Le linee di cellule della prostata sono gentilmente forniti dal Dr. Ling-Yu Wang e Dr. Hsing-Jien Kung al Cancer Center, UC Davis. Questo progetto è sostenuto da NIH concedere galvanotaxis 4R33AI080604.

1. Le celle Culturing prostata <ol><li> Cultura dei PRN-1-1 e pnt2 prostata cellule in 100 piatti mm di coltura in RPMI 1640 supplementato con 10% FBS e antibiotici contro funghi a 37 ° C con il 5% di CO 2. Aggiornare il terreno di coltura ogni giorno fino a quando le cellule raggiungono 80% di confluenza per gli esperimenti galvanotaxis. </li></ol> 2. Montaggio Galvanotaxis Chambers <ol><li> Assemblaggio camere inferiori <ol><li> Pulire una camera galvanotaxis plastica con 2-propanolo. Applicare il sigillante silicio di qualità marine intorno alle aperture circolari sul lato inferiore di una camera con una siringa e attaccare un grande coprioggetti (Figura 1). Usare il lato posteriore di un applicatore di cotone per spingere verso il basso il coprioggetti e pulire la colla in eccesso con tamponi di cotone. Le aperture circolari consentono la corrente elettrica media e scorrano sulle cellule situate tra i due serbatoi medie interne. </li><li> Tagliare un piccolo coprioggetti per fare 6 mm x 25 millimetri distanziali con un pennarello a punta di diamante. </li><li> Capovolgere la camera. Applicare 2 strisce di colla silicio con una siringa e / o una spatola metallica per creare una superficie canale x 25 mm 10 mm per l&#39;adesione delle cellule tra i 2 aperture circolari sul fondo di vetro. Colla 2 pezzi di vetro distanziatore tra le due aperture circolari. Utilizzare il retro degli applicatori di cotone per spingere verso il basso i distanziali e pulire la colla in eccesso con tamponi e / o stuzzicadenti cotone. </li><li> Asciugare la camera per 24 ore fino a quando la colla di silicio è guarito. Mettere a bagno nella camera durante la notte acqua distillata per rimuovere il residuo di acido acetico da colla. Asciugare la camera per un uso immediato, o conservare la camera per un uso successivo in un contenitore pulito. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> Figura 1: Montaggio della camera di fondo galvanotaxis. A) Una grande coprioggetti è attaccato al fondo di una camera pulita. B) Two pezzi di distanziali vetro sono incollati tra le 2 aperture circolari per creare un canale 10 mm mm x 25 per le cellule di allegare. </li></ol></li><li> Semina cellule sulle camere galvanotaxis <ol><li> Preparare il numero richiedere di camere galvanotaxis necessari per l&#39;esperimento. Ogni linea o il trattamento di cellule richiede una sola camera galvanotaxis. Pulire le camere galvanotaxis con 2-propanolo. Lavare le camere sterili con PBS 3 volte e controllare il flusso di liquido tra le due aperture circolari nelle camere. </li><li> Racchiudere le camere sterili in piatti di coltura di cellule e permettere loro di equilibrare a 37 ° C per 15 min. Staccare le cellule della prostata dal loro piatto di coltura utilizzando 5 ml 0,25% tripsina-EDTA a 37 ° C per 5 minuti. Neutralizzare il tripsina-EDTA con 5 ml di 10% FBS in PBS. </li><li> Trasferire le cellule a 15 ml provette e centrifugare le cellule per 5 minuti a 200 xg, 37 ° C. Aspirare il supernatante e risospendere le cellule in 1 ml di terreno di coltura. </li><li> Prendere 20 μl di soluzione di cellule per caricare ad una camera di conteggio e calcolare il numero di cellule. Regolare la concentrazione cellulare di 8 x 10 4 cellule / ml con terreno di coltura. </li><li> Prendete le camere galvanotaxis dalla C incubatore 37 °. Seed 350 ml di sospensione cellulare su ciascuna camera. </li><li> Incubare le cellule nelle camere di notte nel piatto cultura con Kimwipe bagnato o in una camera umida in incubatore a 37 ° C con 5% di CO 2. </li></ol></li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> Figura 2:. Semina le cellule alla camera La camera inferiore viene asciugato e pulito cellule A) della prostata vengono tripsinizzate, contate e trasferiti alla camera e incubate overnight B) Un coprioggetti superiore è collegata a sigillare la camera prima di girare... <ol start="3"><li> Lassembling le camere superiori <ol><li> Montare la camera superiore prima delle riprese. Il microscopio può ospitare l&#39;imaging un&#39;unica camera galvanotaxis contemporaneamente. Warm up 10 ml di terreno di coltura a 37 ° C per ogni camera galvanotaxis. Trasferire una camera galvanotaxis dalle incubatrici ad una piastra 37 ° C il riscaldamento. </li><li> Lavare le cellule con terreno di coltura per rimuovere le cellule separati. Lasciare 400 microlitri di media nella camera. Utilizzare una siringa per applicare grasso alto vuoto sulla parte superiore di entrambi i distanziali vetro. </li><li> Aggiungere una piccola coprioggetti per sigillare la camera. Premere delicatamente verso il basso il coprioggetti con il lato posteriore di un applicatore di cotone. Pulire l&#39;eccesso di terreno con tamponi di cotone. </li><li> Asciugare la superficie di vetro e applicare il grasso alto