La ricerca è sostenuta dal National Institutes of concede Salute AI-52381, CA138623 e Kentucky consiglio Lung Cancer Research e il sostegno istituzionale di James Graham Brown Cancer Center.

Metodologia Descrizione del microscopio Dal vivo gli esperimenti di imaging cellulare eseguita utilizzando TE-FM Epi-Fluorescenza sistema collegato al microscopio invertito Nikon Eclipse TE300. Il microscopio dotato fase di riscaldamento. Un fresco scatto HQ digitale B / N CCD (Roper scientifico) della fotocamera e LAMDA 10-2 filtro ottico changer (società strumento Sutter) è collegata al microscopio. Lunghezze d&#39;onda di eccitazione ed emissione sono controllati con ruote filtro e controllato da Lamba 10-2 regolatore ruota portafiltri, Sutter Instruments Co. 500 ms Tempo di esposizione dovrebbe essere sufficiente per visualizzare RFP o GFP in cellule vive. Controllo hardware e acquisizione delle immagini sono controllati da software Metamorph. La scelta dei filtri per il presente studio sono, set di filtri S480/20x, S525/40m e S565/25x, S620/60m per la GFP e RFP, rispettivamente; EGFP / DsRed dual dicroiche, Tecnologia Chroma. Questa ruota filtro in grado di ospitare fino a sei set di filtri. Microscopio è collegato con regolatore Prima tappa Proscan con bastone Gioia. Tutti questi allegati hardware può essere controllato da un software Metamorph. Il microscopio è inoltre collegato con Micromanipolatori per tenere la micropipette. A. Misurazione leucotrienici B 4 migrazione Regia cellule dendritiche Utilizzando l&#39;impostazione sopra descritta microscopio, abbiamo sviluppato metodi per seguire ligando la migrazione delle cellule dendritiche diretto in tempo reale. Due micromanipolatori (Narishige) sono collegati al microscopio. La chemiotassi di midollo osseo dei topi cellule dendritiche derivate (BMDCs) verso LTB 4 gradiente è stato registrato. Qui, descriviamo i metodi per preparare il micro-pipette con micro-pipetta estrattore, il caricamento del ligando in micro-pipetta e di iniziare l&#39;esperimento chemiotassi sul palco riscaldato del microscopio di monitorare la migrazione direzionale delle cellule. Questo metodo può essere applicato a testare l&#39;efficacia di diversi fattori chemiotattici nell&#39;indurre la migrazione così come effetto degli inibitori della chemiotassi in cellule vive. L&#39;isolamento di BMDCs 21 da topo è stato descritto in diverse pubblicazioni tra cui JOVE 22. 1. Preparazione del BMDCs <ol><li> Targhetta del BMDCs (dopo coltura per 10 giorni) al piatto copertura Fluoro fondo antiscivolo e permettere loro di crescere per 16 ore prima dell&#39;esperimento. </li><li> Lavare le cellule con 3 ml di PBS 1X almeno 2 volte (con l&#39;aggiunta di 3 ml di PBS 1X alla piastra ed aspirando il fuori buffer). </li><li> Aggiungere 2 ml di 1XPBS al piatto. Le cellule sono ora pronti per l&#39;esperimento. Mantenere le cellule in incubatore a 37 ° C prima di sperimentare. </li></ol> Preparazione del ligando caricata micro pipetta: <ol start="4"><li> Fissare i tubi di vetro capillari (vetro di serie, 0,75 mm x 0,4 mm, 6 &quot;, Cat # 625500, AM Systems, INC) sulla verticale micropipetta estrattore (Narishige International USA, Inc). </li><li> Mantenere la temperatura a 52 ° C e permettono di fondere il vetro e tirare fuori per formare punte spigolo vivo. </li><li> Fissare la pipetta sulla micro-pipetta titolare. </li><li> Smussare i bordi grezzi di micro pipetta con Micro forgiare M-900 (Narishige International USA, Inc), osservando al microscopio. </li><li> Preparare almeno il 10-20 pipette per ciascun esperimento. Nota: Il micropipette possono anche essere acquistati da Strumenti di precisione mondiale (TIP μ, T1801TW1F). </li><li> Prendere concentrazione desiderata (500 l di 100 nM LTB 4 in l&#39;esperimento in corso) di legante in 1 ml di tubo effendorf e mantenere la punta micro pipetta nella soluzione e lasciare che la soluzione per entrare nel micro pipetta (il riempimento). </li><li> Prendere 1 ml di legante e nella siringa (1 cc siringa da tubercolina) e lentamente riempire il ligando tramite Micro-Fill ago (28G MF-5, strumenti di precisione mondiale) nella pipetta micro dall&#39;alto. </li><li> Collegare il tubo, che si adatta perfettamente alla pipetta pipette e tubo per l&#39;ago (21 G 11 / 2) collegato al siringa da 1 ml, che è pieno di legante. </li><li> Ora, muovere con cautela il ligando riempito micro-pipetta alla fase microscopio e fissare su micro-manipolatori. </li><li> Applicare una pressione poco da siringa per fare ligando che si sta lentamente liberando dalla punta. </li><li> Portare il BMDCs placcato immaturo al microscopio e tenerli sul piatto riscaldato (37 ° C) palco. </li><li> Togliere il coperchio del piatto fluoro. </li><li> Concentrare le cellule utilizzando immersione 60X olio lente dell&#39;obiettivo. </li><li> Attenzione, far cadere il ligando caricata pipetta usando &#39;grossolana Manuale Manipolatore MN-188NE&#39; per la vicinanza delle cellule. </li><li> Messa a fuoco con la pipetta &#39;Micromanipolatore