Diese Arbeit wurde von der UCSF Abteilung Anästhesie und perioperative Pflege unterstützt.

1. Plating und Wartung von mikrovaskulären Endothelzellen <ol><li> Auftauen und das Wachstum Ihres mikrovaskulären Endothelzellen nach den Herstellern mitgelieferten Anleitung. In diesem Protokoll wurden die Zellen in EGM-2 MV Medien (Lonza) gewachsen, und es wird empfohlen, dass alle Versuche innerhalb von zwei Wochen nach dem Auftauen eine Variation aufgrund Durchgang Nummer minimieren durchgeführt werden. Unsere Experimente mit HMVEC-Lung zwischen Passagen 4 bis 9 durchgeführt. </li><li> Tag 1: Wenn die Zellen 80-90% Konfluenz erreichen, trypsinize und resuspendieren bei 120.000 Zellen pro ml. Teller 36.000 Zellen (0,3 ml) pro Well in 48-Well-, Polystyrol Gewebekulturplatte (n). Fügen Vertiefungen HMVEC die nicht mit Neutrophilen inkubiert werden, um eine Ablesung zu erhalten Hintergrundfluoreszenz. Es sei denn, bei der 48-Well-Platten speziell für Fluoreszenz-Assays, alternierenden Reihen oder Spalten entworfen wird minimieren Licht Kontamination von benachbarten Brunnen. HINWEIS: Wells kann vorher mit Poly-Lysine, Kollagen oder Fibronektin. </li><li> Tag 2: In frischem Medium. </li><li> 3. Tag: Nach Phasenkontrast-Mikroskopie, bestätigen die Zellen gesund sind (dh &quot;Kopfsteinpflaster&quot; Aussehen und wenig Zelltrümmer), und sie sind zu 100% konfluent (Abbildung 1). Wenn die Zellen nicht zu 100% konfluent, verändern die Medien jeden Tag, bis sie bereit sind. </li><li> Sobald die HMVEC Monoschichten bereit für die Behandlung sind, waschen Sie die Zellen einmal mit ausgewogener Hank-Salzlösung (HBSS) und fügen Sie 0,3 ml frisches EGM-2 MV Medien oder Medien, die entzündliche Agonisten in die entsprechenden Vertiefungen. In diesem Protokoll die HMVEC-Lung wurden mit TNF behandelt [100 ng / ml] (PeproTech, New Jersey) für 3 Stunden, da dies eine gut beschriebene Aktivator des ECs 21. HINWEIS: Es wird empfohlen, dass Sie Zeit Ihres Experiments, so dass Ihre aktivierten HMVEC Zellen (Schritt 4.1) und Calcein markierten Neutrophilen (Schritt 3.5) Uhr bereit sind, in der gleichen Zeit zu verwenden. Es führt uns ca. 2,5 Stunden zu isolieren und zu beschriften neutrophils. </li></ol> 2. Neutrophile Isolation mit Polymorphprep <ol><li> Isolieren Neutrophilen, wie zuvor beschrieben 22, oder mit einem ready-made Dichtegradientenlösung zu polymorphkernigen Granulozyten aus Vollblut zu isolieren. Der Einsatz Polymorphprep nach dem Protokoll von Nuzzi ea 2007 Ergebnisse in Ausbeuten von ca. 2-3 x 10 6 Neutrophile pro ml Vollblut 23. Hinweis: Um eine übermäßige Erythrozyten-Lyse, ein Dextran Sedimentation zu vermeiden, um rote Blutkörperchen zu entfernen, kann vor Dichtegradientenzentrifugation 24 durchgeführt werden. </li><li> Bringen Sie die Polymorphprep Dichtegradientenlösung auf RT. </li><li> Sammeln 30 ml Vollblut eines gesunden menschlichen Freiwilligen in Gegenwart eines Antikoagulans wie