vuoto per sigillare il divario tra il coprioggetti e la camera. Utilizzare una spatola metallica per diffondere il grasso. Aggiungere 4 ml del terreno di coltura per i serbatoi interni e controllare il flusso di liquido tra i serbatoi. </li></ol></li><li> Fai agarponti <ol><li> Tagliare un paio di tubi lungo PVC da 2 pollici (503/16 ID x 5/16 OD x 1/16 Muro), capovolgere e inserirli in un bicchiere da 100 ml. </li><li> Misura 200 mg Bacto-agar e aggiungere in un pallone da 50 ml con 10 ml di terreno di coltura per fare 2% gel di agar. Forno a microonde per 30 secondi. Caricare il gel di agar per il tubo di plastica con una pipetta di trasferimento. Lasciare i ponti agar a temperatura ambiente per 10 minuti per solidificare l&#39;agar. </li><li> Aggiungere 2 ml di terreno di coltura per i serbatoi esterni della camera galvanotaxis. Inserire i ponti agar al mezzo interno ed esterno serbatoi per condurre la corrente. </li></ol></li></ol><img alt="Figura 3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> Figura 3:. Filmare le celle della camera Un diagramma per dimostrare l&#39;assemblaggio finale della camera galvanotaxis. La camera è completamente sigillata con grasso alto vuoto. Medium è aggiunto to riempire i serbatoi, e due ponti agar vengono inseriti nella camera. Poi la camera galvanotaxis viene trasferito alla fase microscopio e gli elettrodi sono attaccati per applicare il campo elettrico. Vista laterale della camera galvanotaxis assemblato è mostrato per dimostrare che la corrente elettrica scorre sulle cellule attraverso i ponti agar e lo spazio tra il vetro di copertura. 3. Time-lapse imaging <ol><li> Accendere la camera ambientale a 37 ° C con 5% di CO 2 in aria circolante 2 ore prima di imaging. </li><li> Accendere il microscopio e avviare il software di immagine acquisizione. Calibrare palco microscopio e selezionare l&#39;obiettivo 10X. </li><li> Trasferire le camere galvanotaxis al palco microscopio, fissare la camera con nastro adesivo e concentrarsi sulle cellule. Gli elettrodi galvanotaxis ai serbatoi esterni con catodo sul lato destro. Fissare il filo e gli elettrodi con nastro adesivo. </li><li> Accendere il bo poterex per applicare un campo elettrico alla camera. Misurare la tensione di uscita con il voltmetro attraverso la camera per raggiungere 2,5 volt per la camera lunga 25 mm (100 mV / mm). Mantenere l&#39;intensità di campo durante l&#39;esperimento regolando la corrente di uscita. </li><li> Selezionare 10 punti in tutta la camera per filmare nel software per generare dieci film time-lapse. Impostare le condizioni di acquisizione a intervalli di 10 min per 2 ore. </li><li> Inizio riprese e regolare la corrente di uscita, se necessario. </li><li> Alla fine dell&#39;esperimento, rimuovere la camera dalla fase e fissare le cellule con 95% di alcol. Rompere aperto la camera con una lama di rasoio per pulire e ri-utilizzarlo. </li></ol> 4. Quantificazione di Galvanotaxis <ol><li> Ruotare i filmati time-lapse e ri-orientare il catodo alla parte superiore delle immagini. Esportare i film per il software di monitoraggio delle cellule. </li><li> Monitorare manualmente il (x, y) posizione 10-20 cellule in ciascun time-point da ogni film (Figura 4 A, B).Le distanze di migrazione e gli angoli rispetto alla direzione nord-sud, lo stesso orientamento del campo elettrico, vengono calcolati nel software di monitoraggio cella (Figura 4C). NOTA: La velocità media di migrazione viene convertito dalla distanza di migrazione totale e tempo totale riprese. La direzionalità viene convertito da angoli al valore del coseno. Se le cellule migrano con direzionalità a caso, la media del coseno netto sarà vicino allo zero. Se le cellule migrano direttamente verso il catodo, il valore del coseno sarà +1. Se le cellule migrano direttamente verso l&#39;anodo, il valore del coseno sarà -1. </li></ol><img alt="Figura 4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> Figura 4: Inseguimento cellulare per misurare direzionalità A) Sovrapposizione delle linee di tracciamento con immagini di cellule.. I (x, y) posizioni delle cellule sono Manually rintracciato nei film time-lapse. . Se le cellule migrano caso, il coseno media è vicina a zero B) Tuttavia, se le cellule migrano verso il catodo e l&#39;anodo, il valore del coseno media è vicino a + (catodo) o -. (Anodo) 1.0 C ) La direzionalità è presentato dal valore del coseno, che viene convertito da angoli di migrazione (θ). Il coseno (θ) è uguale al rapporto tra la distanza a (la distanza di migrazione) della distanza b (la distanza proiettata alla direzione del campo elettrico). <ol start="3"><li> Salvare le misurazioni. Importare i dati di un&#39;applicazione database per calcolare i risultati combinati. </li><li> Esportare i dati combinati di velocità media di migrazione e il valore medio del coseno di un foglio di calcolo per tracciare i grafici a barre (Figura 5). </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=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

