<p class="jove_content"&gt; RN wurde teilweise durch ein Marie Curie European Reintegration Grants (ERG) von der Europäischen Gemeinschaft Siebten Rahmenprogramms, Projektnummer 268324 gefördert.</p&gt;

1. Isolation von PMN aus menschlichem Blut durch Dichtezentrifugation <ol><li> Unentschieden 5 9 ml Röhrchen heparinisierter venösen Blut. </li><li> Bereiten Dual-Layer-Ficoll-Gradienten (4 Steigungen für 5 Gefäße Blut) wie folgt: Gießen Sie 12 ml mit einer Dichte von 1,119 g / ml Ficoll-Lösung in einem 50 ml-Tube und sorgfältig Schicht 10 ml mit einer Dichte von 1,077 g / ml Ficoll-Lösung an der Spitze. </li><li> Man verdünnt das Blut mit einem gleichen Volumen von PBS. </li><li> Schicht das verdünnte Blut vorsichtig auf die Dual-Layer-Ficollgradienten; 20-25 ml pro Gradienten. </li><li> Zentrifuge 20 min bei 396 xg in einem Ausschwingrotor, 22 ° C ohne Bremsen. </li><li> Bereiten kalt RPMI mit 0,05% Human Serum Albumin (RPMI-HSA). Auch bereiten 9 ml sterilem entionisiertem H 2 O. Pre-kühlen sowohl die RPMI und H 2 O auf Eis. </li><li> Absaugen der oberen Schicht mit Ficoll Plasma (gelbe Farbe) und PBMC, und die zweite Schicht von Ficoll (weiße Farbe) unter Verwendung einer Vakuumpumpe (Put eine sterile Pipettenspitzedie Pipette). </li><li> Sammeln Sie die PMN in 50 ml-Röhrchen mit einem kleinen Kunststoff-Pipette (1 Tube für die PMN Fraktionen von jeweils 2 Gradienten), und legen Sie sie auf Eis. </li><li> In kaltem RPMI-HSA auf ein Gesamtvolumen von 50 ml und Zentrifuge für 10 min bei 249 xg bei 4 ° C. </li><li> Nehmen Sie den Überstand mit dem Einsatz einer Vakuumpumpe und Wirbel das Pellet vorsichtig (Erythrozyten und Granulozyten). </li><li> In 9 ml sterilem entionisiertem H 2 O und den Timer zu starten. Stoppen Sie den hyper osmotischen Schock nach 30 Sekunden genau, durch Zugabe von 1 ml 10 mal konzentrierter PBS. Hinweis: Die 30 Sekunden sind sehr kritisch. </li><li> In kaltem RPMI-HSA auf ein Gesamtvolumen von 50 ml und Zentrifuge für 10 min bei 249 xg bei 4 ° C. </li><li> Nehmen Sie den Überstand unter Verwendung einer Vakuumpumpe zu sammeln und das Pellet in 1 PMN Rohr mit einem definierten Volumen (1-2 ml) RPMI-HSA. </li><li> Bestimmen Sie die Menge an Zellen und stellen Sie die Konzentration auf 1,10 7 Zellen / ml. Je nach Spender, wird die Ausbeute der isolierten PMN sein betwede 5 x 10 6 und 3 x 10 7 PMN von jedem 9 ml Röhrchen Blut </li></ol> 2. Durchflusszytometrischer Assay für die Bewertung von Calcium Mobilisierung in Human PMNs <ol><li> Wägezellen (5 x 10 6 Zellen / ml) mit 2 uM Fluo-3-AM in RPMI-HSA und Inkubation für 20 min bei Raumtemperatur langsam schaukeln, zum Beispiel unter Verwendung eines Wippschüttler Schüttler, vor Licht geschützt. </li><li> In der Zwischenzeit bereiten eine serielle Verdünnung (z. B. 3-fach) des Reizes bei 10x die Endkonzentration. PSMα3 wird in diesem Protokoll verwendet, aber jede GPCR Reiz, der auf Neutrophilen wirkt eignet. </li><li> Bereiten 10x konzentrierter Inhibitor des Rezeptors in RPMI-HSA. Wenn der Hemmstoff auf den Stimulus (zB HDL auf PSMα3) Pre-Inkubation 25 ul Stimulus wirkt mit einem gleichen Volumen an Inhibitor für 10 min bei Raumtemperatur. </li><li> Waschen der Zellen durch Zugabe von 10 ml RPMI-HSA und Zentrifuge für 249 xg bei Raumtemperatur. Die Zellen bis 5 x10 6 Zellen / ml. </li><li> Kurz vor dem Experiment, verdünnt den Zellen bis 2x10 6 Zellen / ml in RPMI-HSA und 200 ul Zellen pro