Questo lavoro è stato sostenuto dal National Institutes of Health (NIH) concede R01-DK080752 e P50-DK064540. Ringraziamo J. Loughman per il suo impegno nella creazione di questo saggio.

1. Coltura 5637 Cellule epiteliali della vescica Effettuare le seguenti operazioni in una cappa di tessuto-cultura flusso laminare preparato con irradiazione UV e spazzato via giù con il 70% di etanolo. <ol><li> Preparare mezzo RPMI-1640 contenente 10% siero bovino fetale (FBS) [definito RPMI +], filtro sterilizzare usando un filtro di 0,22 micron dimensione dei pori e riscaldare a 37 ° C in un bagno d&#39;acqua. </li><li> Scongelare una esageratamente di 5637 cellule (American Type Culture Collection HTB-9; derivata da carcinoma della vescica) in un bagno d&#39;acqua a 37 °. Trasferire rapidamente le cellule scongelati per un tessuto pallone 75 centimetri 2 di coltura contenente 20 ml di RPMI +. </li><li> Incubare il matraccio a 37 ° C in atmosfera umidificata con 5% di CO 2 fino a quando le cellule sono circa il 95% confluenti (~ 6 x 10 6 cellule per pallone), circa 4 giorni. </li><li> Per sottocultura le cellule 5637: <ol><li> Rimuovere il terreno dal pallone. Lavare le cellule con 10 ml di Dulbecco Phosphate-Buffered Saline (DPBS) a temperatura ambiente. </li><li> Aggiungere 6 ml calda (37 ° C) 0,05% tripsina, soluzione di EDTA 0,02% al pallone, e incubare a 37 ° C con 5% di CO 2 per 15 min. </li><li> Trasferire le cellule in una provetta da 15 ml, e pellet per centrifugazione a 300 xg per 5 min. Rimuovere la soluzione di tripsina / EDTA. </li><li> Risospendere le cellule in 6 ml RPMI + (10 6 cellule / ml), e trasferimento 1 ml (10 6 cellule) in un pallone di 75 centimetri 2 contenente 20 ml di RPMI +. </li></ol></li><li> Ripetere passo 1.4 al sottocultura cellule ogni 4 giorni o quando le cellule raggiungono il 95% di confluenza. </li></ol> 2. Semina e Growing 5.637 celle su supporti permeabili Effettuare le seguenti operazioni in una cappa di coltura di tessuti con tecnica asettica. Per risultati ottimali, utilizzare 5.637 cellule che hanno subito meno di 10 sottoculture. <ol><li> Utilizzando pinze sterili, invertire i supporti permeabili in 25 millimetri piatto di coltura dei tessuti profondi sterile. </li><li&gt; trypsinize un pallone di 5637 cellule di 75 centimetri 2 al 95% di confluenza secondo le fasi 1.4.1-1.4.3. </li><li> Usando una pipetta 1.000 ml, delicatamente risospendere le cellule in ~ 2 ml RPMI + ad una concentrazione di 3 x 10 6 cellule / ml, determinato da cellule contando con un emocitometro. </li><li> Applicare 50 ml di sospensione cellulare (1,5 x 10 5 cellule) per ciascun supporto permeabile senza toccare la membrana. Mettere il coperchio sul piatto, e posizionare con cura il piatto a 37 ° C con 5% di CO 2 per non più di 16 ore. </li><li> Utilizzando pinze sterili, giusti i supporti permeabili in una piastra a 24 pozzetti contenente 0,6 ml di RPMI + per pozzetto. Aggiungere 0,1 ml RPMI + al serbatoio superiore di ciascun supporto permeabile. Incubare a 37 ° C con 5% di CO 2. </li><li> Sostituire il mezzo ogni 2 giorni. Primo, Piano aspirato dal serbatoio superiore seguito dal serbatoio inferiore, e quindi applicare fresco RPMI + in ordine inverso, per il serbatoio inferiore (0,6 ml) e poi il Rese superiorervoir (0.1 ml). </li><li> Sette giorni dopo la semina dei supporti permeabili con 5637 celle, valutare confluenza delle cellule. Riempire il serbatoio superiore con RPMI + (~ 0.35 ml). Le cellule sono sufficientemente confluenti quando il mezzo non equilibrare tra i serbatoi superiore ed inferiore. </li></ol> 3. Preparazione del batterica dell&#39;inoculo <ol><li> Utilizzando una tecnica asettica, aggiungere 20 ml di brodo di coltura appropriato per un pallone da 250 ml con tappo. Utilizzare Luria-Bertani (LB) brodo per E. coli culture e aggiungere antibiotici per il brodo all&#39;occorrenza. </li><li> Utilizzando un ciclo di inoculazione sterile, inoculare il brodo con i batteri provenienti da un glicerolo magazzino o un piatto realizzato. Per i ceppi UPEC, incubare la coltura batterica a 37 ° C per circa 16 ore, senza agitare (per