idraulico Belle MN-188 NE&#39;. </li><li> Applicare una pressione poco da siringa per assicurarsi che ligando sta rilasciando in piatto. </li><li> Impostare i parametri di acquisizione di immagini per l&#39;acquisizione in brillanti depositato (10 esposizione ms) in ogni 15 secondi, per 2 ore utilizzando il software Metamorph. </li><li> Continuare a monitorare ogni 30 min per progression di migrazione verso ligando. </li><li> Se le cellule sono affollate di punta della pipetta, modificare la posizione della punta della piastra per continuare la migrazione di esperimento. </li></ol> B. Misurazione GPCR (BLT1) e proteina citoplasmatica (β-arrestina) Interazioni, traslocazione in cellule vive 1. Preparazione di costrutti di DNA plasmidi I dettagli dei costrutti di DNA plasmidico sono state descritte in Jala et al. (2005) 18. 2. Trasfezione di Human BLT1-RFP e β-arrestina-GFP in cellule RBL-2H3 <ol><li> Mantenere la Rat basofili Leukomia (RBL-2H3) Cells (60 al 70% confluenza) a 37 ° C in atmosfera umidificata del 95% di aria, 5% di CO 2 come colture monostrato in terreni di coltura (DMEM integrato con 15% FBS, 2 mM L-glutammina, 100 U / ml penicillina e 100 mg / ml streptomicina) nel T75 fiaschi. </li><li> Staccare le cellule dal piatto / fiaschi utilizzando 6 mL di tripsina-EDTA (0,05% tripsina, 0,53 mM EDTA) ed incubare per 5 minuti a 37 ° C in atmosfera umidificata del 95% di aria, 5% di CO 2. Agitare delicatamente i flaconi di monitorare l&#39;entità del distacco delle cellule. </li><li> Aggiungere 6 ml di terreni di crescita a pallone contenente 6 ml di tripsina-EDTA e raccogliere le cellule mescolandoli pipettando su e giù più volte. Trasferire le cellule in 15 ml tubi falco. </li><li> Contare le cellule utilizzando un emocitometro e prendere 4 x 10 6 cellule in provette 15 ml centrifuga e centrifugare a 480g per 3 giri min e risospendere in 200 ml di mezzi di trasfezione (DMEM, 20% FBS, 50 HEPES mM). Se si prevede di effettuare 3 trasfezioni, preparare le cellule e dei media tranfection di conseguenza dal numero di cellulare e aumentando il volume del mezzo di trasfezione. </li><li> Preparare i plasmidi da transfettate alle concentrazioni di almeno 1,5 mg / mL. Prepariamo il DNA plasmidico utilizzando DNA Maxi QIAGEN kit plasmide. Aggiungere l&#39;individuo o sia la codifica plasmide DNA umano per BLT1-RFP (25 mcg) e β-arrestin1-GFP (15 mcg) per elettroforesi cuvette sterili, (Bio-Rad # 16552088), che ha distanza tra gli elettrodi 0,4 cm. </li><li> Prendete 200 ml di cellule sopra risospeso e metterli in provette contenenti sia hBLT1-RFP o β-arrestin1-GFP o entrambe insieme e mescolare delicatamente con pipetta da 1 ml sterile. </li><li> Lasciare la cuvette sopra a temperatura ambiente per 10 min. </li><li> Electroporate le cellule in Gene Pulser II con tensione a 250 V e capacità di 500 uF. </li><li> Dopo l&#39;elettroporazione permettere alle cellule rimanere a temperatura ambiente per 10 min. </li><li> Prendere 1 ml di mezzo di crescita regolare e mescolarlo con le cellule elettroporate nella cuvette. </li><li> Distribuire 300 ml di questa miscela in 35 millimetri piatti fondo fibra di vetro cultura (piatto Fluoro, strumenti di precisione Mondiale) contenente 2 ml di terreni di crescita regolare e incubare per 1 ora a 37 ° C in atmosfera umidificata del 95% di aria, 5% CO 2. Permettono alle cellule di aderire al fondo del piatto. </li><li> Sostituire i media dopo 1 ora di incubazione con i media una crescita regolare e permettere loro crescere a 37 ° C in atmosfera umidificata al 95% di aria, 5% CO 2 incubatore per 18-24 ore. </li></ol> 3. Cellulare Imaging in diretta Acquisizione di immagini <ol><li> Dopo 18-24 ore, lavare le cellule 2 o 3 volte con 2 ml di caldo rosso fenolo libero RPMI contenente 10 HEPES mM, pH 7,55 (Aggiungere 2 ml di mezzi di comunicazione calda direttamente alla piastra e media aspirare. Ripetere 2 o 3 volte). Aggiungere 1,8 ml di caldo rosso fenolo libero RPMI contenente 10 mM HEPES. </li><li> Posto 30 millimetri di vetro coprioggetto fondo piatti di coltura contenente RBL-2H3 cellule, transfettate con hBLT1-RFP e β-arrestin1-GFP sul palco microscopio riscaldata (37 ° C). </li><li> Raccogliere le immagini delle cellule con ingrandimento 600x con 60 X obiettivo Nikon Piano Apo 60X/1.4 numerica olio apertura dell&#39;obiettivo immersione (disponibile nel nostro sistema microscopio). </li><li> Mettere una goccia di olio sulla lente 60 x obiettivo e mettere a fuoco le cellule con regolare campo chiaro luce trasmessa toccando il fondo della piastra. </li><li> Per monitorare la fluorescenza delle proteine ​​transfettate, passare da campo luminoso (luce trasmessa) a fluorescenza filtro (RFP filtro, se volete vedere dei recettori o GFP, se volete vedere β-arrestin1). </li><li> Scegliere la cella luminoso e sano, che esprime hBLT1-RFP sulla superficie cellulare utilizzando RFP filtro e catturare l&#39;immagine. </li><li> Poi passare alla GFP filtro e fare in modo che β-arrestina-GFP espresso nel citoplasma e catturare l&#39;immagine. Poi il