FACS-Röhrchen. Das verwässerte PMN sind sehr zerbrechlich. Halten sie zu lange bei dieser Konzentration wird die Auto-Aktivierung führen, das gleiche gilt für kräftiges Schütteln oder Pipettieren. </li><li> Befestigen Sie das Rohr mit dem Durchflusszytometer, warten Sie 3 Sekunden und starten Akquisition. Nach einem festgelegten Zeitraum (zB 8 sec), nehmen Sie den Schlauch und schnell hinzufügen 50 ul Stimulus zur Probe. Sofort wieder anzubringen das Rohr auf dem Probenhalter und weiterhin den Erwerb. </li><li> Beginnen Sie mit der niedrigsten und mit der höchsten Konzentration an Reiz. Waschen Sie die Durchflusszytometer Nadel regelmäßig nach jedem Lauf mit RPMI-HSA. </li><li> Analysieren von Daten mit Durchflusszytometrie-Analyse-Software. Vergleichen Sie die durchschnittliche Fluoreszenz-Signal vor der Zugabe des Stimulus an, dass nach Zugabe des Stimulus und verwenden Sie die entsprechenden positiven und negativen Kontrollen an den AC berechnenMotivation Stärke für jede Verdünnung gemessen. </li></ol> Alternativ, wenn der Inhibitor auf dem Rezeptor vor dem Inkubieren der Zellen mit dem Inhibitor wirkt. 3. Fluoreszenzmikroskopie Analyse von Bakterien GFP Expression nach Phagozytose durch PMN <ol><li> Wachsen S. aureus-Stämme, die ein Reporter Konstrukt von Interesse über Nacht in der Brühe (mit Antibiotika, wenn erforderlich, um die Reporter-Plasmid aufrecht zu erhalten). In diesem Fall enthält ein Stamm MW2 PSMα GFP-Reporter-Konstrukt, 10 verwendet wird, gewachsen in einer 50 ml Plastikröhrchen mit 5 ml LB-Kulturmedium. </li><li> Um alle GFP von O / N Ausdruck entfernen, verdünnt den Kulturen zu OD 660 0,01 und wachsen bis zu einer OD 660 0,1. Redilute diese Kultur 1:30 und überwachen Wachstum bis die Kultur erreicht eine OD 660 von 0,1. Sammeln Sie die Bakterien durch Zentrifugation und waschen einmal in DPBS. Resuspendieren im Maßstab 1:10 des ursprünglichen Volumens auf eine OD 660 von 1,0 oder r erhaltengründlich 5.10 8 CFU / ml. </li><li> Mischen Sie die Bakterien (1.10 7 / ml) mit frisch isolierten PMN (1.10 6 / ml) in RPMI-HSA (10:1) in einem 1,5 ml Mikroröhre und fügen gepoolten menschlichen Serum zu einer Endkonzentration von 10%. Schütteln auf einem Schüttler Plattform für 10 min bei 37 ° C bis Phagozytose stimulieren. Verdünnen Sie die PMN mit Bakterien bis 5.10 5 PMN / ml und 250 ul Pipette in eine Vertiefung einer 8 gut Kammern Deckglas geladen. </li><li> Bild der PMN mit Bakterien auf einem Mikroskop. Ein umgekehrtes Mikroskop mit einer 40X/0.85 NA Ziel ausgestattet funktioniert gut und sollte in einer dunklen Umgebung Kammer werden eingehüllt, um die Umwelt stabil halten bei 37 ° C Erwerben Sie Bilder für eine Reihe von Preset-Positionen der Verwendung der Kamera alle 5-10 min sowohl im Hellfeld und dem GFP Kanal GFP-Produktion in der Zeit folgen. </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>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

In der hier beschriebenen Verfahren einige Schritte sind sehr kritisch. Wir werden diese hier hervorzuheben. Für die Isolierung von Neutrophilen durch Dichtegradientenzentrifugation ist es wichtig, die Schichten während oder nach der Zentrifugationsschritte stören. Wenn Ansaugen der Neutrophilen mit einem Kunststoff-Pipette, stellen Sie sicher nicht, um den Ballon zu quetschen, während in der Schicht von Zellen, wie Ausstoßen von Flüssigkeit werden die Schichten zu stören. Auch eine S...

