<p class="jove_content"&gt; RN a été partiellement financée par une bourse Marie subventions Re-Curie d&#39;intégration européens (ERG) du septième programme-cadre de la Communauté européenne, le numéro de projet 268324.</p&gt;

1. Isolation des PMN du sang humain par centrifugation par densité <ol><li> Dessinez 5 9 tubes ml de sang veineux hépariné. </li><li> Préparer double couche gradients Ficoll (4 gradients de 5 tubes de sang), comme suit: verser 12 ml d&#39;une solution de ficoll de densité 1,119 g / ml dans un tube de 50 ml et la couche soigneusement 10 ml d&#39;une solution de ficoll de densité 1,077 g / ml sur le dessus. </li><li> Diluer le sang avec un volume égal de PBS. </li><li> La couche de sang dilué avec soin sur la couche double gradient Ficoll; 20-25 ml par gradient. </li><li> Centrifuger 20 min à 396 x g dans un rotor à godets oscillants, 22 ° C sans freinage. </li><li> Préparer RPMI froid contenant 0,05% de l&#39;albumine sérique humaine (RPMI-HSA). Préparez aussi 9 ml stérile déminéralisée H 2 O. Pré-cool à la fois le RPMI et H 2 O sur la glace. </li><li> Aspirer la couche de Ficoll supérieure contenant le plasma (de couleur jaune) et des PBMC, et la seconde couche de Ficoll (couleur blanche) avec l&#39;utilisation d&#39;une pompe à vide (mettre une pointe de pipette stérile surla pipette). </li><li> Recueillir les PMN en tubes de 50 ml à l&#39;aide d&#39;une petite pipette en plastique (1 Tube pour les fractions de PMN tous les 2 gradients), et les placer sur la glace. </li><li> Ajouter froid RPMI-HSA pour un volume total de 50 ml et centrifuger pendant 10 min à 249 xg à 4 ° C. </li><li> Enlever le surnageant à l&#39;aide d&#39;une pompe à vide et le culot vortex doucement (érythrocytes et PMN). </li><li> Ajouter 9 ml stérile déminéralisée H 2 O et lancer le chronomètre. Arrêter le choc osmotique hyper après 30 secondes exactement, en ajoutant 1 ml 10 fois concentré PBS. Note: le 30 sec sont très critiques. </li><li> Ajouter froid RPMI-HSA pour un volume total de 50 ml et centrifuger pendant 10 min à 249 xg à 4 ° C. </li><li> Enlever le surnageant à l&#39;aide d&#39;une pompe à vide et de recueillir le PMN culot dans 1 tube avec un volume défini (2.1 ml) de milieu RPMI-HSA. </li><li> Déterminer la quantité de cellules et ajuster la concentration de 1.10 7 cellules / ml. Selon le donateur, le rendement du PMN isolés sont betweFR 5 x 10 6 et 3 x 10 7 PMN de chaque tube 9 ml de sang </li></ol> 2. Débit Assay Cytometric pour l&#39;évaluation de la mobilisation de calcium dans PMN humains <ol><li> Les cellules de charge (5 x 10 6 cellules / ml) avec 2uM Fluo-3-AM dans RPMI-HSA et incuber pendant 20 min à température ambiante bascule lentement, en utilisant par exemple une plate-forme à bascule shaker, à l&#39;abri de la lumière. </li><li> En attendant préparer une dilution en série (par exemple 3 fois) du stimulus à 10x la concentration finale. PSMα3 est utilisé dans ce protocole, mais tout stimulus de GPCR qui agit sur les neutrophiles est approprié. </li><li> Préparer inhibiteur concentré 10x du récepteur dans RPMI-HSA. Lorsque l&#39;inhibiteur agit sur ​​la stimulation (par exemple HDL sur PSMα3) pré-incuber 25 stimulus ul avec un volume égal d&#39;inhibiteur pendant 10 min à température