Die Autoren bedanken sich bei Richard Ruhe für die großzügige Schenkung von anthrolysin O, Michael Caparon für das großzügige Geschenk der SLO Plasmid und Jonathon Franks für die technische Unterstützung bedanken. Diese Arbeit wurde vom NIH Zuschüsse T32CA82084 (PAK) und R01AI072083 (RDS) finanziert.

Ein. Reinigung von Streptolysin O (SLO) <ol><li> Beimpft 20 ml Übernacht-Kultur von BL21 GOLD Zellen mit pBADgIII-SLOhis Plasmid 9 in 0,5 L LB-Brühe und 500 ul 50 mg / ml Ampicillin. Schüttelkultur bei 225 UpM bei 37 ° C bis OD600 = 0,6, in der Regel ~ 1,5 Stunden. Zentrifuge 1 ml Bakterien (als T 0) bei 10.000 xg 5 min auflöst Pellet in 140 ul 1x SDS Probenpuffer / 1 OD und beschallen zu scheren DNA auf Proteinreinheit Analyse. </li><li> Bakterien induzieren unter Zusatz von 5 ml 20% Arabinose zu Kultur, bei 225 rpm bei Raumtemperatur für 3 Stunden geschüttelt. Sammeln Sie 1 ml der Bakterien, zentrifugieren und lösen in 1x SDS Probenpuffer wie oben für T 3 Zeitpunkt für Reinheit Analyse. </li><li> Sammle verbleibenden Bakterien in 500 ml Zentrifugenflasche, drehen 12.000 xg für 12 min bei 4 ° C (8.500 rpm Sorvall GS-3-Rotor). Den Überstand umfüllen, speichern Pellet bei -80 ° C über Nacht oder länger, falls erforderlich. </li><li> Resuspendieren frozen Pellet auf Eis in 10 ml Lyse / Waschpuffer (50 mM NaH 2 PO 4, 300 mM NaCl, 10 mM Imidazol, pH 8,0) mit 400 ul 25% Triton X-100, 100 ul 100 mg / ml Lysozym, 50 ergänzt ul 200 mM Phenylmethylsulfonylfluorid (PMSF). Übertragen des resuspendierten Pellets in 50 ml Oak Ridge Röhre. </li><li> Beschallen Lysat 5 mal für 30 sec auf Eis mit 30 Sekunden-Intervallen. Spin beschallt Lysat in Oak Ridge Röhrchen bei 39.000 xg für 20 min bei 4 ° C (18.000 rpm Sorvall SS-34-Rotor). </li><li> Während das Lysat dreht, waschen 1 ml Ni-NTA-Agarose mit 10 ml Lyse / Waschpuffer und Spin bei 1.200 UpM für 5 min bei 4 ° C. (Alle weiteren Wäschen / Elutionen verwenden Zentrifugation unter diesen Bedingungen). Absaugen zu waschen. </li><li> Fügen bakterielle Überstand aus Schritt 1,5 bis Ni-NTA Agarose, schütteln für 2,5 h bei 4 ° C. Spin zu sammeln Überstand. Kombinieren Sie 30 ul Überstand mit 10 μ 4x SDS-Probenpuffer und sparen für Reinheit Analyse. Waschen Sie die Kugeln 4 Mal in Lyse / Waschpuffer undce in Tris / Salz-Puffer (50 mM Tris, 300 mM NaCl, pH 8,0). </li><li> Eluieren 3 mal durch Zugabe von 1 ml Elutionspuffer (250 mM Imidazol in Tris / Salzpuffer) und 5 ul 1 M Dithiothreit (DTT), um die Kügelchen, Inkubieren auf Eis für 10 min, dreht und Sammeln des Überstands. SLO ist ein sehr Redox-sensitive Protein, und muss auf Eis zu allen Zeiten gehalten werden. Einmal verringert wurde, wird das Protein rasch verlieren, wenn seine Aktivität auf 37 ° C erwärmt, auch nur für kurze Zeit. </li><li> Waschen 500 ul Polymixin-konjugierte Agarose in Elutionspuffer, Spin bei 10.000 xg für 1 min bei 4 ° C und resuspendieren in 