promuovere la produzione di tipo 1 pili). </li><li> Trasferire la coltura batterica in una provetta da centrifuga. Agglomerare le batteri mediante centrifugazione a 8000 xg per 10 min. </li><li> Decantare il surnatante, E risospendere i batteri in PBS a OD 600 = 1.000, equivalente a ~ 10 9 UFC / ml. <ol><li> Per generare uno stimolo batterica ucciso al calore, incubare una aliquota (es ~ 1,5 x 10 8 UFC in 150 microlitri) dei batteri risospese a 55 ° C per 30 min. Piatto una aliquota della sospensione calore ucciso per confermare la morte dei batteri. </li></ol></li><li> Immediatamente prima dell&#39;uso, diluire i batteri risospeso (vivo o ucciso al calore) di 10 volte a 10 8 CFU / ml nel warm privo di siero RPMI [definito RPMI-] in una provetta per microcentrifuga. </li></ol> 4. Isolamento di neutrofili umani da sangue periferico La raccolta di sangue da volontari adulti necessita di revisione anticipo e l&#39;approvazione di un comitato di revisione istituzionale. Indossare dispositivi di protezione adeguati e smaltire materiali pericolosi per evitare l&#39;esposizione al sangue umano. <ol><li> Circa 25 ml di sangue venoso da un adulto sano volunteer deve trarre in 3 provette sterili di sodio-eparina contenente la raccolta del sangue da parte del personale qualificato in salasso. <ol><li> Strettamente avvolgere un laccio emostatico di gomma attorno al braccio, sopra il gomito. </li><li> Disinfettare il sito di ingresso, fossa antecubitale, utilizzando una sterile alcool al 70% pulire. </li><li> Collegare un supporto di tubo di plastica ad un sistema di ago a farfalla dalle ali. </li><li> Inserire l&#39;ago in una vena antecubitale ad un angolo di 30 ° o meno, smusso rivolto verso l&#39;alto. L&#39;ago ha perforato la vena quando un getto di sangue appare nel tubo di plastica. </li><li> Inserire un tubo di raccolta di sangue nel supporto tubo di plastica. Quando il primo tubo di raccolta è pieno, sostituirlo con il secondo tubo di raccolta. Ripetere con il terzo tubo. </li><li> Quando il terzo tubo di raccolta è di circa mezzo pieno, rimuovere il laccio emostatico. </li><li> Coprire il sito di puntura con un batuffolo di cotone sterile e ritirare lentamente l&#39;ago dalla vena. Far scorrere lo schermo protettivo sopra l&#39;ago e il luogo ina rischio biologico sharps container. </li><li> Applicare pressione sul sito di puntura. Coprire la palla sito di puntura e cotone con un bendaggio adesivo. </li><li> Capovolgere delicatamente le provette per il prelievo del sangue per disperdere l&#39;eparina. </li></ol></li></ol> Effettuare le seguenti operazioni in una cappa di coltura di tessuti con tecnica asettica. <ol start="2"><li> Usando una pipetta 10 ml, trasferire delicatamente il sangue ad una, 50 ml sterile tubo conico fresca. Aggiungere un volume equivalente di 3% (w / v) destrano in 0.9% NaCl e mescolare per inversione. Incubare la provetta in posizione verticale a temperatura ambiente per 20 min. </li><li> Senza interrompere lo strato inferiore, aspirare accuratamente lo strato superiore e trasferirlo in una nuova provetta conica da 50 ml. Agglomerare le cellule per centrifugazione a 300 xg per 10 min. Scartare il surnatante. </li><li> Risospendere il pellet di cellule in un volume di 0,9% NaCl equivalente al volume iniziale di sangue. </li><li> Strato 10 ml di soluzione di centrifugazione densità inferiore sospensione cellulare, priservando l&#39;interfaccia tra le due fasi. Centrifugare a 400 xg per 30 min senza freno. Scartare il surnatante. </li><li> Per lisare restanti globuli rossi, risospendere il pellet cellulare in 10 ml freddo 0,2% NaCl. Incubare per 30 secondi, e poi aggiungere subito 10 ml di freddo 1.6% NaCl per ripristinare isotonicità. </li><li> Agglomerare le cellule per centrifugazione a 300 xg per 6 min. Scartare il surnatante. </li><li> Ripetere i passaggi 4,6-4,7 finché non appare il pellet cellulare per essere liberi di globuli rossi, in genere 3 turni di lisi. </li><li> Usando una pipetta 1,000 microlitri, risospendere il pellet cellulare, principalmente PMN, in acqua calda (37 ° C) RPMI-ad una concentrazione di 10 7 