ambiante. </li><li> Laver les cellules en ajoutant 10 ml de RPMI-HSA et centrifugeuse pour 249 g à température ambiante. Remettre les cellules à 5 x10 6 cellules / ml. </li><li> Juste avant l&#39;expérience, diluer les cellules à 2x10 6 cellules / ml dans du RPMI-HSA et ajouter 200 pi de cellules par tube FACS. PMN dilué sont très fragiles. Les garder trop longtemps à cette concentration va conduire à l&#39;activation automatique; la même chose pour secouer vigoureusement ou pipetage. </li><li> Attachez le tube à la cytométrie en flux, attendre 3 secondes et lancer l&#39;acquisition. Après une période de temps fixe (par exemple 8 sec), enlever le tube et ajouter rapidement 50 stimulus ul de l&#39;échantillon. Immédiatement ré-attacher le tube sur le porte-échantillon et de continuer l&#39;acquisition. </li><li> Commencez par le bas et à la fin avec la plus forte concentration de stimulus. Laver l&#39;aiguille de cytomètre de flux régulièrement après chaque course avec RPMI-HSA. </li><li> Analyser des données avec le logiciel d&#39;analyse de cytométrie en flux. Comparer le signal de fluorescence moyenne avant l&#39;ajout du stimulus pour que, après plus de stimulus et d&#39;utiliser les contrôles positifs et négatifs appropriés pour calculer l&#39;acrésistance à la vation mesurée pour chaque dilution. </li></ol> En variante lorsque l&#39;inhibiteur agit sur les récepteurs de pré-incuber les cellules avec l&#39;inhibiteur. 3. Analyse par microscopie de fluorescence de la GFP bactéries expression après phagocytose par les PMN <ol><li> Croître S. souches de Staphylococcus contenant une construction reporter d&#39;intérêt pendant la nuit dans un bouillon (avec des antibiotiques lorsque cela est nécessaire pour maintenir le plasmide rapporteur). Dans ce cas, la souche de MW2 contenant une construction rapporteur GFP PSMα 10 est utilisé, cultivées dans un tube en matière plastique de 50 ml avec 5 ml de milieu de culture LB. </li><li> Pour supprimer tous GFP de l&#39;expression O / N, diluer les cultures à OD 660 0,01 croître et à OD 660 0.1. Redilute ce 1h30 de culture, et de surveiller la croissance jusqu&#39;à ce que la culture atteint une DO660 de 0,1. Recueillir les bactéries par centrifugation et les laver une fois dans DPBS. Reprendre en 1h10 du volume initial pour obtenir une DO 660 de 1,0 ou roughly 5.10 8 ufc / ml. </li><li> Mélanger les bactéries (1.10 7 / ml) avec PMN fraîchement isolés (1.10 6 / ml) dans du RPMI-HSA (10:1) dans un microtube de 1,5 ml et ajouter le sérum humain mis en commun à une concentration finale de 10%. Secouez sur une plate-forme agitation pendant 10 min à 37 ° C pour stimuler la phagocytose. Diluer les PMN chargées de bactéries à 5.10 5 PN / ml et pipette 250 ul dans un puits d&#39;une lamelle bien chambré 8. </li><li> Image PMN avec des bactéries sur un microscope. Un microscope inversé équipé d&#39;un objectif NA 40X/0.85 fonctionne bien et ne doit être enfermé dans une chambre à environnement sombre pour maintenir l&#39;environnement de manière stable à 37 ° C. Acquérir les images pour un certain nombre de positions prédéfinies à l&#39;aide de la caméra toutes les 5-10 min à la fois dans le champ lumineux et le canal GFP de suivre la production de GFP dans le temps. </li></ol>

