Las líneas celulares de próstata se amablemente proporcionados por el Dr. Ling-Yu Wang y Dr. Hsing-Jien Kung en el Centro de Cáncer, la Universidad de California Davis. Este proyecto es apoyado por el NIH subvención galvanotaxia 4R33AI080604.

1. cultivar células de la próstata <ol><li> Cultura los PRN-1-1 y PNT2 próstata células en 100 mm cultura platos en medio RPMI 1640 suplementado con 10% de SFB y antibióticos Antimycotic a 37 ° C con 5% de CO 2. Actualizar el medio de cultivo cada día hasta que las células alcanzan 80% de confluencia para los experimentos galvanotaxia. </li></ol> 2. Composición galvanotaxia Cámaras <ol><li> Montaje de cámaras inferiores <ol><li> Limpie una cámara galvanotaxia plástico con 2-propanol. Aplicar el sellador de silicona de grado marino alrededor de las aberturas circulares en la parte inferior de una cámara con una jeringa y adjuntar una gran cubreobjetos (Figura 1). Use la parte de atrás de un aplicador de algodón para empujar hacia abajo el cubreobjetos y limpie el exceso de pegamento con hisopos de algodón. Las aberturas circulares permiten el medio y la corriente eléctrica fluya sobre las células situadas entre los dos depósitos medianas interiores. </li><li> Corte un pequeño cubreobjetos para hacer 6 mm x 25 espaciadores mm con un marcador de punta de diamante. </li><li> Voltear la cámara. Aplicar 2 rayas de la cola de silicio con una jeringa y / o una espátula de metal para crear un x 25 mm superficie del canal 10 mm para la fijación celular entre las 2 aberturas circulares en el cristal inferior. Pegue 2 piezas de vidrio separador entre las 2 aberturas circulares. Use la parte de atrás de los aplicadores de algodón para empujar hacia abajo los espaciadores y limpie el exceso de pegamento con hisopos y / o palillos de algodón. </li><li> Seque la cámara durante 24 horas hasta que se cure el pegamento de silicona. Remojar la cámara durante la noche en agua destilada para eliminar el residuo de ácido acético a partir de la cola. Seque la cámara para su uso inmediato, o almacenar la cámara para su uso posterior en un recipiente limpio. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> Figura 1: Montaje de la cámara de la parte inferior galvanotaxia. A) Un gran cubreobjetos está unido a la parte inferior de una cámara limpia. B) Two piezas de separadores de cristal se pegan entre las 2 aberturas circulares para crear una 10 mm x 25 mm para el canal de las células se unan. </li></ol></li><li> Siembra de las células en las cámaras galvanotaxia <ol><li> Preparar el número requerir de cámaras galvanotaxia necesarios para el experimento. Cada línea o el tratamiento de células requiere una única cámara galvanotaxia. Limpie las cámaras galvanotaxia con 2-propanol. Lave las cámaras con estériles PBS 3 veces y compruebe el flujo de líquido entre las 2 aberturas circulares en las cámaras. </li><li> Incluya las cámaras en placas de cultivo de células estériles y dejar que se equilibren a 37 ° C durante 15 minutos. Separar las células de la próstata a partir de su plato de cultivo utilizando 5 ml 0,25% de tripsina-EDTA a 37 ° C durante 5 min. Neutralizar la tripsina-EDTA con 5 ml 10% de FBS en PBS. </li><li> Transferir las células a tubos de 15 ml y centrifugar las células durante 5 min a 200 xg, 37 ° C. Aspirar el sobrenadante y volver a suspender las células en 1 ml de medio de cultivo. </li><li> Tome 20 μl de solución de células para cargar a una cámara de recuento y calcular el número de células. Ajustar la concentración de células a 8 x 10 4 células / ml con medio de cultivo. </li><li> Tome las cámaras galvanotaxia de los 37 ° C incubadora. Semillas 350 l de la suspensión celular sobre cada cámara. </li><li> Se incuban las células en las cámaras durante la noche en la placa de cultivo con Kimwipe mojado o en una cámara de humedad en la incubadora a 37 ° C con 5% de 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:. Siembra de las células a la cámara de la cámara inferior se seca y limpia las células A) de próstata se tripsinizaron, se contaron y se transfirieron a la cámara y se incubaron durante la noche B) un cubreobjetos superior está unido a sellar la cámara antes de la filmación... <ol start="3"><li> Lassembling las cámaras superiores <ol><li> Montar la cámara superior antes de la filmación. El microscopio sólo puede acomodar imágenes de una sola cámara galvanotaxia al mismo tiempo. Calentar 10 ml de medio de cultivo a 37 ° C para cada cámara galvanotaxia. Transferir una cámara galvanotaxia de las incubadoras a una placa de calentamiento 37 ° C. </li><li> Enjuagar las células con medio de cultivo para eliminar las células no unidas. Deja 400 l de medio en la cámara. Utilice una jeringa para aplicar grasa de alto vacío en la parte superior de ambos separadores de vidrio. </li><li> Añadir una pequeña cubreobjetos para sellar la cámara. Presione suavemente el cubreobjetos con el lado posterior de un aplicador de algodón. Limpie el medio exceso con hisopos de algodón. </li><li> Seque la superficie del vidrio y aplicar grasa de alto vacío para sellar la brecha entre