The authors have nothing to disclose.

L&#39;analisi della risposta galvanotaxis di una cellula è stato un indicatore funzionale importante per molti migratori o crescita cellulare elabora 27, 28. Qui usiamo una camera di misura con superficie di vetro per filmare due linee di cellule della prostata. Queste linee cellulari hanno dimostrato diversi gradi di galvanotaxis, e ipotizzano che la localizzazione intracellulare o l&#39;attivazione delle proteine ​​galvanotaxis mediare potrebbero creare interferenze durante il processo di generazione d...

Campi elettrici endogeni vengono rilevati in vari tessuti, come la pelle 1, 32, 33 e il cervello 2. Il campo elettrico fisiologica serve funzioni biologiche specifiche, tra dirigere sviluppo embrionale 3, 4, guidando la conseguenza di processi neuronali 5, 6 e promuovere cicatrizzazione corneale epiteliale e 1, 7. In vitro, l&#39;applicazione di un campo elettrico in corrente continua a cellule coltivate imita il campo elettrico fisiologico e induce migrazione cellulare direzionale, o galvanotaxis. Galvanotaxis è stato studiato in fibroblasti, cheratinociti 8 9 pesci, epiteliale umano e cheratinociti corneali 10-12, linfociti 13, neuroblasti 2 e cellule progenitrici neuronali 14. Se esposto al campo applicato, la maggioranza delle cellule studiate migrare direzionalmente verso il catodica (-) poli. Tuttavia, diverse cellule tumorali, tra cui altamente metastaticole cellule del cancro al seno umano e della prostata umano linea di cellule di cancro PC-3M, passare al anodica (+) Polo 15, 16. Diversi meccanismi sono proposti a mediare galvanotaxis o per spiegare la capacità delle cellule di percepire il campo elettrico, tra cui l&#39;attivazione recettori di EGF 12, il canale del sodio epiteliale 17, PI3K e PTEN 18, e il rilascio di ioni calcio 15, 19. Il meccanismo non è ancora completamente compresa ed è possibile che più vie di segnalazione sono coinvolti in galvanotaxis. Il metodo galvanotaxis 2-dimensionale dimostriamo qui è utile per caratterizzare la migrazione direzionale aderenti, cellule mobili, sia per monitorare la migrazione singola cella 10, 12, 17 o migrazione di un foglio di cellule confluenti 18, 20. Questa tecnica è modificato dal Peng e Jaffe 21, e Nishimura et al. 10 con, camere di PVC trasparente su misura, con coversl rimovibileips consentendo il recupero delle cellule facile dopo galvanotaxis per l&#39;analisi secondaria, come l&#39;imaging immuno-fluorescenza. La superficie di vetro delle camere galvanotaxis è ottica compatibile, che consente le riprese ad alto ingrandimento e con cellule fluorescenza marcata. Inoltre permette il disegno sperimentale con modifica della superficie di vetro, ad esempio modificando il rivestimento o di superficie. Distanziatori realizzati No. 1 coprioggetti sono utilizzati nelle camere per minimizzare il flusso di corrente sulle celle; quindi il riscaldamento joule, che è proporzionale al quadrato del flusso di corrente, non surriscaldare le cellule durante l&#39;esperimento. I ponti di collegamento agar impediscono il contatto diretto degli elettrodi con le cellule e prevenire cambiamenti del pH medio o concentrazione di ioni durante galvanotaxis. Due linee cellulari di prostata umana non-cancerogeni sono stati esaminati per la loro risposta galvanotaxis in questo studio. I PRN-1-1 22 e 23 sono entrambi pnt2 SV40 immortalizzate, linee cellulari fattore dipendente dalla crescita esprimono marcatori epiteliali citocheratina 5, 8, 18 e 19 con bassa o nessuna espressione di antigene-specifica della prostata (PSA). Entrambe le linee cellulari mantengono la morfologia poligonale di cellule epiteliali normali, ma cromosoma anomalia è stata osservata in cariotipo 22, 24. Sebbene PRN-1-1 e pnt2 condividono comportamenti simili nella maggior parte degli esperimenti, fanno mostrano differenze nella formazione della struttura e in acinose galvanotaxis. Su una matrice 3-D, Matrigel, i PRN-1-1 cellule formano strutture acinose cave con lumen che assomiglia alla prostata normale della ghiandola tessuto 25. Tuttavia, le cellule pnt2 formano sferoidi solidi senza lumen o polarizzata dell&#39;epitelio 26. Le cellule PRN-1-1 dimostrano anche una risposta galvanotactic superiore alla pnt2 in questo studio. La correlazione tra la formazione della struttura acinar e galvanotaxis in PRN-1-1 suggerisce che i segnali galvanotactic possono svolgere un ruolo nell&#39;organizzazione della prmovimenti tessuto ghiandolare ostate in risposta a campi elettrici endogeni, e fornisce ulteriori caratteristiche di discriminare tra queste linee cellulari 2.

