numéro d&#39;identification et la zone d&#39;objets) des statistiques. Export de toutes les statistiques de surface 1 à un fichier Excel par clic <img alt="Icône" fo:content-width=".1in" src="/files/ftp_upload/52058/52058icon4.jpg" /> (Save). </li></ol>

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No conflicts of interest declared.

Le système endo-lysosomal de cellules de mammifère contrôle les processus physiologiques importants, y compris l&#39;absorption des nutriments, l&#39;hormone de la transduction du signal à médiation par, la surveillance immunitaire et la présentation de l&#39;antigène 17. Jusqu&#39;à présent, divers marqueurs ont été utilisés pour le marquage des études de la voie d&#39;endocytose et de suivi. Par exemple, les sondes sont des sondes d&#39;Lysotracker acidotropic fluorescents mis au point par Mole...

Nanoparticules fluorescentes (IP), y compris IP métalliques, points quantiques, IP, IP polymères de silice, des points de carbone, etc., ont attiré une attention considérable au cours des dernières décennies 1,2. Par rapport à des colorants organiques traditionnels, les IP fluorescents montrent chimiques, des propriétés optiques et mécaniques souhaitables, telles que la résistance forte du signal, la résistance au photoblanchiment et haut de 3,4 biocompatibilité. Ces avantages en font la méthode de choix pour la détection intracellulaire et imagerie des cellules vivantes. En outre, une variété de IP denses aux électrons sont visibles en microscopie électronique (EM), ce qui facilite leur utilisation pour l&#39;analyse microscopique corrélée, qui permet combinaison de suivi en microscopie optique (LM) et une résolution supérieure au niveau ultrastructural avec EM 5 cellules vivantes. Par exemple, les IP or ont été longtemps utilisé efficacement comme biocapteurs pour le diagnostic dans les cellules sensibles vie ainsi que dans le domaine de l&#39;immuno-marquage 6. S récentses études indiquent que les IP or avec la taille et de forme différentes peuvent être facilement absorption par une grande variété de lignées de cellules et de transporter régulièrement par la voie endosomale, ont donc grand potentiel étant appliquée pour le suivi du transport des vésicules intracellulaires et l&#39;étiquetage du système endo-lysosomale 7,8 . Les pathogènes microbiens, tels que Salmonella enterica, Shigella flexneri et Listeria monocytogenes, ont développé des mécanismes différents pour envahir les cellules hôtes non-phagocytaires neuf. Après avoir été intériorisé, les agents pathogènes, soit localisées dans le cytosol ou séquestrés dans les compartiments membranaires, interagissent beaucoup avec leur environnement d&#39;accueil et les moduler pour favoriser leur propre survie 10. Par exemple, Salmonella enterica réside et se multiplie dans un phagosome intracellulaire appelé la vacuole contenant Salmonella (SCV) lors de l&#39;infection 11. Le SCV maturationtrafics vers l&#39;appareil de Golgi, subissant interactions continues avec la voie d&#39;endocytose, et induit la formation de vastes structures tubulaires, tels que Salmonella filaments induites (SIF), le tri tubules nexine, Salmonella induite porteuses de sécrétion des protéines de la membrane 3 (SCAMP3) tubules, etc. . 12-14. Étudier comment ces agents pathogènes bactériens manipuler voies cellule hôte est essentielle pour comprendre les maladies infectieuses. Ici, NP or-BSA-rhodamine ont été utilisés comme marqueurs de fluide pour marquer le système endo-lysosomal cellulaire hôte et l&#39;agent pathogène gastro-intestinal Salmonella enterica sérovar Typhimurium humain (Salmonella) a été utilisé comme une bactérie modèle pour étudier les interactions de l&#39;agent pathogène avec le accueillir voie d&#39;endocytose. Intracellulaires IP or BSA-rhodamine dans les cellules et les cellules non infectées infectés par Salmonella WT ou souches mutantes ont été imagées par un microscope confocal à balayage laser (CLSM).Puis logiciels Imaris a été utilisée pour quantifier la distribution des IP, ce qui indique que l&#39;infection à Salmonella induit extrême réarrangement des endosomes / lysosomes. Après la description de cette méthode, des expériences analogues peuvent être conçus pour suivre devenir à long terme des IP intériorisés et d&#39;étudier l&#39;influence de diverses substances exogènes ou facteurs endogènes sur la voie d&#39;endocytose des cellules eucaryotes.