Heparin, Citrat oder EDTA. Das Blut sollte innerhalb von 2 Stunden verwendet werden, und hielt zwischen 18-22 ° C. </li><li> Schicht 5 ml Vollblut in 5 ml der Dichtegradient-Lösung in einem 15 ml konischen Röhrchen (Abbildung 2A). Vermeiden Änderung Volumina von Blut und die Dichtegradientenlösung da dies die anschließende Zentrifugation Bedingungen ändern kann. </li><li> Zentrifugation bei 450 × g für 30 min bei 18-22 ° C. Vergewissern Sie sich, langsam zu beschleunigen und auf der Zentrifuge (dh schalten Sie die Zentrifuge Bremse), und das Gleichgewicht der Rohre gut um Vibrationen zu vermeiden zu helfen, reduzieren die Aktivierung der Neutrophilen und verhindern, dass die Vermischung der Schichten. Längere Zentrifugationszeiten oder höher Zentrifugalkraft wird in der unteren Schicht Leukozyten, die neutrophilen Granulozyten enthält führen, um weiter nach unten wandern in Richtung zu den pelletierten roten Blutkörperchen. </li><li> Absaugen des Plasmas und oberen Leukozyten enthaltende Bande PBMCs die Kontamination der Schicht, die Neutrophilen (2B) zu verhindern. </li><li> Verwenden Sie einen 1-2 ml serologische Pipette, um die untere Schicht, die Neutrophile Leukozyten zu entfernen. Kombinieren der Neutrophilen-Schichten in 30 ml PBS (ohne Ca 2 + und Mg 2 +) in einem 50 mlkonischen Rohr. </li><li> Bringen Sie die Lautstärke von bis zu 50 ml mit PBS (ohne Ca 2 + und Mg 2 +) und Zentrifuge bei 450 g für 10 min bei 18-22 ° C. </li></ol> Hinweis: Die Schritte 2.9 und 2.10 sind optional, aber sehr zu empfehlen. <ol start="9"><li> Saugen Sie den Überstand. Resuspendieren der Neutrophilen in 8 ml sterilem Wasser für 30 Sekunden, um rote Blutkörperchen zu lysieren, sofort fügen Sie die Zellsuspension in 2 ml 5x PBS (ohne Ca 2 + und Mg 2 +) und vorsichtig von Hand mischen. NICHT inkubieren Zellen in Wasser für mehr als 30 Sekunden da erhebliche Neutrophilen Tod auftreten können. </li><li> Zentrifugation bei 250 × g für 5 min bei 18-22 ° C. </li><li> Die Zellen werden einmal mit 10 ml PBS (ohne Ca 2 + und Mg 2 +) und erneut zentrifugiert bei 250 × g für 5 min bei 18-22 ° C. </li><li> Resuspendieren der Neutrophilen in 10 ml RPMI-1640 (ohne Phenolrot) und zählen die lebenden Zellen unter Verwendung des TrypanblauFärbeausschlussverfahren Verfahren. Vermeiden längere Zelldichten größer als 5 x 10 6 pro ml, da dies zu einer Aktivierung der Neutrophilen führen kann. </li></ol> 3. Neutrophile Labeling mit Calcein AM <ol><li> 6 x 10 5 Neutrophilen pro Vertiefung einer 48-Well-Platte verwendet. </li><li> Nach dem Zählen, übertragen Sie die resuspendierten Zellen in eine 50 ml konischen Röhrchen und verdünnen die Neutrophilen auf 2-4 x 10 6 Zellen pro ml mit Phenolrot-freiem RPMI-1640. </li><li> In Calcein AM Stammlösung bis 3 um (dh 2,98 ul Calcein AM stock (1 mg / ml) pro ml Medium) und gut mischen durch leichtes schnippen die Röhre. Mehr als 5 um Calcein AM wird in Leck aus den Neutrophilen