cellule / ml, determinato per conteggio delle cellule utilizzando un emocitometro. Conservare le cellule a 37 ° C fino all&#39;utilizzo. Tipicamente, PMN vitalità e purezza sono&gt; 99% come valutato dal trypan blu e la visualizzazione della morfologia nucleare dopo colorazione, rispettivamente. </li></ol> 5. Transuroepithelial neutrofili migrazionedire Effettuare le seguenti operazioni in una cappa di coltura di tessuti con tecnica asettica. Utilizzare solo supporti permeabili cuscinetto confluenti 5637 strati di cellule, come determinato al punto 2.7. Le piastre a 24 pozzetti contenenti RPMI-possono essere preparati in anticipo e conservati a 37 ° C con 5% di CO 2 fino al momento dell&#39;uso. <ol><li> Aliquota 1 ml caldo RPMI-per bene in un 24-pozzetti; preparare 3 pozzetti per il supporto permeabile. </li><li> Aspirare il mezzo dai serbatoi superiore ed inferiore dei supporti permeabili. </li><li> Utilizzando pinze sterili, trasferire i supporti permeabili alla piastra 24 pozzetti preparato al punto 5.1. Lavare i supporti permeabili tre volte trasferendo i supporti da pozzetto a pozzetto. </li><li> Se un inoculo batterico deve essere utilizzato, invertire i supporti permeabili in 25 mm piatto di coltura dei tessuti profondi sterile. Se un chemiotattico (ad esempio, N-Formyl-Met-Leu-Phe (fMLF) o IL-8), deve essere utilizzato, passare al punto 5.6. </li><li> Per gli esperimenti con bact vivo o uccisostimoli Erial, inoculare la parte apicale di ciascun supporto permeabile con 60 microlitri RPMI-(infezione finto) o con l&#39;inoculo batterico (6 x 10 6 CFU) preparato al punto 3.5. Posizionare il coperchio sul piatto e incubare a 37 ° C con 5% di CO 2 per 1 ora. </li><li> Aggiungere 0,6 ml di RPMI-per bene ad una piastra a 24 pozzetti basso attaccamento; preparare 1 e per il supporto permeabile. Preparare 3 pozzetti contenenti 0,5 ml RPMI-per enumerare ingresso PMN. <ol><li> Se un fattore chemiotattico viene utilizzato al posto di batteri, aggiungere il fattore chemiotattico per 0,6 ml RPMI-nella piastra a 24 pozzetti preparato al punto 5.6. </li></ol></li><li> Destra supporti permeabili nella piastra a 24 pozzetti low-attacco. </li><li> Aggiungere 0,1 ml PMN (10 6 PMN), preparato nella fase 4.9, al serbatoio superiore (lato basolaterale) di ciascun supporto permeabile. Aggiungere 100 microlitri PMN direttamente nei pozzetti contenenti 500 ml RPMI-. Incubare a 37 ° C con 5% di CO 2 per 1 ora. </li><li> Utilizzando pinze sterili, raschiare delicatamente lamembrana del supporto permeabile contro il bordo del pozzo per rimuovere PMN supplementare dal lato apicale e poi smaltire il supporto. </li><li> Raccogliere PMN raschiando delicatamente la parte inferiore di ogni bene con la punta della pipetta 1.000 microlitri, e trasferire le sospensioni PMN per microcentrifuga tubi. </li><li> Enumerare PMN utilizzando un emocitometro. </li><li> Per calcolare il numero totale di PMN nel serbatoio inferiore, moltiplicare il numero di PMN in 1 mm 2 (100 nl) da 6.000. I dati possono essere presentate come percentuale di input di PMN, o come numeri PMN normalizzati 10 6 ingresso PMN. </li></ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+the+human+leukocyte+enzymes+myeloperoxidase+and+eosinophil+peroxidase.">Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase.</a> J. Immunol. Methods. 126, 125-133 (1990).</li><li>Lee, W. Y., Chin, A. C., Voss, S., Parkos, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+neutrophil+transepithelial+migration.">In vitro neutrophil transepithelial migration.</a> Methods Mol. 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Usando una linea di cellule epiteliali della vescica colta e preparata isolato PMN umani, abbiamo stabilito un modello in vitro di migrazione dei neutrofili transuroepithelial. Questo modello è stato determinante nel cominciare per analizzare la complessità della risposta immunitaria innata durante l&#39;infezione del tratto urinario (UTI), un&#39;infezione batterica molto comune in genere causata da UPEC 9. Durante l&#39;infezione della vescica o cistite, assunzione di PMN al lume della vescica è...