el cubreobjetos y la cámara. Use una espátula de metal para extender la grasa. Añadir 4 ml de medio de cultivo para los depósitos internos y comprobar el flujo de líquido entre los embalses. </li></ol></li><li> Haga agarpuentes <ol><li> Corte un par de tubos de PVC de 2 pulgadas de largo (503/16 ID x 5.16 OD x 1/16 Wall), dar la vuelta e insertarlos en un vaso de 100 ml. </li><li> Mide 200 mg Bacto-Agar y agregarlo en un matraz de 50 ml con medio de cultivo de 10 ml de hacer 2% en gel de agar. Microondas durante 30 segundos. Cargue el gel de agar para el tubo de plástico con una pipeta de transferencia. Deje los puentes de agar a temperatura ambiente durante 10 min para solidificar el agar. </li><li> Añadir 2 ml de medio de cultivo a los depósitos exteriores de la cámara galvanotaxia. Inserte los puentes de agar a los embalses medianas interior y exterior para conducir la corriente. </li></ol></li></ol><img alt="Figura 3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> Figura 3:. El rodaje de las células en la cámara de un diagrama para demostrar el montaje final de la cámara de galvanotaxia. La cámara está completamente sellado con grasa de alto vacío. El medio se añadió to llenar los depósitos, y dos puentes de agar se insertan en la cámara. A continuación, la cámara de galvanotaxia se transfiere a la platina del microscopio y los electrodos están unidos a aplicar el campo eléctrico. Vista lateral de la cámara de galvanotaxia ensamblado se muestra para demostrar que la corriente eléctrica fluye sobre las células a través de los puentes de agar y el espacio entre el vidrio de cubierta. Imaging 3. Time-lapse <ol><li> Encender la cámara ambiental a 37 ° C con 5% de CO2 en el aire que circula 2 hr antes de la imagen. </li><li> Encienda el microscopio e iniciar el software de adquisición de imagen. Calibrar la platina del microscopio y seleccione el objetivo de 10X. </li><li> Transferir las cámaras galvanotaxia a la platina del microscopio, asegure la cámara con cinta y se centran en las células. Introducir los electrodos galvanotaxia a los depósitos exteriores con cátodo en el lado derecho. Fije el cable y los electrodos con cinta. </li><li> Encienda el bo poderx para aplicar un campo eléctrico a la cámara. Mida el voltaje de salida con el voltímetro a través de la cámara de alcanzar 2,5 voltios para la cámara de 25 mm de largo (100 mV / mm). Mantener la intensidad de campo durante el experimento mediante el ajuste de la corriente de salida. </li><li> Seleccione 10 puntos a través de la cámara para filmar en el software para generar diez películas a intervalos. Configurar las condiciones de adquisición a intervalos de 10 minutos durante 2 horas. </li><li> Iniciar el rodaje y ajustar la corriente de salida, según sea necesario. </li><li> Al final del experimento, retire la cámara desde el escenario y fijar las células con 95% de alcohol. Rompa la cámara con una hoja de afeitar para limpiar y volver a usarlo. </li></ol> 4. Cuantificación de Galvanotaxis <ol><li> Gire las películas a intervalos y reorientar el cátodo a la parte superior de las imágenes. Exportar las películas para el software de seguimiento de la célula. </li><li> Rastrear manualmente el (x, y), la posición de 10 a 20 células en cada punto de tiempo de cada película (Figura 4 A, B).Las distancias de migración y los ángulos relativos a la dirección norte-sur, la misma orientación del campo eléctrico, se calcularán en el software de seguimiento de la célula (Figura 4C). NOTA: La velocidad media de migración se convierte de la distancia de migración total y el tiempo total de rodaje. La direccionalidad se convierte de los ángulos en el valor del coseno. Si las células migran con direccionalidad al azar, el promedio de coseno neto será cercano a cero. Si las células migran directamente hacia el cátodo, el valor del coseno será 1. Si las células migran directamente hacia el ánodo, el valor del coseno será -1. </li></ol><img alt="Figura 4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> Figura 4: seguimiento de la célula para medir la direccionalidad A) Superposición de las líneas de seguimiento con imágenes de células.. Los (x, y) posiciones de las células son manually rastreado en las películas a intervalos. . Si las células migran al azar, el coseno promedio es cercano a cero B) Sin embargo, si las células migran hacia el cátodo o el ánodo, el valor del coseno media es de cerca de + (cátodo) o -. (Ánodo) 1,0 C ) La direccionalidad es presentado por el valor del coseno, que se convierte a partir de los ángulos de migración (θ). El coseno (θ) es igual a la relación de la distancia a (la distancia de migración) la distancia b (la distancia proyectada a la dirección del campo eléctrico). <ol start="3"><li> Guarde las mediciones. Importe los datos a una aplicación de base de datos para el cálculo de los resultados combinados. </li><li> Exportar los datos combinados de la velocidad media de migración y el valor promedio del coseno de una hoja de cálculo para trazar los gráficos de barras (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.

