Questo lavoro è stato sostenuto dal Dipartimento UCSF di Cura Anestesia e perioperatoria.

1. Placcatura e manutenzione di microvascolari cellule endoteliali <ol><li> Scongelare e far crescere le cellule endoteliali microvascolari secondo i produttori fornite istruzioni. In questo protocollo, le cellule sono state coltivate in EGM-2 MV multimediale (Lonza), e si raccomanda che tutti gli esperimenti vengono eseguiti entro due settimane lo scongelamento per ridurre al minimo qualsiasi variazione dovuta al numero di passaggio. I nostri esperimenti con HMVEC-Lung vengono eseguiti tra passaggi 4-9. </li><li> Giorno 1: Quando le cellule raggiungono 80-90% di confluenza, trypsinize e sospendere nuovamente a 120.000 cellule per ml. Piastra 36.000 cellule (0,3 ml) per pozzetto in 48 pozzetti, piastra di coltura tissutale polistirene (s). Includere pozzi di HMVEC che non saranno incubate con neutrofili in modo da ottenere una lettura della fluorescenza di fondo. A meno che utilizzando piastre 48 pozzetti appositamente progettati per le analisi di fluorescenza, alternando righe o colonne ridurrà al minimo la contaminazione leggera da pozzi vicini. NOTA: Wells può essere pre-rivestito con poli-LYSIne, collagene o fibronectina. </li><li> 2 ° giorno: Aggiungi mezzi freschi. </li><li> Giorno 3: In contrasto di fase, confermano le cellule sono sane (cioè l&#39;aspetto &quot;acciottolato&quot; e poco detriti cellulari), e sono al 100% confluenti (Figura 1). Se le cellule non sono al 100% confluenti, cambiare i media ogni giorno fino a quando non sono pronti. </li><li> Una volta che i monostrati HMVEC sono pronti per il trattamento, lavare le cellule una volta con soluzione salina bilanciata di Hank (HBSS) e aggiungere 0,3 ml di fresca EGM-2 supporti MV o supporti contenenti agonisti infiammatoria nei rispettivi pozzetti. In questo protocollo il HMVEC-Lung state trattate con TNFa [100 ng / ml] (Peprotech, New Jersey) per 3 ore poiché questo è un attivatore ben descritto di EC 21. NOTA: Si raccomanda di tempo l&#39;esperimento in modo che le vostre cellule HMVEC attivati ​​(Passo 4.1) e calceina AM neutrofili etichettati (Passo 3.5) sono pronti per l&#39;uso, allo stesso tempo. Ci vogliono circa 2,5 ore per isolare ed etichettare neutrophils. </li></ol> 2. Isolamento dei neutrofili utilizzando Polymorphprep <ol><li> Isolare i neutrofili come descritto in precedenza 22, o con una soluzione pronta gradiente di densità di isolare granulociti polimorfonucleati dal sangue intero. Impiegando Polymorphprep seguendo il protocollo di Nuzzi et.al. risultati 2007 dei rendimenti di circa 2-3 x 10 6 neutrofili per ml di sangue intero 23. NOTA: per evitare un eccessivo lisi degli eritrociti, una sedimentazione destrano per rimuovere i globuli rossi possono essere svolte prima di centrifugazione in gradiente di densità 24. </li><li> Portare la soluzione gradiente di densità Polymorphprep a RT. </li><li> Raccogliere 30 ml di sangue