500 ul. Endotoxin führen, mit 200 ul Perlen jeder Elution und schütteln 4 ° C für 30 min. Spin bei 10.000 xg für 1 min bei 4 ° C, und speichern Sie den Überstand. Kombinieren Sie 30 ul jeder Elution mit 10 ul 4x SDS-Probenpuffer für die SDS-PAGE-Analyse. </li><li> Testen der Reinheit Elutionen durch SDS-PAGE. Kochen Sie die Proben für die Gel-Analyse bei 95 ° C gesichert für 5 min und Last 10 ul auf einem 10% Gel. Stain gel mit Coomassie zu visualisieren Proteins (Abbildung 2A). SLO ist 69 kDa. Durchführen einer Bradford Assay zur Proteinkonzentration zu bestimmen (typische Ausbeute beträgt 4 mg / ml für die ersten zwei Elutionen, sondern kann durch Bakterienstamm und Plasmid verwendet variieren). </li><li> Testen der hämolytischen Aktivität jedes Elution (siehe unten). Pool Elutionen wo Reinheit, Konzentration und hämolytische Aktivität zufriedenstellend sind. Aliquot SLO in Einweg-Aliquots (typischerweise 5-10 ul) auf Trockeneis und bei -80 ° C einfrieren </li></ol> 2. Hämolytische Assay <ol><li> Waschen Schaf rote Blutkörperchen (Erythrozyten) in RBC Assay-Puffer (0,3% Rinderserumalbumin (BSA), 2 mM CaCl 2, 10 mM Hepes, pH 7,4), Spin bei 1.200 UpM für 5 min und resuspendiert in 2,5% in RBCs 10 ml RBC Assay-Puffer. </li><li> Fügen Sie 10 ul RBC-Assay-Puffer / Well in 96-Well-V-Bodenplatte auf Eis. Serienmäßig verdünnen jeder Elution in zweifacher Ausfertigung. Typische Bereiche liegen Verdünnung 1:1.000 bis 1:512,000. Inklusive 3 Brunnen ohne Toxin und 3 Brunnen mit 10ul 2% Triton X-100 als minimal und maximal Vertiefungen sind. </li><li> Abdeckplatte und bei 37 ° C 30 min. Zentrifuge bei 1.200 rpm für 5 min. Übertragen 70 ul Überstand in einem flachen Boden 96 Well-Platte. Lesen A 405. Eine Einheit ist die Verdünnung des Toxins für 50% Lyse der Erythrozyten benötigt. Toxinaktivität ist 1 Einheit * Verdünnung factor/0.01 ml. </li></ol> 3. Zelle Lytic Assay <ol><li> Ernten Sie die Zellen, count, Spin. Resuspendieren bei 2x10 6 / ml in Puffer R / B (RPMI mit 2 mM CaCl 2, 0,5% BSA) und 20 ug / ml Propidiumiodid. Je 100 ul Zellen auf eine 96-Well-V-Bodenplatte. </li><li> Serienmäßig verdünnen SLO bis zur endgültigen Konzentration im Puffer R / B 2x, mit 100 ul Toxin oder 100 ul Puffer R / B-Zellen. Inkubieren 5 min 37 ° C. Typische Endkonzentrationen Bereich von 2000 U / ml bis 31,25 U / ml. </li><li> Führen Zellen auf Durchflusszytometer und sammeln Daten mit Filtern für Phycoerythrin (PE). A one-log Verschiebung stellt transient permeabilisiert cells während eine 3 log Verschiebung zeigt toten Zellen 4. Berechnen Sie die spezifische Lyse der Zellen durch Subtrahieren der Prozentsatz an toten Zellen in der Kontrolle (% PI hohen ctl) von der experimentellen (% PI hohen exp), wie folgt: spezifische Lyse = (% PI hohen exp -% PI hohen ctl) / (100 -% PI hohen ctl) * 100 </li></ol> 4. Mikropipette Lieferung von Toxin zu Zellen in Kulturschalen <ol><li> Einen Tag vor der Experiment Platte 2x10 5 Makrophagen auf einem mit Kollagen beschichteten Glas-Boden 35 mm-Schale. </li><li> Schalten Mikroskop und Mikroinjektor. Erlauben Heiztisch Zeit auf 37 ° C erwärmen Die Wahl des Mikroskops in der Regel davon abhängt, was lokal verfügbar ist. Das Mikroskop braucht eine umgekehrte Stufe, eine Stufe der Lage Erhitzen der Speisen bis 37 ° C, Anregungs / Emissions Filterwürfel geeigneter für den Farbstoff gewählt, und der Raum den Anschluss der Mikroinjektorkopfs. A Bertrandlinseist hilfreich für die Mikroinjektion, aber nicht erforderlich. Der Computer Ansteuerung des Mikroskops muss ausreichend Speicher zu sammeln und zu speichern. </li><li> Etikett Zellen für 30 min mit Farbstoff bei 37 ° C. Je nach dem Assay kann Markierung mit 5 ul Fura2 Uhr in 1 ml PBS oder 2 ul Calcein AM in 1 ml vollständige Medien erfolgen. Für Fura2 wird Anregung / Emission für 340/510 nm und 380/510 nm gesammelt und das Verhältnis von 340/380 bestimmt, Signale Calciumflusses. Für Calcein ist Anregung / Emission 495/515 nm, während Ethidiumhomodimer ist 525/620 nm und APC ist 650/660 nm. Andere Etiketten können auch gewählt werden. </li><li> Waschen der Zellen mit PBS gewaschen und in 1 ml RPMI ergänzt mit 2 mM CaCl 2. Montieren Sie am Mikroskop. </li><li> Verdünnte Toxin und Dextran in Wasser und Zentrifuge bei 20.000 × g für 10 min 4 ° C Typischerweise wird 1 ul SLO und 4 ul 10 mg / ml Dextran-555 in 6 ul Wasser verdünnt. Dextran oder andere fluoreszierende Flüssigkeit-Phasen-Molekül wird verwendet, um sicherzustellen, dass die Femto-Spitze nicht verstopft ist, und injects wie gewünscht. </li><li> Legen Femto-Tipp von hinten mit 0,07 ul der verdünnten Toxin mit einem Microloader. </li><li> Legen Femto-Spitze auf Mikroinjektor. Stellen Sie den Winkel der Injektor so, dass die Spitze sitzen über der Mitte der Zellen mit Raum in alle Richtungen bewegen. Löschen z-Grenze. Injection-Einstellungen sollten für 0,5 sec werden Injizieren bei 120 psi mit 20 psi Gegendruck. Untere Spitze, bis sie das Medium eintritt. </li><li> Verwenden des Bertrand-Linse, in der Mitte die Spitze und folgen der Spitze, wie es näher an den Zellen erniedrigt wird. Sobald Sie den Fokus verlieren, wechseln wieder zur normalen Optiken. Die Nadel Schatten sollte auf dem Gebiet offensichtlich. Konzentrieren Sie sich über den Zellen und Senken der Nadel bis es scharf ist. Der Fokus wieder auf die Zellen und sorgfältig bringen die Nadel benachbart zu einer Zelle. Stellen Sie die z-Limit für die Injektion. Commence Bildgebung, bewegen Spitze in die gewünschte Position, zu injizieren, um bei der gewünschten Toxin Zeitpunkt freizugeben. Heben Sie die Nadel, um eine neue Region von Zellen bewegen und zu injizieren. Nadel zur Ausgangsposition, um unerwünschte zu verhindernToxin Leckage von der Nadel. </li></ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