El análisis de la respuesta galvanotaxia de una célula ha sido un indicador funcional importante para muchos migratoria o crecimiento celular procesa 27, 28. Aquí se utiliza una cámara de medida con la superficie de vidrio a la película dos líneas celulares de próstata. Estas líneas celulares demostraron diferentes grados de galvanotaxia, y especulan que la localización intracelular o la activación de las proteínas galvanotaxia-mediación pueden ser interferidas durante el proceso de generación de...

Campos eléctricos endógenos se detectan en varios tejidos, como la piel 1, 32, 33 y el cerebro 2. El campo eléctrico fisiológica sirve funciones biológicas específicas, incluyendo la dirección de desarrollo del embrión 3, 4, guiando la consecuencia de los procesos neuronales 5, 6 y la promoción de cierre de la herida epitelial corneal y 1, 7. In vitro, la aplicación de un campo eléctrico de corriente directa a las células cultivadas imita el campo eléctrico fisiológica e induce la migración celular direccional, o galvanotaxia. Galvanotaxis se ha estudiado en fibroblastos, queratinocitos 8 de pescado 9, epitelial humano y queratinocitos corneales 10-12, linfocitos 13, neuroblastos 2, y células progenitoras neuronales 14. Cuando se expone al campo aplicado, la mayoría de las células estudiadas migran direccionalmente hacia el catódica polo (-). Sin embargo, varias células cancerosas, incluyendo altamente metastásicocélulas de cáncer de mama humano y la próstata humana línea celular de cáncer PC-3M, se mueven a la anódica polo (+) 15, 16. Se proponen varios mecanismos para mediar galvanotaxia o para explicar la capacidad de las células para detectar el campo eléctrico, incluyendo la activación de los receptores de EGF 12, el canal de sodio epitelial 17, PI3K y PTEN 18, y la liberación de iones de calcio 15, 19. El mecanismo no es todavía plenamente comprendida y es posible que múltiples vías de señalización están implicadas en galvanotaxia. El método galvanotaxia 2-dimensional se demuestra aquí es útil para caracterizar la migración direccional de adherente, células móviles, ya sea para controlar la migración de células individuales 10, 12, 17 o la migración de una hoja de células confluentes 18, 20. Esta técnica se modifica desde Peng y Jaffe 21 y Nishimura et al. 10 con, cámaras de PVC claras a medida, con coversl extraíbleips permitiendo la recuperación de células fácil después galvanotaxia para el análisis secundario, como las imágenes de inmunofluorescencia. La superficie de vidrio de las cámaras galvanotaxia es compatible con óptica, que permite la grabación a alta magnificación y con células marcadas con fluorescencia. También permite que el diseño experimental con la modificación de la superficie de vidrio, tales como cambiar el revestimiento de la superficie o cargos. Los espaciadores hechos de No. 1 cubreobjetos se utilizan en las cámaras para minimizar el flujo de corriente a través de las células; por lo tanto el calentamiento por efecto Joule, que es proporcional al cuadrado del flujo de corriente, no sobrecalentar las células durante el experimento. Los puentes de conexión de agar evitar el contacto directo de los electrodos con las células y prevenir el cambio del pH del medio o la concentración de iones durante galvanotaxia. Dos no tumorigénicas líneas celulares de próstata humano se examinaron por su respuesta galvanotaxia en este estudio. Los PRN-1-1 22 y PNT2 23 son ambos SV40 inmortalizadas, dependientes de factor de crecimiento de líneas celulares que expresan los marcadores epiteliales citoqueratina 5, 8, 18 y 19 con baja o ninguna expresión de la específica de antígeno de la próstata (PSA). Ambas líneas celulares mantienen la morfología poligonal de las células epiteliales normales, pero anomalía cromosómica se observó en el cariotipo 22, 24. Aunque PRN-1-1 y PNT2 comparten comportamientos similares en la mayoría de los experimentos, muestran diferencias en la formación de la estructura acinar y en galvanotaxia. En una matriz de 3-D, Matrigel, los PRN-1-1 células acinares formar estructuras huecas con lúmenes se asemejan a la tejido de la glándula próstata normal 25. Sin embargo, las células PNT2 formar esferoides sólidos sin un lumen o epitelio polarizado 26. Las células PRN-1-1 también demuestran una respuesta galvanotactic más alta que la PNT2 en el estudio actual. La correlación entre la formación de la estructura acinar y galvanotaxia en PRN-1-1 sugiere que las señales galvanotactic pueden jugar un papel en la organización de la prmovimientos de tejido de la glándula ostate en respuesta a campos eléctricos endógenos, y ofrece características adicionales para discriminar entre estas 2 líneas celulares.