<ol>
	<li>Rigby, K. M., DeLeo, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+in+innate+host+defense+against+Staphylococcus+aureus+infections.">Neutrophils in innate host defense against Staphylococcus aureus infections.</a> Semin. Immunopathol. 34, 237-259 (2012).</li><li>Voyich, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Mechanisms+Used+by+Staphylococcus+aureus+to+Avoid+Destruction+by+Human+Neutrophils.">Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils.</a> The Journal of Immunology. 175, 3907-3919 (2005).</li><li>Kobayashi, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+neutrophil+destruction+following+phagocytosis+of+Staphylococcus+aureus.">Rapid neutrophil destruction following phagocytosis of Staphylococcus aureus.</a> J. Innate Immun. 2, 560-575 (2010).</li><li>DeLeo, F. R., Otto, M., Kreiswirth, B. N., Chambers, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-associated+meticillin-resistant+Staphylococcus+aureus.">Community-associated meticillin-resistant Staphylococcus aureus.</a> The Lancet. 375, 1557 (2010).</li><li>David, M. Z., Daum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community-Associated+Methicillin-Resistant+Staphylococcus+aureus:+Epidemiology+and+Clinical+Consequences+of+an+Emerging+Epidemic.">Community-Associated Methicillin-Resistant Staphylococcus aureus: Epidemiology and Clinical Consequences of an Emerging Epidemic.</a> Clinical Microbiology Reviews. 23, 616-687 (2010).</li><li>Kretschmer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Formyl+Peptide+Receptor+2+Senses+Highly+Pathogenic+Staphylococcus+aureus.">Human Formyl Peptide Receptor 2 Senses Highly Pathogenic Staphylococcus aureus.</a> Cell Host &amp; Microbe. 7, 463 (2010).</li><li>Prat, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homolog+of+formyl+peptide+receptor-like+1+(FPRL1)+inhibitor+from+Staphylococcus+aureus+(FPRL1+inhibitory+protein)+that+inhibits+FPRL1+and+FPR.">A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR.</a> J. Immunol. 183, 6569-6578 (2009).</li><li>Prat, C., Bestebroer, J., de Haas, C. J. C., van Strijp, J. A. G., van Kessel, K. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Staphylococcal+Anti-Inflammatory+Protein+That+Antagonizes+the+Formyl+Peptide+Receptor-Like+1.">A New Staphylococcal Anti-Inflammatory Protein That Antagonizes the Formyl Peptide Receptor-Like 1.</a> The Journal of Immunology. 177, 8017-8026 (2006).</li><li>de Haas, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+inhibitory+protein+of+Staphylococcus+aureus,+a+bacterial+antiinflammatory+agent.">Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.</a> The Journal of Experimental Medicine. 199, 687-695 (2004).</li><li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Staphylococcal+Phenol+Soluble+Modulins+by+Serum+Lipoprotein+Particles.">Inactivation of Staphylococcal Phenol Soluble Modulins by Serum Lipoprotein Particles.</a> PLoS Pathog. 8, e1002606 (2012).</li><li>Kubica, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Potential+New+Pathway+for+Staphylococcus+aureus+Dissemination:+The+Silent+Survival+of+S.+aureus+Phagocytosed+by+Human+Monocyte-Derived+Macrophages.">A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages.</a> PLoS ONE. 3, e1409 (2008).</li><li>Surewaard, B. G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+alpha-phenol+soluble+modulins+contribute+to+neutrophil+lysis+after+phagocytosis.">Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis.</a> Cellular Microbiology. , (2013).</li><li>Queck, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAIII-Independent+Target+Gene+Control+by+the+agr+Quorum-Sensing+System:+Insight+into+the+Evolution+of+Virulence+Regulation+in+Staphylococcus+aureus.">RNAIII-Independent Target Gene Control by the agr Quorum-Sensing System: Insight into the Evolution of Virulence Regulation in Staphylococcus aureus.</a> Molecular Cell. 32, 150 (2008).</li><li>Baban, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Bacterial+Imaging+In+Vivo.">Bioluminescent Bacterial Imaging In Vivo.</a> J. Vis. Exp. (69), e4318 (2012).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Dans les procédés décrits ici quelques étapes sont très critiques. Nous allons mettre en évidence ces ici. Pour l&#39;isolement des neutrophiles par centrifugation en gradient de densité, il est important de ne pas perturber les couches pendant ou après les étapes de centrifugation. Lors de l&#39;aspiration des neutrophiles à l&#39;aide d&#39;une pipette en plastique, assurez-vous de ne pas presser le ballon alors que dans la couche de cellules, comme éjecter un liquide va perturb...