führen. Hinweis: Non-fluoreszierendem Calcein AM wird innerhalb der Zellen durch endogene Esterasen gespalten, um das stark fluoreszierende Molekül Calcein (dh tote Zellen nicht fluoreszieren) zu produzieren. </li><li> Decken Sie die Röhrchen mit Alufolie und Inkubation for 30 min bei 37 ° C im Dunkeln. Hinweis: Längere Inkubationszeiten in mehr Calcein führen freigesetzt aus den Neutrophilen im Laufe der Adhäsionsassay. </li><li> Zentrifugation bei 450 × g für 5 min bei 18-22 ° C und wäscht die Neutrophilen 2x mit 10 ml RPMI-1640 (ohne Phenolrot, vorgewärmt auf 37 ° C). Nach Resuspendieren der Zellen für die zweite waschen, zuerst über die Neutrophilen obwohl eine 40 uM Sterilfilter Klumpen zu entfernen und dann zentrifugiert erneut bei 450 xg für 5 min bei 18-22 ° C. </li><li> Resuspendieren der Neutrophilen in RPMI 1640 (ohne Phenolrot, vorgewärmt auf 37 ° C) mit 2 x 10 6 pro ml Neutrophilen. </li></ol> 4. Neutrophile Binding Assay <ol><li> Waschen Sie die HMVEC Monoschichten 3x mit 0,5 ml steril filtriert (0,22 um) RPMI 1640 (ohne Phenolrot) mit 3% BSA pro Well. </li><li> Saugen Sie die Medien, und fügen Sie 6 x 10 5 Calcein-markierten Neutrophilen (0,3 ml Zellsuspension) Oder 0,3 ml RPMI 1640 (ohne Phenolrot) ohne Neutrophilen, in die entsprechenden Vertiefungen der 48-Well-Platte. Hinweis: Dies ist das Äquivalent von 8 x 10 5 Neutrophilen pro cm 2. </li><li> Inkubation für 20 min bei 37 ° C, 5% CO 2-Inkubator. </li><li> Insgesamt Neutrophile Fluoreszenz (Vorwäsche): Messen Sie die Fluoreszenzintensität jeder Vertiefung mit einer Anregungswellenlänge von 485 nm und einer Emissionswellenlänge von 520 nm (Fluoresceinfilter set) in einem FLUOstar, oder gleichwertig, Spektralphotometer. Diesen Schritt unmittelbar vor der Inkubation Endpunkt Anmerkung: In diesem Protokoll Calcein Anregung und Detektion des emittierten Lichts wurden von der Oberseite der nicht abgedeckten Vertiefungen durchgeführt, aber beide können auch aus dem Boden der Vertiefungen vorgenommen werden.. </li><li> Waschen der Vertiefungen mit HMVEC Monoschichten und Neutrophilen 5x mit 0,5 ml pro Vertiefung von PBS (w / Ca 2 + und Mg 2 +). Entfernen Sie die Medien und Waschungendurch Umdrehen der Platte, und klopfte sanft auf Küchenpapier entfernen Sie die restliche Flüssigkeit. </li><li> In wieder 0,3 ml RPMI 1640 (ohne Phenolrot) pro Vertiefung. </li><li> Adherent Neutrophile Fluoreszenz (POST wash): Messen Sie die Fluoreszenzintensität jedes gut wie in Schritt 4.4. </li><li> Bestimmen Sie die Einhaltung Prozent und relative Einhaltung pro Well: </li></ol> Hinweis: In diesem Stadium, Bilder der Calcein-markierten Neutrophilen kann unter Verwendung eines Fluoreszenz-Mikroskop mit Standard-fähig Fluorescein Filtern ausgestattet, erhalten werden. Die Bilder in Abbildung 4 wurden an einem Olympus IX51 inversen Mikroskop mit einer Kamera Retiga 2000R (Q Imaging) mit Q-Capture-Software Pro7 (Q Imaging) ausgestattet erhalten.