<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

El campo eléctrico fisiológica sirve funciones biológicas específicas, tales como la dirección de la migración celular en el desarrollo del embrión, consecuencia neuronal y la curación de la herida epitelial. La aplicación de un campo eléctrico de corriente directa a las células cultivadas in vitro induce la migración celular direccional, o galvanotaxia. El método galvanotaxia 2-dimensional se demuestra aquí se modifica con cámaras de poli por encargo (cloruro de vinilo) (PVC), superficie de vidrio, electrodos de platino y el uso de una etapa motorizada en la que se crean imágenes de las células. Las cámaras de PVC y electrodos de platino presentan baja citotoxicidad y son asequibles y re-usable. La superficie de vidrio y la platina del microscopio motorizado mejorar la calidad de las imágenes y permiten posibles modificaciones a la superficie de vidrio y tratamientos para las células. Filmamos la galvanotaxia de dos líneas de células no tumorigénicas SV40 inmortalizado próstata, PRN-1-1 y PNT2. Estas dos líneas celulares muestran velocidades de migración similares y ambos migran haciael cátodo, pero muestran un grado diferente de direccionalidad en galvanotaxia. Los resultados obtenidos a través de este protocolo sugerir que los PRN-1.1 y las líneas celulares PNT2 pueden tener diferentes características intrínsecas que gobiernan sus respuestas migratorias direccionales.

Dos líneas de células de la próstata (PRN-1-1 y PNT2) se investigaron con este método. Las células en ambas líneas migran a velocidades similares de 1,0 +/- 0,3 micras / min durante el transcurso de 2 horas (figura 5A). Sin embargo, la direccionalidad al campo eléctrico es 0,7 +/- 0,3-PRN para la línea 1-1, y 0,2 +/- 0,8 para la línea PNT2 (Figura 5B). Los resultados muestran una diferencia significativa en la galvanotaxia de estas dos líneas celulares (p &lt;0,01, 100 célula...

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.

Utilizando diseño personalizado galvanotaxia Cámaras para Estudiar direccional migración de las células de la próstata

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 de Cartagena UDEC

Research

JoVE Journal

Biology

9.1K vistas.  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.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

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.

Análisis cuantitativo de la migración desordenada de las células mediante microscopía de Time-lapse vídeo

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.

Preparación Individual Drosophila Huevos Cámaras de Imágenes en directo

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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

El uso de células Caco-2 para el Estudio de lípidos Transporte por el 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.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

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.

La utilización de receptores de etiquetado pHluorin de Seguimiento de localización subcelular y el Tráfico

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

Filamentos intermedios y microtúbulos con microscopía de reconstrucción óptica estocástica directo 2-dimensional de imágenes

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.