Il movimento delle cellule in tutto il corpo, spesso su lunghe distanze, è necessaria per la crescita e lo sviluppo, la guarigione della ferita, e la risposta immunitaria. La migrazione cellulare è complesso e richiede il coordinamento di molti processi diversi, tra cascate di segnalazione e il riassetto dei componenti del citoscheletro. Le cellule possono muoversi in modo casuale (chemochinesi) così come verso gradienti chimici definiti (chemiotassi). Molte tecniche sono state sviluppate per studiare la migrazione delle cellule in vitro. La più antica e più comune tecnica, la camera di Boyden, costituito da un sistema a due camera verticale quando una sostanza chemiotattico viene posta nella camera inferiore e cellule di interesse vengono inseriti nella camera superiore 1. Il movimento delle cellule attraverso il filtro permeabile, con pori di dimensioni definite, che separa le due camere è monitorato. Altre tecniche sono state sviluppate per studiare la migrazione cellulare, compresa la camera Zigmond 2 e la camera di Dunn3. Questi approcci collettivi hanno dato una visione significativa nel movimento di molti tipi cellulari diversi. Oltre a interrogare i principi fondamentali della chemochinesi e chemiotassi, saggi a due camere hanno facilitato l&#39;indagine della migrazione cellulare attraverso componenti della matrice extracellulare e di entrambi gli strati di cellule endoteliali ed epiteliali. Un vantaggio dei sistemi a due camere su altre tecniche è che la membrana porosa può essere rivestito con proteine ​​come il collagene o fibrinogeno e migrazione cellulare attraverso una barriera a matrice extracellulare può essere valutata. Inoltre, linee cellulari coltivate possono essere coltivate e differenziati sui supporti permeabili. Per studiare il movimento delle cellule attraverso una barriera endoteliale, cellule endoteliali coltivate vengono seminate e coltivate nel serbatoio superiore dei supporti permeabili. Cellule mobili, come le cellule immunitarie, vengono aggiunti al serbatoio superiore e la migrazione nel serbatoio inferiore di tutti i enbarriera endoteliale in direzione apicale a basolaterale fisiologica viene osservata. Questo modello è stato prezioso per comprendere stravaso di cellule immunitarie dal flusso sanguigno. In contrasto transendoteliale migrazione, il movimento delle cellule attraverso una barriera epiteliale verifica in genere nella direzione basolaterale-to-apicale. Per modellare questi eventi in vitro, i ricercatori seme e crescono cellule epiteliali coltivate sul lato inferiore dei supporti permeabili. Cellule mobili vengono aggiunti al serbatoio superiore e la migrazione attraverso la barriera epiteliale, che rappresenta la direzione basolaterale-to-apicale, viene monitorato. Tali modelli di migrazione transepiteliale hanno contribuito in modo significativo alla nostra comprensione delle risposte infiammatorie a superfici mucose, in particolare quelli del polmone e intestino 4,5. Al contrario, il traffico delle cellule immunitarie attraverso l&#39;epitelio delle vie urinarie ha ricevuto molta meno attenzione. Per promuovere il nostro understanding delle risposte immunitarie innate nel tratto urinario durante l&#39;infezione con uropatogeni Escherichia coli (UPEC), abbiamo sviluppato un test in vitro, la transuroepithelial test di migrazione dei neutrofili, che consente di indagine di leucociti polimorfonucleati (PMN, neutrofili) movimento attraverso una barriera epiteliale della vescica 6 -8. Come con altri modelli a due camere di migrazione transepiteliale, colture di cellule epiteliali umane della vescica sono coltivate sul lato inferiore di un supporto permeabile e formano strati epiteliali confluenti. Neutrofili umani, isolate da sangue venoso, vengono applicati sul lato basolaterale dello strato epiteliale, e migrazione attraverso l&#39;epitelio nella direzione basolaterale-to-apicale fisiologicamente rilevanti viene quantificato in risposta a infezione con diversi ceppi di E. coli o la presenza di molecole chemiotattici. Molta ricerca era concentrata sul movimento dei neutrofili, sia chemochinesi e chemiotassi, in assenza di altri tipi di cellule. Il transuroepithelial neutrofili test di migrazione è vantaggioso in quanto tiene conto di complesse interazioni fra le cellule epiteliali della vescica e le cellule del sistema immunitario durante l&#39;infezione. Questo trattabili modello in vitro ha il potenziale per consentire l&#39;esame approfondito delle risposte immunitarie a superfici uroepithelial.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Transwell Inserts (i.e.&#160;permeable supports)</td>
			<td>Corning</td>
			<td>3472</td>
			<td>6.5 mm, 3 &#956;m pore, polyester membrane insert</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>366480</td>
			<td>16 mm x 100 mm x 10 ml green cap tubes containing sodium heparin</td>
		</tr>
		<tr>
			<td>BD Safety-Lok Blood Collection and Infusion Set</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>367281</td>
			<td>21 G x 0.75 in needle x 12 in tubing</td>
		</tr>
		<tr>
			<td>BD Vacutainer one-use, nonstackable holder</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364815</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D4876</td>
			<td>From Leuconostoc mesenteroides</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density centrifugation solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>24-well, clear flat bottom</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLF)</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td>Reconstituted in DMSO to 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human CXCL8/IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td>Reconstituted in PBS to 100 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