<ol>
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U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., 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+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, 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+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, G., 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=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, 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=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, 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=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., 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=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

Autoren haben nichts zu offenbaren.

Die wichtigsten Schritte für eine erfolgreiche Neutrophilen / Mikrogefäßendothelzellen Zelladhäsionsassay sind: 1) der niedrigen Passage-Nummer (&lt;9), gesunde Endothelzellen Verwenden; 2) Pflege der isolierten Neutrophilen bei einer geringen Dichte (dh &lt;5 x 10 6 Zellen / ml) und mit ihnen innerhalb von zwei Stunden der Isolation, und 3) Fastidious Waschen der Calcein AM-markierten Neutrophilen und die Nutzung der Calcein AM-markierten Neutrophilen rechtzeitig EG Verschmutzung und Verlust von...

Da das vaskuläre Endothel ist in direktem Kontakt mit dem zirkulierenden Blut wird in ausgezeichneter Lage, eine schnelle Entzündungsreaktion während der Infektion oder Verletzung zu initiieren. Endothelzellen (ECs) express pattern recognition receptors, die eine Vielzahl von konservierten bakteriellen Komponenten und Gefahr Moleküle erkennen und Rezeptoren für Host Entzündungsmediatoren wie TNFa. Die Aktivierung dieser Rezeptoren führt ECs, Zytokine (z. B. IL-6, IL-8, CXCL1 und CCL2) sezernieren, und Adhäsionsmoleküle (zB E / P-Selectin, VCAM-1 und ICAM-1) in ihrer Zelloberfläche 1 hochreguliert , 2. Diese Moleküle alle erleichtern die Lokalisierung von Leukozyten an Stellen der Infektion und Verletzungen, um das Heer der Infektionserreger zu löschen und die Reparatur von Gewebe 3,4 initiieren. Neutrophilen Die Reaktion auf eine Infektion beinhaltet eine gut koordinierte Zusammenspiel zwischen dem vaskulären Endothel und dem antwortenden Neutrophilen. Nach Aktivierung EG, IL-8 abgesondert und bildet einen intravaskulären Gradienten auf das Endothel, die Neutrophilen erlaubt, nach Hause, um den Ort der Infektion oder Verletzung 5,6. E / P-Selektine vermitteln Neutrophilen-Abscheidung und-rollen durch relativ schwache Assoziationen mit glycomolecules auf der neutrophilen Zelloberfläche. Diese Wechselwirkungen, zusammen mit IL-8-Bindung an seinen verwandten Rezeptoren, erleichtern die robust, Integrin-vermittelte Bindung und eventuellen Verhaftung von Neutrophilen am Endothel Zelloberfläche 7-10. Nach Verhaftung kann Neutrophile wandern aus dem Gefäßsystem zu den Austragungsorten der Infektion direkt eliminieren Krankheitserreger erzeugen Neutrophilen extrazellulären Fallen, um die Verbreitung von Krankheitserregern zu verhindern, die Wundheilung fördern und lassen weitere Faktoren, die andere Leukozyten wie Monozyten, Makrophagen und dendritischen rekrutieren Zellen 11-17. Beschrieben in diesem Bericht ist ein in vitro-Methode, um die Einhaltung der neutrophilen MicroV quantifizierenascular ECs nach Aktivierung durch den Host Entzündungsmediatoren TNF. Dieser Test wurde entwickelt, um die Aktivierung von Endothelzellen und nicht für Neutrophile zu bewerten. Primäre humane Neutrophile sind zunächst getrennt mit Dichtegradiententrennung, und werden dann mit Calcein acetoxymethyl (AM) markiert. Esterasen innerhalb der lebenden Zellen hydrolysieren Calcein AM zu stark fluoreszierenden Calcein Molekül mit einer Anregung von 492-495 nm und die Emission von 513-516 nm 18. Die Fluoreszenz-markierten Neutrophilen werden dann mit EC Monoschichten inkubiert und nicht-adhärente Neutrophile werden anschließend entfernt. Die Fluoreszenz der verbleibenden gebundenen Neutrophilen wird dann unter Verwendung eines Fluoreszenz-Spektralphotometer, berechnet und als Prozentsatz der gesamten Neutrophilen Fluoreszenz Eingang pro Vertiefung. Dieses Verfahren hat den zusätzlichen Vorteil, dass die gebundenen Calcein-markierten Neutrophilen in Spektrofotometrie direkt verwendet werden kann, visualisiert mittels Fluoreszenzmikroskopie, um eine qualitative aus EG ac lesen gebenMotivation. Da dieser Test unter statischen Bedingungen durchgeführt wird, werden nur die ersten Ereignisse, die sehr in der neutrophilen Adhäsionskaskade auftreten beurteilt werden. Dies wird in diesem Bericht mit E-Selektin blockierende Antikörper gegen diese Neutrophilen Einhaltung TNF-behandelten menschlichen Lunge mikrovaskulären EC (HMVEC-Lung) Monoschichten wird drastisch reduziert, wenn die Interaktion mit E-Selektin gestört zeigen bestätigt. Neben TNF, haben wir erfolgreich diesen Test verwendet werden, um das Ausmaß der menschlichen Nabelschnur-EC (HUVEC) Aktivierung durch den Toll-like-Rezeptor-1/2-Agonisten peptidoglycan Lipoprotein-assoziierte (PAL), Murein-Lipoprotein (MLP) und Pam3Cys und bestimmen HMVEC-Lung Aktivierung durch Pam3Cys 19,20. Darüber hinaus haben wir erfolgreich verwendet diesen Test mit Kinase-Inhibitoren und nach RNAi-vermittelter Knockdown von Oberfläche und zytoplasmatische Proteine ​​in HMVEC-Lung, was darauf hindeutet, dass diese Methode kompatibel mit einer Vielzahl von biochemischen und ist screening Assays 20. Zusammenfassend stellt dieser Test ein leicht, reproduzierbar, funktioneller Weise zu verwenden, um das Ausmaß der EG-Aktivierung durch Entzündungsmediatoren in vitro zu übersetzen.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