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Die hier beschriebenen Techniken ermöglichen die Untersuchung der Reaktionen von Immunzellen, um bakterielle Toxine. Der wichtigste Schritt ist die Handhabung und Dosierung des Toxins. Toxinaktivität kann äußerst variabel, sogar zwischen verschiedenen Aliquots derselben Zubereitung, aufgrund ihrer Zerbrechlichkeit. Dies erfordert entweder eine Prüfung jedes einzelnen Aliquot des Toxins gegen eine Referenz Zelllinie oder RBCs oder mit Toxin Steigungen. Toxin Gradienten, wie Mikropipette geliefert, damit das gesamte ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Ni-NTA Agarose </td><td> Qiagen </td><td> 30210 </td><td></td></tr><tr><td> Polymixin-Agarose </td><td> Sigma </td><td> P1411-5ML </td><td></td></tr><tr><td> Zeba Desalt Spin col </td><td> Fischer </td><td> PI-89891 </td><td></td></tr><tr><td> Schafe RBCs </td><td> Fischer </td><td> 50-415-688 </td><td></td></tr><tr><td> pBADgIII-SLO </td><td> N / A </td><td> N / A </td><td> siehe Lit. 9 </td></tr><tr><td> Cy5 monoreaktiven Farbstoff </td><td> GE Healthcare </td><td> PA25001 </td><td></td></tr><tr><td> Fura2-AM </td><td> Life Technologies </td><td> F1221 </td><td></td></tr><tr><td> Calcein AM / Ethidium Homodimer </td><td> Life Technologies </td><td> L3224 </td><td></td></tr><tr><td> Anti-CD11c-APC </td><td> BD Biosciences </td><td> 550261 </td><td></td></tr><tr><td> Kollagen-beschichteten Glas-bottom Gericht </td><td> Mattek </td><td> P35GCol-1.5-10-C </td><td></td></tr><tr><td> Femto-Tipp II </td><td> Fischer </td><td> E5242957000 </td><td></td></tr><tr><td> Microloader </td><td> Fischer </td><td> E5242956003 </td><td></td></tr><tr><td> Dextran Alexa 555 </td><td> Life Technologies </td><td> D34679 </td><td></td></tr><tr><td> InjectMan NI 2 </td><td> Eppendorf </td><td> 920000029 </td><td></td></tr><tr><td> FemtoJet </td><td> Eppendorf </td><td> 5247 000.013 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabelle 1. List und Quelle der spezifischen Reagenzien und Plaent benötigt. Besondere Geräte und Reagenzien in diesem Protokoll verwendet, zusammen mit Unternehmen und Katalog-Nummer aufgelistet. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Puffer </td><td> Zusammensetzung </td><td> Schritt </td></tr><tr><td> Lyse / Wash </td><td> 50 mM NaH 2 PO 4 300 mM NaCl 10 mM Imidazol, pH 8,0 </td><td> 1,4 </td></tr><tr><td> Tris / Salz </td><td> 50 mM Tris, pH 8,0 300 mM NaCl </td><td> 1,7 </td></tr><tr><td> Elutionspuffer </td><td> 50 mM Tris, pH 8,0 300 mM NaCl 250 mM Imidazol </td><td> 1,8 </td></tr><tr><td> RBC Assay-Puffer </td><td> 0,3% BSA 2 mM CaCl 2 10 mM HEPES, pH 7,4 </td><td> 2,1 </td></tr><tr><td> Puffer R / B </td><td> Zellkulturmedium RPMI 2 mM CaCl 2 0,5% BSA </td><td&gt; 3,1 </td></tr></tbody></table></td></tr><tr><td></td><td></td><td></td><td> Tabelle 2. Liste von Puffern in diesem Protokoll verwendet. Die verwendeten Puffer sind die Zusammensetzung und der erste Schritt bei der sie in dem Protokoll verwendet werden aufgelistet. </td></tr></tbody></table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Bakterielle Toxine binden an Cholesterin in Membranen, Bildung von Poren, die auf Undichtigkeiten des Zellinhalts und Einströmen von Material von der äußeren Umgebung ermöglichen. Die Zelle kann entweder ins Insult, die Wirkmembran Reparaturprozesse erfordert wiederherzustellen, oder sterben abhängig von der Menge des Toxins Belichtung und ein Zelltyp. Darüber hinaus führen diese Toxine starke entzündliche Reaktionen in infizierten Wirten durch Aktivierung von Immunzellen, einschließlich Makrophagen, die ein Array von pro-inflammatorischen Zytokinen 2 zu erzeugen. Viele Gram-positive Bakterien produzieren Cholesterin bindenden Toxine, die gezeigt haben, um ihre Virulenz durch weitgehend charakterisierten Mechanismen beitragen. Morphologischen Veränderungen in der Plasmamembran von Zellen, die diese Toxine umfassen ihre Sequestrierung in Cholesterin angereicherten Oberflächenprotrusionen, die in den extrazellulären Raum vergossen werden kann, was auf eine intrinsische zelluläre AbwehrMechanismus 3,4. Dieser Vorgang geschieht für alle Zellen in Abwesenheit einer metabolischen Aktivität und visualisiert werden kann, nachdem mit EM chemischen Fixierung 4. In Immunzellen wie Makrophagen, die Entzündung als Reaktion auf Exposition Toxin vermitteln, induziert werden Membranvesikel vorgeschlagen, Zytokine der IL-1-Familie enthalten und kann sowohl für vergießen Toxin und Weiterleitung dieser proinflammatorischen Zytokine 5,6,7. Eine Verbindung zwischen IL-1β Freisetzung und einer bestimmten Art von Zelltod genannt hat pyroptosis vorgeschlagen worden, da beide Caspase-1-abhängiger Prozesse sind 8. So sortieren Sie die Komplexität dieser Makrophagen Antwort, die Toxinbindung, vergießen Membranvesikel Zytokinfreisetzung und potenziell Zelltod enthält, haben wir die Kennzeichnung Techniken und Fluoreszenzmikroskopie Methoden, die für Echtzeit-Visualisierung von Toxin-Zell-Interaktionen ermöglichen, einschließlich entwickelt Messungen der Dysfunktion und Tod (Abbildung 1). Verwendendes Live Cell Imaging ist notwendig wegen der Beschränkungen in anderen Techniken. Biochemische Ansätze nicht lösen können auftretenden Effekte in einzelnen Zellen, während Durchflusszytometrie nicht bieten hohe Auflösung, Echtzeit-Visualisierung von einzelnen Zellen. Die hier beschriebenen Methoden können zur kinetischen Analyse der Antworten durch andere Reize mit komplexen phänotypischen Veränderungen in Zellen induziert aufgebracht werden.