colore pseudo entrambe le immagini, prima che il tempo trascorso catturare immagini cellule vive per assicurarsi che sanguinano attraverso di fluorescenza non sta che si verificano tra GFP e RFP. </li><li> Una volta che la scelta del vostro cellulare fatto, impostare i parametri in base alle proprie finalità utilizzando il software (software Metamorph in questo caso). </li><li> Nella dimostrazione corrente, sedici bit sono le immagini acquisite con il sira impostata binning a 1 X 1 combinato con 60 Piano di X obiettivo Nikon Apo 60X/1.4 numerica olio apertura dell&#39;obiettivo immersione. </li><li> Raccogliere le immagini a fluorescenza per la richiesta di offerta e fluorocromi GFP simultaneamente a 30 sec intervallo di tempo (se le interazioni traslocazione / sono troppo veloce, si possono ridurre gli intervalli di tempo) di queste ruote con filtro controllato dal software Metamorph. </li><li> I tempi di esposizione della fotocamera impostata a 1000 msec per la GFP e RFP. (Impostare i tempi di esposizione specifici a seconda dell&#39;intensità di fluorescenza GFP e RFP). </li><li> In primo luogo il ritiro delle immagini per 1 minuto, senza l&#39;aggiunta di ligando, che serve come punto 0 tempo. </li><li> Aggiungere i 200 ml di legante (dal magazzino di 10 micron ad una concentrazione finale di 1 mM) dopo 1 min senza disturbare la piastra / o la posizione della cella. </li><li> Le immagini a fluorescenza sono raccolti per 60 minuti dopo l&#39;aggiunta del legante e memorizzati come immagini TIFF con ordine crescente dei nomi dei file. </li><li> Tutti questi file possono essere effettuate come file stack con immagini di fluorescenza singoli / combinati con data e ora utilizzando il software. </li></ol> Processo di immagini e quantificazione della fluorescenza di proteine ​​e localizzazione <ol><li> Acquisire le immagini con i media pianura (senza cellule) con filtri RFP e fluorescente GFP e salvarli come immagini di sfondo. Usare queste immagini per sottrarre le immagini reali dati ottenuti in precedenza. </li><li> Preparare la pila di immagini singolarmente per le immagini GFP e RFP. </li><li> Selezionare / definire le diverse regioni (superficie vs citosolico) per misurare l&#39;intensità di fluorescenza di GFP e RFP (BLT1 e β-arrestina traslocazione) </li><li> Tracciare la fluorescenza importo come la funzione del tempo. Questo grafico fornirà le informazioni relative cinetica di traslocazione di data molecola. </li></ol> Rappresentante Risultati A. Misurazione leucotrienici B 4 migrazione Regia cellule dendritiche Il metodo descritto in questo protocollo è stato utilizzato per determinare la migrazione delle cellule dendritiche verso leucotriene B 4 (Figura 1 e video allegato 1) 21. Siamo in grado di applicare la metodologia simile a determinare la capacità chemiotattica di cellule di un ligando particolare in cellule vive. <img src="/files/ftp_upload/2315/2315fig1.jpg" alt="Figura 1" /> Figura 1. Midollo osseo del mouse delle cellule dendritiche derivate migrazione verso il 100 nM LTB 4. Video 1. Dal vivo la migrazione delle cellule di BMDCs verso LTB 4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Boddulrui_Video1.AVI">Clicca qui per vedere il video</a> . B. Misurazione GPCR (BLT1) e proteina citoplasmatica (β-arrestina) Interazioni, traslocazione in cellule vive Al termine di questa procedura, si possono ottenere le seguenti informazioni in eventi GPCR segnalazione. <ol class="lroman"><li> La localizzazione dei GPCR e proteine ​​citosoliche e nucleo (Figura 2). </li><li> Interazione ligando indotta di superficie GPCR (BLT1 in questo caso) con la proteina citoplasmatica (β-arrestina in questo caso) (Fig. 3). </li><li> Cinetica di internalizzazione recettoriale e β-arrestina traslocazione alla membrana seguita da internalizzazione insieme con i recettori (Figura 4) (Jala et al. 2005) 18. </li><li> Si può determinare la dipendenza ligando stato di attivazione dei recettori in cellule vive (in allegato il video 2) e determinare motivi critici o processi coinvolti nella attivazione del recettore (Jala et al. 2005) 18. </li></ol><img src="/files/ftp_upload/2315/2315fig2.jpg" alt="Figura 2" /> Figura 2. Espressione di BLT1-RFP (A), β-arrestina-GFP (B), pNuc-CFP (C) in cellule RBL-2H3. Colore immagine combinata mostrato nel pannello D. <img src="/files/ftp_upload/2315/2315fig3.jpg" alt="Figura 3" /> Figura 3. Traslocazione ligando del recettore indotta e β-arrestina. Scansione linea di intensità fluorescente nelle cellule sono mostrati. <img src="/files/ftp_upload/2315/2315fig4.jpg" alt="Figura 4" /> Figura 4. Cinetica di internalizzazione recettoriale e β-arrestina traslocazione dopo l&#39;aggiunta di 1 mM LTB 4. L&#39;intensità di fluorescenza è stata misurata in funzione del tempo a membrana e luoghi citosolico della cellula. Video 2 immagini dal vivo di cellule che esprimono BLT1-RFP e β-arrestina-GFP sul aggiunta di 1 mM LTB 4. <a href="https://www.jove.com/files/ftp_upload/2315/Jala and Bodduluri_Video2.AVI">Clicca qui per guardare il video</a>