intero da un volontario umano sano in presenza di un anticoagulante come l&#39;eparina, citrato o EDTA. Il sangue deve essere utilizzato entro 2 ore, e mantenuta tra 18-22 ° C. </li><li> Strato 5 ml di sangue intero in 5 ml della soluzione di gradiente di densità in un tubo da 15 ml (Figura 2A). Evitare di alterare i volumi di sangue e la soluzione gradiente di densità in quanto ciò potrebbe cambiare le successive condizioni di centrifugazione. </li><li> Centrifugare a 450 xg per 30 minuti a 18-22 ° C. Assicurati di accelerare lentamente su e giù per la centrifuga (cioè spegnere il freno centrifuga), e bilanciare i tubi bene per evitare che le vibrazioni per contribuire a ridurre l&#39;attivazione dei neutrofili e prevenire la miscelazione di strati. Tempi più lunghi di centrifugazione o di forza centrifuga maggiore si tradurrà nello strato leucocitaria inferiore, che contiene i neutrofili, a migrare più in basso verso i globuli rossi pellet. </li><li> Aspirare il plasma e leucociti banda superiore contenente PBMC di impedire la contaminazione dello strato contenente neutrofili (Figura 2B). </li><li> Utilizzare un 1-2 ml pipetta sierologica per rimuovere lo strato inferiore contenente leucociti neutrofili. Unire i livelli di neutrofili in 30 ml di PBS (senza Ca 2 + e Mg 2 +) in 50 mltubo conico. </li><li> Portare il volume a 50 ml con PBS (senza Ca 2 + e Mg 2 +) e centrifugare a 450 xg per 10 minuti a 18-22 ° C. </li></ol> Nota: I punti 2.9 e 2.10 sono opzionali, ma altamente raccomandato. <ol start="9"><li> Aspirare il surnatante. Risospendere i neutrofili in 8 ml di acqua sterile per 30 sec per la lisi dei globuli rossi, aggiungere immediatamente la sospensione di cellule per 2 ml di PBS 5x (senza Ca 2 + e Mg 2 +) e mescolare delicatamente con le mani. Non incubare le cellule in acqua per più di 30 secondi dal momento significativo morte neutrofili può verificarsi. </li><li> Centrifugare a 250 xg per 5 minuti a 18-22 ° C. </li><li> Lavare le cellule una volta con 10 ml di PBS (senza Ca 2 + e Mg 2 +) e centrifugare nuovamente a 250 xg per 5 minuti a 18-22 ° C. </li><li> Risospendere i neutrofili in 10 ml di RPMI-1640 (senza rosso fenolo) e contare le cellule vive usando il blu tripanotingere metodo di esclusione. Evitare periodi prolungati di densità cellulari maggiori di 5 x 10 6 per ml poiché questo può causare l&#39;attivazione dei neutrofili. </li></ol> 3. Neutrofili Etichettatura con Calcein AM <ol><li> 6 x 10 5 neutrofili sono utilizzati per pozzetto di una piastra a 48 pozzetti. </li><li> Dopo il conteggio, trasferire le cellule risospese ad una provetta da 50 ml e diluire i neutrofili a 2-4 x 10 6 cellule per ml con fenolo aneritro RPMI-1640. </li><li> Aggiungi calceina AM soluzione madre di 3 micron (cioè 2,98 ml di calceina AM madre (1 mg / ml) per ml di media) e mescolare bene muovendo delicatamente il tubo. Superiore a 5 micron calceina AM si tradurrà in perdite dai neutrofili. Nota: non