quantitative assessment of human neutrophil migration across a cultured bladder epithelium

Il reclutamento di cellule immuni dalla periferia al sito di infiammazione è un passo essenziale nella risposta immunitaria innata in qualsiasi superficie mucosa. Durante l&#39;infezione della vescica urinaria, leucociti polimorfonucleati (PMN; neutrofili) migrano dal sangue e attraversano l&#39;epitelio vescicale. Mancata soluzione infezione in assenza di una risposta neutrofila dimostra l&#39;importanza di PMN in difesa vescica. Per facilitare la colonizzazione dell&#39;epitelio vescicale, uropatogeni Escherichia coli (UPEC), l&#39;agente eziologico della maggior parte delle infezioni delle vie urinarie (IVU), smorzare la risposta infiammatoria acuta utilizzando una varietà di meccanismi parzialmente definiti. Per approfondire l&#39;interazione tra ospite e patogeno batterico, abbiamo sviluppato un modello in vitro di questo aspetto della risposta immunitaria innata di UPEC. Nel transuroepithelial saggio migrazione dei neutrofili, una variazione sul camera di Boyden, epith vescica coltaElial cellule sono coltivate a confluenza sul lato inferiore di un supporto permeabile. PMN sono isolate da sangue venoso umano e vengono applicati al lato basolaterale degli strati di cellule epiteliali della vescica. Migrazione PMN rappresenta la direzione basolaterale-to-apicale fisiologicamente pertinente in seguito a infezione batterica o molecole chemiotattici viene enumerato con un emocitometro. Questo modello può essere utilizzato per analizzare le interazioni tra UPEC e cellule eucariotiche nonché per interrogare i requisiti molecolari per l&#39;attraversamento di vescica epiteli da PMN. Il modello di migrazione dei neutrofili transuroepithelial sarà ulteriormente la nostra comprensione della risposta infiammatoria iniziale UPEC nella vescica.