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H., Del Prete, A., Bock, C. B., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+leukotriene+B4+receptors+BLT1+and+BLT2:+a+critical+role+for+BLT1+in+collagen-induced+arthritis+in+mice.">Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice.</a> J Immunol. 176, 6254-6261 (2006).</li><li>Kim, N. D., Chou, R. C., Seung, E., Tager, A. M., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+requirement+for+the+leukotriene+B4+receptor+BLT1+for+neutrophil+recruitment+in+inflammatory+arthritis.">A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis.</a> J Exp Med. 203, 829-835 (2006).</li><li>Jala, V. R., Shao, W. H., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-independent+beta-arrestin+translocation+and+internalization+of+leukotriene+B4+receptors.">Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors.</a> J Biol Chem. 280, 4880-4887 (2005).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+G+protein-coupled+receptor+signaling+in+live+cells.">Real-time analysis of G protein-coupled receptor signaling in live cells.</a> Methods Mol Biol. 332, 159-165 (2006).</li><li>Del Prete, A., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+and+adaptive+immune+response+by+leukotriene+B4+receptors:+a+role+for+LTB4+in+up-regulation+of+CCR7+expression+and+function.">Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function.</a> Blood. 109, 626-631 (2007).</li><li>Salogni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+A+induces+dendritic+cell+migration+through+the+polarized+release+of+CXC+chemokine+ligands+12+and+14.">Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.</a> Blood. 113, 5848-5856 (2009).</li><li>Boudreau, J., Koshy, S., Cummings, D., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+myeloid+dendritic+cells+from+bone+marrow+precursors.">Culture of myeloid dendritic cells from bone marrow precursors.</a> J Vis Exp. , (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;imaging cellulare Live è un potente strumento per dimostrare la funzione e le interazioni delle proteine ​​specifiche che si verificano in tempo reale. I metodi descritti in questo manoscritto mostrano chiaramente che LTB 4 può provocare una rapida migrazione delle cellule dendritiche. Questi metodi non solo ampliare gli aspetti della funzione di LTB 4 tipi di cellule diverse, consentono metodi simili da applicare ad una varietà di altre chemochine e prova la loro efficacia come agenti...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