fluorescente calceina AM viene scissa nelle cellule dalle esterasi endogene per produrre la molecola altamente fluorescente calceina (vale a dire le cellule morte non fluorescenza). </li><li> Coprire il tubo con un foglio di alluminio e incubare for 30 min a 37 ° C al buio. Nota: i tempi di incubazione più lunghi possono tradursi in maggiore calceina essere liberata dai neutrofili nel corso del saggio di adesione. </li><li> Centrifugare a 450 xg per 5 minuti a 18-22 ° C, e lavare i neutrofili 2x con 10 ml di RPMI-1640 (senza rosso fenolo; pre-riscaldato a 37 ° C). Dopo risospendere le cellule per il secondo lavaggio, prima passare i neutrofili con un filtro sterile 40 pM per eliminare grumi e quindi si centrifuga di nuovo a 450 xg per 5 minuti a 18-22 ° C. </li><li> Risospendere i neutrofili in RPMI 1640 (senza rosso fenolo; pre-riscaldato a 37 ° C) a 2 x 10 6 neutrofili per ml. </li></ol> 4. Neutrofili Binding Assay <ol><li> Lavare i monostrati HMVEC 3x con 0,5 ml di sterilizzata per filtrazione (0,22 micron) RPMI 1640 (senza rosso fenolo) contenente il 3% di BSA per pozzetto. </li><li> Aspirare i media e aggiungere 6 x 10 5 neutrofili calceina-etichettati (0,3 ml di sospensione cellulare), Oppure 0,3 ml di RPMI 1640 (senza rosso fenolo) senza neutrofili, nei rispettivi pozzetti della 48 pozzetti. Nota: Questo è l&#39;equivalente di 8 x 10 5 neutrofili per cm 2. </li><li> Incubare per 20 min a 37 ° C, 5% CO 2 incubatore. </li><li> Totale dei neutrofili Fluorescenza (PRE lavaggio): Misurare l&#39;intensità di fluorescenza di ciascun bene con una lunghezza d&#39;onda di eccitazione di 485 nm e una lunghezza d&#39;onda di 520 nm (fluoresceina filtro impostato) in una FLUOstar, o equivalente, spettrofotometro di emissione. Eseguire questo passaggio appena prima del punto finale incubazione Nota: In questo protocollo, calceina eccitazione e rilevamento della luce emessa state eseguite dalla sommità dei pozzi scoperti, tuttavia, entrambi possono essere eseguite anche dal fondo dei pozzetti.. </li><li> Lavare i pozzetti contenenti monostrati HMVEC e neutrofili 5x con 0,5 ml per pozzetto di PBS (w / Ca 2 + e Mg 2 +). Rimuovere il supporto e lavaggiinvertendo la piastra, e picchiettando delicatamente su carta assorbente per eliminare il liquido rimasto. </li><li> Aggiungi posteriore 0,3 ml di RPMI 1640 (senza rosso fenolo) per pozzetto. </li><li> Aderente neutrofili Fluorescenza POST (lavaggio): Misurare l&#39;intensità di fluorescenza di ogni nonché al punto 4.4. </li><li> Determinare l&#39;aderenza per cento e l&#39;adesione relativa per bene: </li></ol> Nota: In questa fase, le immagini dei neutrofili calceina-etichettati possono essere ottenuti utilizzando un microscopio capace fluorescenza dotato di filtri fluoresceina standard. Le immagini in figura 4 sono stati ottenuti su una Olympus IX51 microscopio invertito dotato di una fotocamera 2000R Retiga (Q Imaging) con Q-Capture Software Pro7 (Q Imaging).