Neutrophile (PMN) sind professionelle Fresszellen, die eine wichtige Rolle spielen bei der angeborenen Immunantwort gegen Staphylococcus aureus 1. Der ständige Kampf zwischen Wirt und Mikrobe hat zu einem Wettrüsten der beiden führte. Kürzlich, community-associated (CA) Stämme von Methicillin-resistenten S. aureus (MRSA) sind entstanden, die zu sein scheinen sehr effizient in Umgehung der neutrophilen Tötung 2,3. Übermäßige Phenol löslich modulin (PSMs) Produktion von CA-MRSA hat mit höherer Virulenz 4,5 in Verbindung gebracht. Humanneutrophilen können diese PSMs über FPR2 die Aktivierung dieses G-Protein-gekoppelten Rezeptor 6 führen zu erkennen. Eines der frühesten Ereignisse ist die Mobilisierung von intrazellulären Speichern von Calcium (Ca 2 +). Ca 2 + wirkt als sekundärer Botenstoff für eine Vielzahl von Effektor-Funktionen von PMNs einschließlich Degranulation und Phagozytose 7. Daher Ca 2 + ist ein sehr empfindlicher Indikator für dieFunktionsfähigkeit PSMs zu PMN aktivieren. Um die Auswirkungen von PSM auf Neutrophilen studieren, werden frische Neutrophilen isoliert und mit einem Farbstoff, der auf Calcium Mobilisierung fluoresziert geladen. In einem Experiment Durchflusszytometrie diese rasche Mobilisierung gemessen werden kann. Mit diesem Verfahren ist es möglich, die direkte Auswirkungen von toxischen und anderen Komponenten auf Neutrophilen zu untersuchen und festzustellen, die niedrigste Konzentration, bei der diese aktiv sind. Für uns ist es ein sehr nützliches Werkzeug, um die Wirkung von vielen Proteinen von S. produziert studieren aureus in Immunevasion wie FPR2 hemmende Protein (FLIPR) 8, FLIPR-like 7 und Chemotaxis hemmende Protein von Staphylococcus aureus (CHIPS) 9 beteiligt. All diese Proteine ​​haben gezeigt, dass das Calcium-Mobilisierung in Neutrophilen durch Bindung an den Rezeptor erkennt den Agonisten zu hemmen. Vor kurzem hat unsere Gruppe beschrieben, dass PSMs funktionell gehemmt durch Serumlipoproteine ​​10 &lt;/ Sup&gt;. Diese Lipoproteine ​​sind reichlich vorhanden im Blut und menschlichem Gewebe, was darauf hinweist, dass ihre Funktion ausüben PSMs vor allem in der intrazellulären Umgebung. Die Verfügbarkeit der Calciummobilisierung Assay erlaubt uns die präzise Messung der Wirkung von Serum-Lipoproteine ​​auf Aktivierung von Neutrophilen durch den PSM durch die erhebliche Hemmung durch sehr geringe Konzentrationen an Serum angegeben. Da die PSMs funktionell gehemmt durch Serum, stellten wir die Hypothese, dass es eine wichtige Funktion für PSMs als intrazelluläre Toxine. Wir haben daher versucht, die Rolle der PSMs nach Phagozytose bestimmen. Um die Expression der PSMs in den interzellulären Raum zu untersuchen, haben wir Reporter Fusionen von dem Promotor des Operons psmα GFP konstruiert. Wenn diese Reporter Stämme von S. aureus durch Neutrophile wurden phagozytiert wurde die Induktion der Expression mittels Fluoreszenzmikroskopie beobachtet 10. Offensichtlich kann diese technique ermöglicht die Untersuchung der Expression einer Vielzahl von Genen in S. aureus oder anderen Erregern nach Phagozytose. Da für S. aureus Überleben in der interzellulären Nische ist sehr wichtig, um das angeborene Immunsystem 11 10 12 zu überwinden, die Untersuchung der Rolle von Genen in dieser Nische aktiviert ist von großer Bedeutung für das Verständnis ihrer Virulenz.