Recettori accoppiati alla proteina G (GPCR) appartengono alla famiglia delle sette proteine ​​transmembrana e mediare la trasduzione di segnali extracellulari alle risposte intracellulari. GPCR controllo diverse funzioni biologiche come la chemiotassi, rilascio del calcio intracellulare, regolazione genica in modo ligando dipendente tramite eterotrimeriche proteine ​​G 1-2. Di legame induce una serie di cambiamenti conformazionali che portano all&#39;attivazione di eterotrimeriche G-proteine ​​che modulano i livelli di secondi messaggeri come adenosina monofosfato ciclico (cAMP), inositolo trifosfato (IP3) e glicerolo diacyl (DG). In concomitanza con l&#39;attivazione del legame con il ligando del recettore avvia anche una serie di eventi per attenuare il recettore di segnalazione tramite desensibilizzazione, sequestro e / o internalizzazione. Il processo di desensibilizzazione dei GPCR avviene attraverso la fosforilazione del recettore per G-proteina chinasi del recettore (GRKs) e vincolante successiva β-arrestine 3. β-arrestine sono proteine ​​citosoliche e traslocare a membrana su attivazione GPCR, legandosi ai recettori fosforilati (la maggior parte dei casi) ci internalizzazione del recettore, facilitando 4-6. Leucotriene B 4 (LTB 4) è un pro-infiammatorie molecola lipidica derivati ​​dal percorso di acido arachidonico e media tramite le sue azioni GPCR, LTB 4 recettore 1 (BLT1, un recettore ad alta affinità) e LTB 4 recettore 2 (BLT2, un recettore a bassa affinità ) 7-9. Il 4-BLT1 LTB percorso ha dimostrato di essere critici in diverse malattie infiammatorie tra cui, l&#39;asma, l&#39;artrite e l&#39;aterosclerosi 10-17. Nel documento descrive le metodologie sviluppate per il monitoraggio LTB4-indotto migrazione dei leucociti e le interazioni di BLT1 con β-arrestina e traslocazione dei recettori in cellule vive utilizzando tecniche di imaging microscopia 18-19. Il midollo osseo le cellule dendritiche derivate da C57BL / 6 topi sono state isolate e coltivate come descritto in precedenza 20-21. Queste cellule sono state testate dal vivo nei metodi di imaging cellulare per dimostrare LTB 4 migrazione cellulare indotta. Il BLT1 umano è stato taggati con rosso proteina fluorescente (BLT1-RFP) a C-terminale e β-arrestin1 taggati con proteina fluorescente verde (β-arr-GFP) e transfettate entrambi i plasmidi in Rat basofili Leukomia (RBL-2H3) linee cellulari 18-19. La cinetica di interazione tra queste proteine ​​e localizzazione sono stati monitorati utilizzando vivere videomicroscopia cellulare. Le metodologie nel documento corrente descrivono l&#39;uso di tecniche microscopiche per studiare le risposte funzionali di G recettori accoppiati alle proteine ​​in cellule vive. Nel documento descrive inoltre l&#39;utilizzo di software Metamorph per quantificare l&#39;intensità di fluorescenza per determinare la cinetica dei recettori e le interazioni proteina citoplasmatica.