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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>

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Le fasi più critiche per una neutrofili di successo / endoteliale microvascolare saggio di adesione cellulare sono: 1) Uso di basso numero di passaggio (&lt;9), cellule endoteliali sane; 2) Il mantenimento dei neutrofili isolati a bassa densità (cioè &lt;5 x 10 6 cellule / ml) e il loro utilizzo in due ore di isolamento, e 3) il lavaggio Fastidious dei neutrofili calceina AM-etichettati e l&#39;uso dei neutrofili calceina AM-etichettati in modo tempestivo per ridurre al minimo la contaminazione CE...

Poiché l&#39;endotelio vascolare è in contatto diretto con il sangue circolante, esso è sitato ad iniziare una risposta infiammatoria rapido durante infezioni o lesioni. Le cellule endoteliali (EC) recettori di pattern recognition espresso che riconoscono una varietà di componenti batteriche conservati e molecole pericolo, e recettori per Host mediatori infiammatori, come TNFa. Attivazione di questi recettori induce EC per secernere citochine (es IL-6, IL-8, CXCL1 e CCL2), e per upregulate molecole di adesione (ad esempio E / P-selectina, VCAM-1 e ICAM-1) alla loro superficie cellulare 1 , 2. Queste molecole tutto facilitano la localizzazione dei leucociti ai siti di infezione e lesioni per chiarire l&#39;host di agenti infettivi e avviare la riparazione tissutale 3,4. La risposta dei neutrofili all&#39;infezione comporta un&#39;interazione ben coordinato tra l&#39;endotelio vascolare e la risposta neutrofili. All&#39;attivazione EC, IL-8 è secreto e forma un gradiente intravascolare sull&#39;endotelio che permette di neutrofili casa al sito di infezione o lesioni 5,6. E / P-selectine mediata neutrofili cattura e rotolare attraverso le associazioni relativamente deboli con glycomolecules sulla superficie cellulare dei neutrofili. Queste interazioni, insieme con IL-8 legame ai suoi recettori cognate, facilitano il robusto, attaccamento integrina-mediata ed eventuale arresto dei neutrofili sulla superficie delle cellule endoteliali 7-10. Dopo l&#39;arresto, i neutrofili possono migrare fuori della vascolarizzazione ai siti specifici di infezione di eliminare direttamente i patogeni, generare neutrofili trappole extracellulari per prevenire la diffusione di agenti patogeni, promuovere la guarigione delle ferite e rilasciare fattori aggiuntivi che reclutano leucociti altri come monociti, macrofagi e dendritiche cellule 11-17. Descritte nella presente relazione è un metodo in vitro per quantificare l&#39;adesione dei neutrofili alle microVascular I CE dopo l&#39;attivazione da parte del mediatore infiammatorio ospite TNFa. Questo test è stato progettato per valutare l&#39;attivazione delle cellule endoteliali, e non neutrofili. Neutrofili umani primari sono stato isolato utilizzando separazione gradiente di densità, e sono quindi etichettati con calceina acetossimetil (AM). Esterasi all&#39;interno delle cellule in diretta idrolizzano calceina AM alla molecola calceina altamente fluorescente con una eccitazione di 492-495 nm ed emissione di 513-516 nm 18. I neutrofili fluorescenza marcata vengono poi incubate con monostrati CE, e neutrofili non aderenti sono successivamente rimossi. La fluorescenza dei rimanenti, neutrofili legato viene quindi misurata mediante uno spettrofotometro a fluorescenza, e calcolata come percentuale del totale dei neutrofili ingresso fluorescenza per pozzetto. Questo metodo ha il vantaggio aggiuntivo che i neutrofili calceina-etichettati vincolati utilizzati in spettrofotometria possono essere visualizzati direttamente utilizzando la microscopia a fluorescenza per dare una lettura più qualitativa di ac CEtivazione. Dal momento che questo test viene eseguito in condizioni statiche, saranno valutati solo gli eventi molto iniziali che si verificano nella adesione cascata neutrofili. Ciò è confermato in questo rapporto utilizzando anticorpi bloccanti E-selectina per dimostrare che l&#39;adesione dei neutrofili alle TNFa-trattato polmone umano microvascolare CE (HMVEC-Lung) monostrati si riduce drasticamente quando l&#39;interazione con la E-selectina è perturbato. Oltre a TNFa, abbiamo utilizzato con successo questo test per determinare l&#39;entità della vena ombelicale CE attivazione umana (HUVEC) dal recettore Toll-like mezzo agonisti lipoproteine ​​peptidoglicano-associato (PAL), murein lipoproteina (MLP) e Pam3Cys e attivazione HMVEC-Lung da Pam3Cys 19,20. Inoltre, abbiamo utilizzato con successo questo test con inibitori delle chinasi e dopo knockdown RNAi-mediata di superficie e proteine ​​citoplasmatiche in HMVEC-Lung, suggerendo che questa metodologia è compatibile con una varietà di biochimica e screening saggi 20. In sintesi, questo saggio fornisce un facile da usare, modo riproducibile, più funzionale per accedere alla misura dell&#39;attivazione CE da mediatori infiammatori in vitro.