<table border="0" cellpadding="0" cellspacing="0" width="774">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pRNS-1-1 prostate cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>Lee et. al., (1994)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNT2 prostate cells</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">95012613-1VL</td>
			<td>Berthon et al., (1995)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium and solutions</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">11875-093</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premium</td>
			<td style="width:117px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% in PBS, warm up to 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Antibiotic-Antimycotic (100x)</td>
			<td style="width:117px;">Life Technologies</td>
			<td style="width:119px;">15240</td>
			<td>add 5mL to 500mL medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-propanol</td>
			<td style="width:117px;">VWR</td>
			<td>BDH1133-5GL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:117px;">-</td>
			<td style="width:119px;">-</td>
			<td>137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4 and 1.5mM KH2PO4 in 1000mL of H2O, pH to 7.4 and autoclaved, warm up to 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td>warm up to 37&#176;C before use, treat cells for 3-5 min at 37&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galvanotaxis device</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis chambers</td>
			<td style="width:117px;">Precision Plastics Inc, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed (1/4&#34; X 2&#34; X 3.5&#34;), non-toxic, clear PVC chambers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis electrodes</td>
			<td style="width:117px;">UCD electric shop</td>
			<td style="width:119px;"></td>
			<td>platinum coiled electrodes with flexable cords</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis power box</td>
			<td style="width:117px;">Substrate Engineering, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed DC power output with voltmeter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, Large, 45X50mm-No. 1.5</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">12-544-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, small, 25X25mm, No. 1</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">3307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond point marker</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Marine grade silicon sealer, clear</td>
			<td style="width:117px;">3M</td>
			<td style="width:119px;">051135-08019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease</td>
			<td style="width:117px;">Dow Corning</td>
			<td style="width:119px;">2021846-0807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6mL syringe</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">05-561-64</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nichiryo Syringe, 1.5mL</td>
			<td style="width:117px;">Nichiryo</td>
			<td style="width:119px;">SG-M</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton applicators</td>
			<td style="width:117px;">Purtian Medical Products</td>
			<td style="width:119px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qtips</td>
			<td style="width:117px;">Johnson &#38; Johnson</td>
			<td style="width:119px;">729389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene 180 PVC tubing&#160;</td>
			<td style="width:117px;">Nalgene</td>
			<td style="width:119px;">8000-9030</td>
			<td>503/16 ID x 5/16 OD x 1/16 Wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-Agar</td>
			<td style="width:117px;">Difco</td>
			<td style="width:119px;">0140-01</td>
			<td>make 2% agar solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Razor Blade</td>
			<td style="width:117px;">Personna</td>
			<td style="width:119px;">74-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipments and Software</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop Centrifuge</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td>operated with an A-4-62 rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer Auto T4</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">Auto T4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellometer counting chambers</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">CHT4-SD100-002</td>
			<td>load 20uL cell solutions to count</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Culture Temp Warming plate</td>
			<td style="width:117px;">Bel-Art Scienceware</td>
			<td style="width:119px;">370150000</td>
			<td>to keep the galvanotaxis chambers at 37&#176;C&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eclipse TE-2000 microscope with motorized stage and environmental chamber</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plan Fluor 10X/0.30 objective len</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retiga EX CCD camera&#160;</td>
			<td style="width:117px;">Qimaging</td>
			<td style="width:119px;"></td>
			<td>Cooled CCD camara, mono-color, 12-bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compressed air with 5% CO2&#160;</td>
			<td style="width:117px;">Airgas</td>
			<td style="width:119px;"></td>
			<td>special order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volocity 6.3</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Image acquiring software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Improvision OpenLab 5.5.2</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Cell tracking software and customized to measure migration angles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FileMaker Pro Advanced, 8.0</td>
			<td style="width:117px;">FileMaker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft Excel 2008 for Mac</td>
			<td style="width:117px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