<table border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:193px;">Name of the Material/Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:149px;">Catalog Number</td>
			<td style="width:176px;">Comments/ Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gold chloride</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tri-sodium citrate</td>
			<td>Sigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NHS-Rhodamine</td>
			<td>Pierce</td>
			<td>46406</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin</td>
			<td>Applichem</td>
			<td>A1492</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamcyin</td>
			<td>Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>Roth</td>
			<td>6344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-well chamber slides</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>tissue culture treated, sterile</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris Software</td>
			<td>Bitplane</td>
			<td>version 7.6</td>
			<td>various configurations available</td>
		</tr>
	</tbody>
</table>

application of fluorescent nanoparticles to study remodeling of the endo-lysosomal system by intracellular bacteria

Nanoparticules fluorescentes (IP) avec des propriétés optiques et mécaniques et chimiques souhaitables, sont des outils prometteurs pour étiqueter organites intracellulaires. Ici, nous introduisons une méthode utilisant or-BSA-rhodamine IP d&#39;étiqueter le système de cellules eucaryotes endo-lysosomale et surveiller manipulations des voies cellulaires de l&#39;hôte par le pathogène intracellulaire Salmonella enterica. Les IP ont été facilement internalisées par les cellules HeLa et localisées à la fin des endosomes / lysosomes. Infection à Salmonella réarrangement des vésicules et l&#39;accumulation des IP dans les structures membranaires Salmonelle induites induit. Nous avons déployé le progiciel Imaris pour des analyses quantitatives des images de microscopie confocale. Le nombre d&#39;objets et de leur distribution de taille dans les cellules non infectées étaient distinctes de celles dans les cellules infectées par Salmonella, indique une très remodelage du système endo-lysosomale par WT Salmonella.

IP or ont été générés par une méthode bien établie par la réduction de l&#39;acide chloroaurique par le citrate et l&#39;acide tannique. Comme le montre la Figure 2A, les PN synthétisés or étaient quasi-sphérique avec une taille d&#39;environ 10 nm. BSA-revêtement et de la rhodamine-étiquetage ne ​​ont pas d&#39;influence sur la morphologie ou la taille (figure 2B). Il a été rapporté que les IP or peuvent être facilement absorbés par d...

Watch this Scientific Journal Video about Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria at JoVE.com

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

Cet article décrit les méthodes pour la synthèse et le marquage fluorescent de nanoparticules (NP). Les IP ont été appliquées dans des expériences pulse-chase d&#39;étiqueter le système endo-lysosomale des cellules eucaryotes. La manipulation du système d&#39;endo-lysosomal par les activités de l&#39;agent pathogène intracellulaire Salmonella enterica ont été suivis par imagerie des cellules vivantes et quantifiée.

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 ...

application-fluorescent-nanoparticles-to-study-remodeling-endo

Research

JoVE Journal

Immunology and Infection

9.9K vues.  Universität Osnabrück.  Cet article décrit les méthodes pour la synthèse et le marquage fluorescent de nanoparticules (NP). Les IP ont été appliquées dans des expériences pulse-chase d'étiqueter le système endo-lysosomale des cellules eucaryotes. La manipulation du système d'endo-lysosomal par les activités de l'agent pathogène intracellulaire Salmonella enterica ont été suivis par imagerie des cellules vivantes et quantifiée. 

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

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Détection multiplex de bactéries dans les échantillons complexes cliniques et environnementaux à l&#39;aide d&#39;oligonucléotides couplés microsphères fluorescentes

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Recherche

Immunologie et infection

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

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

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 ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Simples mesures de cellules de rupture Vacuolar causées par des pathogènes intracellulaires

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Mise en place d&#39;un<em&gt; In vitro</em&gt; Système d&#39;étudier le comportement intracellulaire de<em&gt; Candida glabrata</em&gt; En droits macrophages THP-1

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

L&#39;application d&#39;un système d&#39;expression inductible pour étudier les interférences de facteurs de virulence bactérienne avec signalisation intracellulaire

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Développement d&#39;un ADN Système économique Livraison par « Acufection » et son application à la recherche de la peau

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Système d&#39;efficacité et de dépistage des inhibiteurs Cytotoxicité ciblage intracellulaire<em&gt; Mycobacterium tuberculosis</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

Marquage immunohistochimique Fluorescent multiplexé, l’imagerie et l’analyse des échantillons histologique du lymphome

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

Modèles de colonisation vaginale murine par des bactéries de culture anaérobie

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...