Il transuroepithelial saggio migrazione dei neutrofili consente la valutazione quantitativa della migrazione PMN umano di tutti gli strati cellulari epiteliali della vescica coltivate in risposta a vari stimoli (Figura 1B). Mentre il protocollo è semplice, ci sono una serie di variabili che possono influenzare la migrazione PMN e conseguentemente influenzare la riproducibilità del saggio. Occorre adottare misure volte durante la preparazione dei supporti permeabili e PMN per ridurre la variabilità tr...

Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Abbiamo sviluppato un modello in vitro che imita un componente importante della risposta infiammatoria acuta durante l&#39;infezione della vescica con uropatogeni Escherichia coli. Il transuroepithelial test di migrazione dei neutrofili consente la valutazione quantitativa della migrazione dei neutrofili umana attraverso vescica epiteli, coltivate su supporti permeabili, in risposta a infezioni batteriche o di sostanze chemoattractant.

La valutazione quantitativa della migrazione dei neutrofili Umana Attraverso una coltivate vescica Epitelio

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

quantitative-assessment-human-neutrophil-migration-across-cultured

Research

JoVE Journal

Immunology and Infection

14.9K Views.  Washington University School of Medicine.  Abbiamo sviluppato un modello in vitro che imita un componente importante della risposta infiammatoria acuta durante l'infezione della vescica con uropatogeni Escherichia coli. Il transuroepithelial test di migrazione dei neutrofili consente la valutazione quantitativa della migrazione dei neutrofili umana attraverso vescica epiteli, coltivate su supporti permeabili, in risposta a infezioni batteriche o di sostanze chemoattractant. 

Video: Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Ricerca

Immunologia e infezioni

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

Monitoraggio Crawling neutrofili intraluminale, migrazione e chemiotassi transendoteliale in tessuto al microscopio video intravitale

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Una valutazione quantitativa della migrazione cellulare con il Test Phagokinetic motilità Traccia

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

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 ...

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 ...

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 ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Sviluppo e identificazione di una sottopopolazione romanzo di Human neutrofili di derivazione fagociti Giant<em&gt; In Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In Vitro Canini trappola extracellulari del neutrofilo formazione: Analisi dinamica e quantitativa mediante microscopia a fluorescenza

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Valutazione della migrazione dei linfociti in un sistema di trasmigrazione Ex Vivo

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Valutazione funzionale della permeabilità intestinale e della migrazione transepiteliale neutrofila nei topi utilizzando un modello standardizzato di loop intestinale

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...