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with Î²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

Questo documento descrive la metodologia per determinare la risposta chemiotattica dei leucociti ai ligandi specifici e identificare le interazioni tra recettori di superficie della cellula e le proteine ​​citosoliche utilizzando vivere tecniche di imaging cellulare.

In tempo reale immagini di leucotriene B 4 Migrazione cellulare mediata e BLT1 Interazioni con β-arrestina

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

real-time-imaging-leukotriene-b4-mediated-cell-migration-blt1

Research

JoVE Journal

Immunology and Infection

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

12.7K Views.  University of Louisville. Questo documento descrive la metodologia per determinare la risposta chemiotattica dei leucociti ai ligandi specifici e identificare le interazioni tra recettori di superficie della cellula e le proteine ​​citosoliche utilizzando vivere tecniche di imaging cellulare.

Video: Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Tempo reale Imaging in diretta di cellule T Signaling formazione del complesso

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Ricerca

Immunologia e infezioni

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoraggio migrazione delle cellule dendritiche usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

In tempo reale di immagine degli eterotipica piastrine-neutrofili interazioni sull&#39;endotelio Attivato durante l&#39;infiammazione vascolare e la formazione di trombi in topi vivi

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

In tempo reale Imaging di cellule mieloidi Dynamics in<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinale tumori da Spinning Disk Microscopia confocale

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

Imaging in tempo reale e quantificazione dello sviluppo di Biofilm fungine utilizza un sistema bifase di flusso a ricircolo

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

Modello di malattia microfluidico in vitro per studiare le interazioni sangue-endoteliali intere e le dinamiche del coagulo di sangue in tempo reale

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Monitoraggio della sopravvivenza del virus influenzale al di fuori dell'ospite utilizzando l'analisi cellulare in tempo reale

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Quantificazione in tempo reale delle specie reattive dell'ossigeno nei neutrofili infettati da Escherichia Coli meningitica

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...