<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

Wir präsentieren Methoden, um die Wirkung von Phenol löslich modulins (PSM) und andere Toxine und Staphylococcus aureus auf Neutrophilen sezerniert studieren. Um die Auswirkungen der PSM auf Neutrophilen wir isolieren frisch Neutrophilen mittels Dichtegradientenzentrifugation studieren. Diese Neutrophilen werden mit einem Farbstoff, der auf Calcium-Mobilisierung fluoresziert geladen. Die Aktivierung von Neutrophilen durch PSMs initiiert eine rasche und vorübergehende Erhöhung der freien intrazellulären Calcium-Konzentration. In einem Experiment Durchflusszytometrie diese rasche Mobilisation kann durch Überwachung der Fluoreszenz eines vorinstallierten Farbstoff, der auf die erhöhte Konzentration an freiem Ca 2 + reagiert gemessen werden. Mit dieser Methode können wir bestimmen, die PSM-Konzentration notwendig, um die Neutrophilen zu aktivieren, und messen die Auswirkungen von spezifischen und allgemeinen Inhibitoren der Aktivierung von Neutrophilen. Um die Expression der PSMs in den intrazellulären Raum, w untersuchene konstruiert haben Reporter Fusionen der Promotor des PSMα Operon zu GFP. Wenn diese Reporter Stämme von S. aureus durch Neutrophile phagozytiert werden, kann die Induktion der Expression mittels Fluoreszenzmikroskopie beobachtet werden.

Durchflusszytometrische Assay zur Beurteilung der Mobilisierung von Kalzium im menschlichen PMNs Inkubieren Neutrophilen mit einer Konzentration Reihe synthetischer PSMα3 wodurch eine schnelle Aktivierung durch Calcium-Fluss, der durch eine Erhöhung des Signals im FL-1 gezeigt, wird gemessen. Pre-Inkubation von synthetischen PSMα3 mit 0,01%, 0,1% oder 1% humanem Serum erheblich behindert die Fähigkeit, Kalzium Flüsse (Abbildung 1) zu entlocken. An...

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

Wir präsentieren Methoden, um die Wirkung von PSM und andere Giftstoffe ausgeschieden durch studieren<em&gt; Staphylococcus aureus</em&gt; Auf Neutrophilen mittels Durchflusszytometrie und Fluoreszenzmikroskopie.

Studieren Wechselwirkungen von<em&gt; Staphylococcus aureus</em&gt; Mit Neutrophilen durch Durchflusszytometrie und Mikroskopie Zeitraffer

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 Aufrufe.  University Medical Center Utrecht. Wir präsentieren Methoden, um die Wirkung von PSM und andere Giftstoffe ausgeschieden durch studieren Staphylococcus aureus Auf Neutrophilen mittels Durchflusszytometrie und Fluoreszenzmikroskopie.

Serie: 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.

Die Verwendung von Drip Flow und Rotating Disk-Reaktoren für 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 ...

Forschung

Immunologie und Infektion

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.

Subkutane Infektion von Methicillin-resistenten 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 ...

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.

Staphylococcus aureus Wachstum mit menschlichen Hämoglobins als Eisenquelle

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

Eine einfache und effiziente Methode, um Nuclear Factor Aktivierung in menschlichen Neutrophilen Erkennen von Durchflusszytometrie

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.

Detektion von fluoreszierenden Nanopartikel Wechselwirkungen mit primären Immun-Zell-Subpopulationen durch Durchflusszytometrie

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.

Abbilden Neutrophilen und Monozyten in Mesenterialvenen von Intravitalmikroskopie an narkotisierten Ratten in Echtzeit

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.

Genotypisierung<em&gt; Staphylococcus aureus</em&gt; Durch ribosomalen 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.

Methodik für die Studie des horizontalen Gentransfers in<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 3D-Bildgebung der Phagozytose durch Makrophagen der Maus

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.

Ein Mausmodell zu beurteilen, angeborene Immunantwort auf Staphylococcus Aureus -Infektion

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