Il campo elettrico fisiologico serve funzioni biologiche specifiche, come dirigere la migrazione delle cellule in fase di sviluppo embrionale, escrescenza neuronale e epiteliale guarigione della ferita. L&#39;applicazione di un campo elettrico in corrente continua a cellule coltivate in vitro induce migrazione cellulare direzionale, o galvanotaxis. Il metodo galvanotaxis 2-dimensionale dimostriamo qui viene modificato con poli misura (cloruro di vinile) (PVC) camere, superficie di vetro, elettrodi di platino e l&#39;uso di uno stadio motorizzato su cui vengono esposte le cellule. Le camere di PVC e elettrodi di platino presentano bassa citotossicità e sono accessibili e riutilizzabili. La superficie di vetro e la fase di microscopio motorizzato migliorare la qualità delle immagini e consentono possibili modifiche alla superficie di vetro e trattamenti alle cellule. Abbiamo girato la galvanotaxis di due linee di cellule tumorali non-SV40-immortalata prostata, PRN-1-1 e pnt2. Queste due linee cellulari mostrano velocità di migrazione simili ed entrambe migrano versoil catodo, ma lo fanno mostrano un diverso grado di direzionalità in galvanotaxis. I risultati ottenuti con questo protocollo suggerire che i PRN-1-1 e le linee cellulari pnt2 possono avere diverse caratteristiche intrinseche che regolano le loro risposte migratori direzionali.

Due linee di cellule della prostata (PRN-1-1 e pnt2) sono stati studiati con questo metodo. Cellule in entrambe le linee migrano a velocità simili di 1,0 +/- 0,3 micron / min nel corso di 2 ore (Figura 5A). Tuttavia, la direzionalità al campo elettrico è di 0,7 +/- 0,3 per l&#39;PRN-1-1 riga e 0,2 +/- 0,8 per la linea pnt2 (Figura 5B). I risultati mostrano una differenza significativa nella galvanotaxis di queste due linee cellulari (p &lt;0.01, 100 cellule sono state seguiti), sugge...

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Utilizzando a disegno Galvanotaxis Chambers per studiare la migrazione direzionale di cellule della prostata

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

utilizing-custom-designed-galvanotaxis-chambers-to-study-directional

Research

JoVE Journal

Biology

9.1K Views.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Video: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Una cellula-cellula Macromolecole saggio di trasporto In Planta Utilizzando Bombardamento Biolistic

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Analisi quantitativa della migrazione casuale di celle usando Time-lapse Microscopia Video

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparazione individuale Drosophila Egg Chambers per l&#39;imaging dal vivo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall&#39;intestino

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizzando Recettori pHluorin-tag per il monitoraggio subcellulare localizzazione e il traffico

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

I filamenti intermedi e microtubuli con microscopia di ricostruzione ottico stocastico diretto 2-dimensionale di imaging

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.