Typischerweise 10 7 -10 8 U / ml SLO kann mit einer Proteinkonzentration von 4 mg / ml erzielt werden. Die Menge des Toxins zur Zelllyse benötigt je nach Zelltyp, aber üblicherweise 125 bis 500 U / ml SLO (2B). Zelltypen wie Makrophagen widerstandsfähiger (4000 U / ml), obwohl andere (vor allem T-Zelllinien) empfindlicher sind. Diese Sensitivitäten mit handelsüblichen SLO entsprechen. Toxinaktivität abnimmt etwa 2-fach mit jedem Frost-Tau, so hämolytischen Assays mit jeder C...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Verfahren zur Reinigung des Cholesterins Bindung Toxin Streptolysin O aus rekombinanten<em&gt; E. coli</em&gt; Und Visualisierung von Toxin Bindung an eukaryotischen Zellen leben beschrieben. Lokalisierte Abgabe von Toxin induziert eine schnelle und komplexe Veränderungen in Zielzellen enthüllen neue Aspekte des Toxins Biologie.

Visualisierung der bakteriellen Toxin induzierten Reaktionen mit Live Cell Fluoreszenzmikroskopie

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

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JoVE Journal

Immunology and Infection

11.7K Aufrufe.  University of Pittsburgh School of Medicine. Verfahren zur Reinigung des Cholesterins Bindung Toxin Streptolysin O aus rekombinanten E. coli Und Visualisierung von Toxin Bindung an eukaryotischen Zellen leben beschrieben. Lokalisierte Abgabe von Toxin induziert eine schnelle und komplexe Veränderungen in Zielzellen enthüllen neue Aspekte des Toxins Biologie.

Serie: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

Visualizing Zell-Zell-Transfer von HIV unter Verwendung von fluoreszierenden Klone von HIV und Live-konfokale Mikroskopie

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

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Immunologie und Infektion

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Die Beurteilung stomatäre Response to Bakterienzellen Live mit Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

Live Cell Imaging von Bacillus subtilis Und Streptococcus pneumoniae Verwendung von Automated Zeitraffer-Mikroskopie

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Live-cell Video Mikroskopie Pilzpathogenentwicklung Phagozytose

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Visualisierung von Protein-DNA-Wechselwirkungen in der lebenden Bakterienzellen Verwendung Photoactivated Einzelmolekül-Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Mess physiologischen Reaktionen von<em&gt; Drosophila</emNeurone&gt; Sensory zu Lipid Pheromone Verwendung von Live-Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantifizierung der Efferocytosis durch einzellige Fluoreszenz-Mikroskopie

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.