Die vaskuläre Endothel spielt eine wesentliche Rolle bei der entzündlichen Reaktion. In der akuten Phase der Entzündung werden Endothelzellen (ECs) von Host-Vermittler oder direkt durch konservierte mikrobielle Komponenten oder Host-derived Gefahr Moleküle aktiviert. Activated ECs express Zytokine, Chemokine und Adhäsionsmoleküle, mobilisieren, aktivieren und behalten Leukozyten an den Ort der Infektion oder Verletzung. Neutrophile sind die ersten Leukozyten, um anzukommen, und sich an das Endothel durch eine Vielzahl von Adhäsionsmolekülen auf den Oberflächen der beiden Zellen. Die wichtigsten Funktionen von Neutrophilen sind direkt beseitigen mikrobiellen Bedrohungen, die Förderung der Rekrutierung von anderen Leukozyten durch die Freisetzung von zusätzlichen Faktoren, und initiieren Wundheilung. Daher ist die Einstellung und Befestigung des Endothels ein kritischer Schritt in der Einleitung der entzündlichen Reaktion. In diesem Bericht beschreiben wir eine in-vitro-Assay mit neutrophilen AdhäsionCalcein primären humanen Neutrophilen AM-markierten, um das Ausmaß der mikrovaskulären endothelialen Zell-Aktivierung unter statischen Bedingungen zu quantifizieren. Dieses Verfahren hat den zusätzlichen Vorteil, dass die gleichen Proben mittels Fluoreszenz-Spektrophotometrie quantifiziert auch direkt visualisiert werden mittels Fluoreszenzmikroskopie für eine qualitative Bewertung der Neutrophilen-Bindung.

Um eine zuverlässige, reproduzierbare Ergebnisse mit einem Neutrophilen Bindungs-Assay erhalten, ist es wichtig, dass die Gesundheit und das Konfluenz der mikrovaskulären Endothelzellen optimal am Tag des Tests sind, wie bei HMVEC-Lung in Abbildung 1 dargestellt. Darüber hinaus ist es unerlässlich, dass niedrige Durchgang Nummer mikrovaskulären ECs verwendet werden (dh weniger als 9 Stellen), und entsprechend, empfehlen wir die Durchführung aller Experimente innerhalb von zwei Wochen nach...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophile Einhaltung der aktivierten Endothel an den Standorten der Infektion ist ein integraler Bestandteil des Gastgebers Entzündungsreaktion. Beschrieben in diesem Bericht ist ein neutrophilen Bindungs-Assay, die für das erlaubt<em&gt; In vitro</em&gt; Quantifizierung von primären humanen Neutrophilen-Bindung an Endothelzellen durch Entzündungsmediatoren unter statischen Bedingungen aktiviert.

Quantitative<em&gt; In-vitro-</em&gt; Assay auf Neutrophile Haftung auf Aktiviert primären humanen mikrovaskulären Endothelzellen unter statischen Bedingungen messen

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

Research

JoVE Journal

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

17.0K Aufrufe.  University of California, San Francisco. Neutrophile Einhaltung der aktivierten Endothel an den Standorten der Infektion ist ein integraler Bestandteil des Gastgebers Entzündungsreaktion. Beschrieben in diesem Bericht ist ein neutrophilen Bindungs-Assay, die für das erlaubt In vitro Quantifizierung von primären humanen Neutrophilen-Bindung an Endothelzellen durch Entzündungsmediatoren unter statischen Bedingungen aktiviert.

Serie: Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

In-vitro-Assay der bakteriellen Adhäsion auf Mammalian Epithelzellen

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Forschung

Immunologie und Infektion

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Isolierung von humanen Nabelschnur-Endothelzellen und ihre Verwendung in der Studie von neutrophilen Transmigration unter Strömungsbedingungen

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Ein<em&gt; In-vitro-</em&gt; Co-Infektion Modell zum Studium<em&gt; Plasmodium falciparum</em&gt; HIV-1-Wechselwirkungen in primären menschlichen Monozyten abgeleitete Immunzellen

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Coculture Assay zur Bewertung pathogeninduzierter Neutrophiler Transepithelmigration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Ein Flow-Assay zur Haftung Leukozyten-Rekrutierung Human Lebersinus Endothel unter den Bedingungen der Schubspannung Studieren

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Humanen neutrophilen Flusskammer Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Statische Haftung Assay für das Studium der Integrin-Aktivierung in T-Lymphozyten

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Echtzeit-Bildgebung von Zell-Zell-Endothelial Junctions Während Neutrophile Transmigration unter physiologischen Fluss

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In-vitro- und In-vivo-Modell, um die bakterielle Adhäsion an der Gefäßwand unter Strömungsbedingungen Studieren

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Test der Haftung unter Scherspannung für das Studium der T-Lymphozyten-Adhäsions-Molekül-Wechselwirkungen

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...