Neutrophiles (PMN) sont des phagocytes professionnels qui jouent un rôle clé dans la réponse immunitaire innée contre le Staphylococcus aureus 1. La bataille constante entre l&#39;hôte et microbe a conduit à une course aux armements des deux. Récemment, la communauté-associés souches (CA) de résistant à la méticilline S. aureus (MRSA) ont vu le jour qui semble être très efficace dans la neutralisation des neutrophiles meurtre 2,3. Modulin (PSM), la production soluble phénol excessif de CA-MRSA a été associée avec une plus grande virulence 4,5. Neutrophiles humains peuvent reconnaître ces PSM via FPR2 qui conduisent à l&#39;activation de cette protéine-G récepteur couplé à 6. Un des premiers événements est la mobilisation des réserves intracellulaires de calcium (Ca 2 +). Ca 2 + agit comme un messager secondaire pour une variété de fonctions effectrices des PMN dont la dégranulation et la phagocytose 7. Par conséquent Ca 2 + est un indicateur très sensible de lala capacité fonctionnelle du PSM pour activer PMN. Pour étudier les effets de PSM sur les neutrophiles, les neutrophiles frais sont isolées et chargées avec un colorant fluorescent qui lors de la mobilisation du calcium. Dans une expérience de cytométrie en flux cette mobilisation rapide peut être mesurée. En utilisant cette méthode, il est possible d&#39;étudier les effets directs des composants toxiques et d&#39;autres sur les neutrophiles, et de déterminer la concentration minimale à laquelle ceux-ci sont actifs. Pour nous, c&#39;est un outil très utile pour étudier l&#39;effet de nombreuses protéines produites par S. aureus impliquée dans l&#39;évasion immunitaire, comme FPR2 protéine inhibitrice (FlipR) 8, FlipR-like 7 et Chemotaxis protéine inhibitrice de Staphylococcus aureus (CHIPS) 9. Toutes ces protéines ont été montré pour empêcher la mobilisation du calcium dans les neutrophiles en se liant au récepteur reconnaissant l&#39;agoniste. Récemment, notre groupe a indiqué que les MSP sont fonctionnellement inhibés par les lipoprotéines du sérum 10 &lt;/ Sup&gt;. Ces lipoprotéines sont abondamment présents dans le sang et les tissus humains, ce qui indique que les MSP exercent leur fonction principalement dans le milieu intracellulaire. La disponibilité du test de mobilisation du calcium permis de mesurer précisément l&#39;effet des lipoprotéines du sérum sur l&#39;activation des neutrophiles par les PSM, indiquée par l&#39;inhibition considérable par de très faibles concentrations de sérum. Depuis les PSM sont fonctionnellement inhibée par le sérum, nous avons émis l&#39;hypothèse qu&#39;il existe une fonction importante pour PSM que les toxines intracellulaires. Nous avons donc cherché à déterminer le rôle de PSM après phagocytose. Pour étudier l&#39;expression des MPS dans l&#39;espace intercellulaire, nous avons construit des fusions rapporteurs du promoteur de l&#39;opéron psmα à la GFP. Lorsque ces souches rapporteuses de S. aureus ont été phagocytés par les neutrophiles, l&#39;induction de l&#39;expression était observable en utilisant la microscopie à fluorescence 10. Évidemment, cette technique permet l&#39;étude de l&#39;expression d&#39;un grand nombre de gènes de S. aureus ou d&#39;autres agents pathogènes après phagocytose. Étant donné que pour S. aureus survivre dans la niche intercellulaire est très important pour surmonter le système immunitaire inné 11 10 12, étudier le rôle des gènes activés dans ce créneau est très pertinente pour la compréhension de sa virulence.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Fluo-3, AM</td>
			<td>Molecular Probes / Life Technologies</td>
			<td>F-1241</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-5442-03</td>
			<td>density 1.077 g/ml</td>
		</tr>
		<tr>
			<td>Histopaque</td>
			<td>Sigma</td>
			<td>11191</td>
			<td>density 1.119 g/ml</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco, Life Technologies</td>
			<td>52400-025</td>
			<td>contains 25 mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>Leica TCS SP5 microscope</td>
			<td>Leica Microsystems, The Netherlands</td>
			<td>TCS SP5</td>
			<td>objective: HCX PL APO 40X/0.85</td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td>FACSCalibur</td>
			<td>Very important that the tube can be removed and replaced during the measurement process</td>
		</tr>
	</tbody>
</table>