<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

L&#39;endotelio vascolare svolge un ruolo fondamentale nella risposta infiammatoria. Durante la fase acuta dell&#39;infiammazione, cellule endoteliali (EC) sono attivati ​​da mediatori ospitanti o direttamente da componenti microbici conservati o molecole pericolo ospite-derivati. Attivati ​​ecs esprimere citochine, chemochine e molecole di adesione che mobilitano, attivano e mantengono leucociti nel sito di infezione o lesioni. Neutrofili sono i primi ad arrivare leucociti, e aderire alla endotelio attraverso una varietà di molecole di adesione presenti sulle superfici di entrambe le celle. Le principali funzioni di neutrofili sono eliminare direttamente minacce microbiche, promuovere l&#39;assunzione di altri leucociti attraverso il rilascio di fattori aggiuntivi, e avviare riparazione della ferita. Pertanto, il loro reclutamento e attaccamento alla endotelio è un passaggio fondamentale nell&#39;iniziazione della risposta infiammatoria. In questo rapporto, descriviamo un test di adesione in vitro usando neutrofilicalceina AM-etichettato neutrofili umani primari per quantificare il grado di attivazione delle cellule endoteliali microvascolari in condizioni statiche. Questo metodo ha il vantaggio aggiuntivo che gli stessi campioni quantizzati mediante spettrofotometria a fluorescenza possono anche essere visualizzati direttamente utilizzando la microscopia a fluorescenza per una valutazione più qualitativa di legame neutrofili.

Per ottenere risultati affidabili e riproducibili usando un saggio di legame neutrofili, è essenziale che la salute e la confluenza delle cellule endoteliali microvascolari sono ottimali nel giorno del saggio, come illustrato con HMVEC-Lung in Figura 1. Inoltre, è indispensabile che passaggio basso numero microvascolare EC vengono utilizzati (cioè meno di 9 brani), e di conseguenza, si consiglia di eseguire tutti gli esperimenti entro due settimane di scongelamento. La salute dei neutrofili ...

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

L&#39;adesione dei neutrofili all&#39;endotelio attivato nei siti di infezione è una componente integrale della risposta infiammatoria dell&#39;ospite. Descritto in questo rapporto è un saggio di legame dei neutrofili che permette la<em&gt; In vitro</em&gt; Quantificazione di neutrofilo umana primaria legame alle cellule endoteliali attivate da mediatori infiammatori in condizioni statiche.

Quantitative<em&gt; In vitro</em&gt; Assay misurare Adesione neutrofili attivati ​​per primarie umane endoteliali microvascolari in condizioni statiche

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 Views.  University of California, San Francisco. L'adesione dei neutrofili all'endotelio attivato nei siti di infezione è una componente integrale della risposta infiammatoria dell'ospite. Descritto in questo rapporto è un saggio di legame dei neutrofili che permette la In vitro Quantificazione di neutrofilo umana primaria legame alle cellule endoteliali attivate da mediatori infiammatori in condizioni statiche.

Video: 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 di Adesione batterica su mammiferi cellule epiteliali

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

Ricerca

Immunologia e infezioni

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.

Isolamento di cellule umane endoteliali della vena ombelicale e il loro uso nello studio della trasmigrazione dei neutrofili in condizioni di flusso

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.

Un<em&gt; In vitro</em&gt; Co-infezione da modello per lo studio<em&gt; Plasmodium falciparum</em&gt;-HIV-1 in Interazioni umane Le cellule immunitarie primarie derivate da monociti

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 Test di cocultura per valutare la migrazione transeteliale neutrofila indotta da agenti patogeni

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.

Un flusso di Adesione saggio per studiare Leucocyte reclutamento a epatici umani sinusoidali Endotelio in condizioni di stress Shear

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.

Umano camera di flusso neutrofili saggio

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.

Static Adesione saggio per lo Studio della integrina attivazione nei linfociti T

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.

In tempo reale Imaging del endoteliali giunzioni cellula-cellula Durante neutrofili Trasmigrazione Sotto fisiologico flusso

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 e in vivo modello per studiare l&#39;adesione batterica alla parete del serbatoio in condizioni di flusso

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.

Saggio di adesione Sotto Shear stress per lo studio di linfociti T-Adhesion Molecule Interazioni

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