Name: utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells
Uploaded: 2014-12-07T14:00:00.000+00:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

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>

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R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imposition+of+a+physiologic+DC+electric+field+alters+the+migratory+response+of+human+keratinocytes+on+extracellular+matrix+molecules.">Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.</a> J Invest Dermatol. 106 (4), 642-646 (1996).</li><li>Feng, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+migration+of+neural+stem+cells+derived+from+human+embryonic+stem+cells+by+an+electric+field.">Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.</a> Stem Cells. 30 (2), 349-355 (2012).</li><li>Mukerjee, E. V., Isseroff, R. R., Nuccitelli, R., Collins, S. D., Smith, R. 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 di linee cellulari immortali, determinando la differenza osservata nella risposta galvanotaxis. Rispetto ad altri disegni da camera galvanotaxis realizzati in capsule di Petri 27, 28, ci sono diversi vantaggi per il design attuale. In primo luogo, la superficie del vetro è adatto per l&#39;imaging e la coprioggetti con le cellule possono essere recuperate dopo galvanotaxis per immunocolorazione. In questo studio abbiamo utilizzato un basso ingrandimento obiettivo 10X per monitorare un gruppo di celle. Tuttavia, con these ottica migliorata vetro, è possibile filmare una singola cella a più alto ingrandimento con un obiettivo di olio 60X, per tracciare l&#39;estensione veloce e retrazione lamellipodial all&#39;avanguardia delle celle 17, o inseguimento cellule marcate con proteine ​​fluorescenti. In secondo luogo, la superficie di vetro può essere modificato con rivestimenti proteici matrice 10, 12 o modellata con micro-canali. Trattamenti chimici sono possibile pre trattare le cellule prima di girare 29 anche. Rivestimento Matrix non è richiesto per le linee cellulari prostata usato qui, e il rivestimento matrice è dipendente dal tipo cellulare. Ad esempio, occorre un rivestimento collagene I per la galvanotaxis di cheratinociti umani 30, ma occorre un rivestimento laminina per la galvanotaxis delle cellule staminali neurali umane 31. Abbiamo testato galvanotaxis cheratinociti umani sul collagene I, collagene IV, fibronectina e laminina. Diverse proteine ​​della matrice alterate velocità di migrazione delle cellule, ma non hanno cambiato i migrati catodica<html> su in galvanotaxis 29. In terzo luogo, gli elettrodi di platino modificati sono inerti nel campo elettrico. Rispetto agli elettrodi di argento come usati in altri metodi galvanotaxis 27, essi non abbattere o rilasciano ioni metallici al mezzo, che riduce la citotossicità in galvanotaxis. Quarto, possiamo cambiare gli intervalli di telaio cattura, che ci aiuta a monitorare la migrazione delle cellule sia veloce o lento. Il microscopio motorizzato offre anche la possibilità di acquisire più filmati provenienti da aree diverse in una camera. Un esperimento di controllo senza il campo elettrico applicato è importante per determinare se il campo elettrico altera la velocità migratoria delle cellule. Tuttavia, le camere galvanotaxis attuali sono limitate alla migrazione cellulare riprese per 4 ore a causa del piccolo volume di terreno nei serbatoi. Con una versione ingrandita del disegno della camera, sarebbe possibile filmare cellule per un periodo di tempo più lungo. <html> ve_content &quot;&gt; Alcuni problemi potenziali nel metodo includono:. 1) le cellule potrebbero muoiono durante l&#39;assemblaggio della camera se diventano essiccato Poiché vi è un piccolo volume di terreno nella camera (400 microlitri), è importante evitare di intrappolare aria bolle nella camera e per mantenere un liquido che copre le cellule durante la fase di risciacquo cella. 2) La camera potrebbe fuoriuscire durante l&#39;imaging, che ridurrebbe la corrente che passa attraverso la camera. Il flusso di liquido nella camera deve essere attentamente controllati prima che sia trasferito al palco microscopio e grasso più alto vuoto applicato, se necessario. 3) e sbavature grasso per alto vuoto può interferire con le riprese. La superficie di vetro può essere pulito con il 95% di alcool per eliminare i grassi. 4) Fase di deriva o spostamento delle camere su una fase di microscopio motorizzato potrebbe falsare le misurazioni della migrazione direzionale. Pertanto, è importante proteggere le camere galvanotaxis con supporto fase adeguata e nastro etichettatura. S copi &quot;&gt; In generale, il metodo galvanotaxis è utile a rivelare la direzionalità intrinseca di cellule mobili in un campo elettrico applicato, che ci aiuterà a capire meglio la migrazione direzionale negli organismi.

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><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></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), suggerendo che hanno diversi meccanismi di segnalazione cellulare che provocano diverse risposte a stimoli direzionali posizionali. <img alt="Figura 5" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig5highres.jpg" width="500" /> Figura 5: Galvanotaxis delle cellule della prostata. A) Le velocità di migrazione di PRN-1-1 e linee pnt2 sono simili quando le cellule sono esposte al campo elettrico.B) Tuttavia, la linea PRN-1-1 mostrato una elevata direzionalità verso catodo (coseno = 0.7), mentre la linea pnt2 mostrato una bassa direzionalità (coseno = 0,2) nel campo elettrico.

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

Universidad San Sebastian

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

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Di campo elettrico controllato migrazione diretta di cellule progenitrici neurali in ambienti 2D e 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Ricerca

Bioingegneria

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Valutazione della migrazione delle cellule staminali del cancro mediante compartimentazione dispositivi microfluidici e Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicina

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Il comportamento migratorio di cellule generate in Culture Eminenza gangliari

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Neuroscienze

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Analisi Live Cell Imaging Studio Glioblastoma cerebrale tumore cellule staminali migrazione e invasione

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Ricerca sul cancro

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Analisi della migrazione cresta neurale cellulare Trunk con un Modified Zigmond Assay Camera

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

Studio della Migrazione cellulare in canali microfabbricati

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Un test Galvanotaxis Analisi della Cinetica precursori neurali migrazione cellulare in un campo applicato esternamente diretto Corrente elettrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Planar gradiente di diffusione del sistema per Indagare Chemiotassi in un 3D collagene Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Misura della chemiotassi cellulari con ECIS / Taxi

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Biologia

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Imaging G protein-coupled recettore-mediata chemiotassi ed i suoi eventi di segnalazione nelle cellule HL60 neutrofili-like

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...