studying interactions of staphylococcus aureus with neutrophils by flow cytometry and time lapse microscopy

Nous présentons les méthodes pour étudier l&#39;effet de modulins solubles phénol (PSM) et d&#39;autres toxines produites et sécrétées par Staphylococcus aureus sur les neutrophiles. Pour étudier les effets des MPS sur les neutrophiles nous isolons neutrophiles frais en utilisant un gradient de densité centrifugation. Ces neutrophiles sont chargées avec un colorant fluorescent qui lors de la mobilisation du calcium. L&#39;activation des neutrophiles par les MSP lance une augmentation rapide et transitoire de la concentration de calcium intracellulaire libre. Dans une expérience de cytométrie en flux cette mobilisation rapide peut être mesurée en surveillant la fluorescence d&#39;un colorant pré-chargé qui réagit à l&#39;augmentation de la concentration de Ca 2 + libre. En utilisant cette méthode, nous pouvons déterminer la concentration de PSM nécessaire d&#39;activer le neutrophiles, et de mesurer les effets des inhibiteurs spécifiques et générales de l&#39;activation des neutrophiles. Pour étudier l&#39;expression des MPS dans l&#39;espace intracellulaire, we ont construit fusions rapporteurs du promoteur de l&#39;opéron PSMα à la GFP. Lorsque ces souches rapporteuses de S. aureus sont phagocytés par les neutrophiles, l&#39;induction de l&#39;expression peut être observée en utilisant la microscopie à fluorescence.

Débit test de cytométrie pour l&#39;évaluation de la mobilisation du calcium dans les PMN humains Incuber des neutrophiles avec une série de concentrations de PSMα3 synthétiques résultant en une activation rapide, tel que mesuré par le flux de calcium, qui est représenté par une augmentation du signal dans FL-1. La pré-incubation de synthèse PSMα3 avec 0,01%, 0,1% ou 1% de sérum humain inhibé de manière significative la capacité de susciter des flux de calcium (Figure 1...

Watch this Scientific Journal Video about Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy at JoVE.com

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Nous présentons les méthodes pour étudier l&#39;effet du PSM et d&#39;autres toxines sécrétées par<em&gt; Staphylococcus aureus</em&gt; Sur les neutrophiles en utilisant la cytométrie en flux et la microscopie de fluorescence.

L&#39;étude des interactions de<em&gt; Staphylococcus aureus</em&gt; Avec des neutrophiles par cytométrie en flux et microscopie Time Lapse

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

studying-interactions-staphylococcus-aureus-with-neutrophils-flow

Research

JoVE Journal

Immunology and Infection

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

13.0K vues.  University Medical Center Utrecht. Nous présentons les méthodes pour étudier l'effet du PSM et d'autres toxines sécrétées par Staphylococcus aureus Sur les neutrophiles en utilisant la cytométrie en flux et la microscopie de fluorescence.

Vidéo: Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

L&#39;utilisation des flux de goutte et des réacteurs de disque rotatif pour Staphylococcus aureus Analyse du biofilm

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Recherche

Immunologie et infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Infection sous-cutanée du Staphylococcus aureus résistant à la méthicilline (SARM)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Croissance Staphylococcus aureus en utilisant l&#39;hémoglobine humaine comme source de fer

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Une méthode simple et efficace pour détecter l&#39;activation du facteur nucléaire dans les neutrophiles humains par cytométrie en flux

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Détection de nanoparticules fluorescentes Interactions avec immunitaire primaire sous-populations de cellules par cytométrie en flux

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagerie neutrophiles et des monocytes dans les veines mésentérique par Microscopie intravitale sur des souris anesthésiées en temps réel

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

génotypage des<em&gt; Staphylococcus aureus</em&gt; par Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Méthodologie de l&#39;étude du transfert horizontal de gènes dans<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Time-lapse imagerie 3D de phagocytose par les Macrophages de souris

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Un modèle de souris pour évaluer réponse immunitaire innée à l’infection par Staphylococcus aureus

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...