Los autores desean agradecer a Glen Slocum (Colegio Médico de Wisconsin) y Colleen A. Lavin (Nikon Instruments Inc) para una excelente asistencia técnica con los experimentos de microscopía. Este estudio fue financiado por los Institutos Nacionales de Salud otorga R01 HL108880 (AS), R01 DK095029 (OPO) y K99 HL116603 (con TSP), la Fundación Nacional del Riñón IG1724 (con TSP), Asociación Americana del Corazón 13GRNT16220002 (OPO) y Ben J. Lipps de Becas de Investigación de la Sociedad Americana de Nefrología (DVI).

Los procedimientos experimentales se describen a continuación fueron aprobados por el Comité de Cuidado y Uso de Animales institucional en el Colegio Médico de Wisconsin y la Universidad de Texas Health Science Center en Houston y estaban de acuerdo con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio. La Figura 1 demuestra principales etapas del aislamiento de tejido y procedimiento de procesamiento. En pocas palabras, los riñones de ratas PCK o SD se utilizan para el aislamiento Manual de monocapas epiteliales de los conductos colectores, ya sea a partir de los túbulos o quistes no marcado saludables. Aquí estudiamos riñones de 4-16 semanas de edad ratas PCK 10,13. 1. Aislamiento del renales Quistes y Tubos de conexión (CNT) / conductos colectores (CD) Segmentos <ol><li> Anestesiar animal experimental con isoflurano (5% de inducción, 1,5 a 2,5% de mantenimiento) / O de grado médico 2 u otro método aprobado. Los animales deben ser monitoreados continuamente para asegurar le adecuadavel de la anestesia. Reacción tasa y puntera pizca respiratoria estable se utiliza para confirmar la anestesia adecuada. </li><li> Realizar laparotomía y lavar los riñones con tampón fosfato salino (PBS) a través de la aorta abdominal 17. <ol><li> Preparar un catéter tubo de polietileno (PE50) y conectarlo a una bomba de jeringa llena con PBS. Cortar la piel y la pared abdominal a lo largo de la línea alba, a continuación, hacer una incisión abdominal transversal a través del abdomen de lado a lado y cambiar los órganos abdominales con hisopos de algodón obtener acceso a la aorta descendente que con ramificación arterias mesentéricas y celíacos. Humedezca los órganos internos con solución salina caliente durante los procedimientos adicionales para evitar su sequedad. </li><li> Coloque una ligadura (# 1) alrededor de las arterias mesentéricas y celíacos y otra ligadura (# 2) alrededor de la aorta por debajo del diafragma (no atar). Separar suavemente a. abdominalis por embotar la disección de los tejidos conectivos alrededor de ella con unas pinzas finas y colocar dos ligaduras ~ 3mm por debajo de la arteria renal izquierda (# 3) y por encima de la bifurcación arterias ilíacas (# 4). </li><li> Ate ligadura # 4 y adjuntar una pinza recipiente alrededor de la aorta entre la arteria renal izquierda y ligadura # 3. Hacer una incisión entre la abrazadera y la ligadura # 4 de sondar la aorta, utilice ligadura # 3 para fijar firmemente el catéter. </li><li> Suelte la abrazadera y asegurarse de que el pulso de la sangre es visible en el catéter para asegurar una correcta instalación. Iniciar la perfusión a una velocidad de 6 ml / min, atar ligaduras # 1 y # 2, y se cortó la vena renal izquierda. Continuar enjuagando durante 1-2 minutos hasta que los órganos están completamente blanqueadas. </li><li> Cortar los vasos sanguíneos renales, la uretra y los tejidos conectivos circundantes y adiposo con unas tijeras para recoger los riñones. A continuación, hacer un corto desgarro en la cápsula renal y la cáscara de los riñones para decapsulate ellos. Coloca los riñones en helado de PBS. La eutanasia se confirma mediante una toracotomía. </li></ol></li><li> Preparar 5 x 5 mm chips de vidrio cubierta revestidas con poli-L-lisina. Corte un gla cubiertass con un lápiz de diamante y el lugar aproximadamente 20 l de solución de 0,01% esterilizada por filtración de poli-L-lisina en cada chip de vidrio. Preparar solución salina y dos pares de pinzas de relojero para la disección. </li><li> Cortar los riñones con una hoja de afeitar a lo largo del plano frontal en rodajas de ~ 1-2 mm de espesor. Coloque una de las rebanadas bajo un microscopio estereoscópico. Aislamiento de tejidos debe hacerse en solución salina enfriada con hielo. </li><li> Busque quistes bajo un microscopio estereoscópico como cavidades redondo (Figura 2); sus paredes contienen a menudo la red prominente de vasos sanguíneos proliferado. Utilizando unas pinzas de punta fina diseccionar la capa epitelial interna de un quiste tan delgada como sea posible para obtener una zona de monocapa. Adjuntar a un chip de vidrio cubierto con poli-L-lisina. Sticky superficie de poli-L-lisina permite al investigador para posicionar firmemente la capa interna del quiste en el vidrio. Exponer el quiste con la parte apical hasta facilitar el acceso de las pipetas (Figura 2B). </li><li> Utilice PCK o rata SD niñorebanadas centrales ney contienen papila para el aislamiento de no dilatadas CNT / CCDsegments 18-21. NOTA: los tejidos papilares son más duraderas que la corteza y mediante la celebración de bandas tubulares papilares con fórceps y desgarrando a lo largo de eje radial de la rebanada puede escindirse en sectores delgadas. Túbulos individuales son visibles y pueden ser sacados para ser colocados en los chips de vidrio cubierta. </li><li> Identificar los segmentos de CNT / CCD por bifurcaciones, una mayor transparencia de los túbulos proximales y grandes células prominentemente visibles (Figura 2C). Coloque el chip de vidrio cubierta con túbulos unidos bajo un microscopio equipado con micromanipuladores adecuados para micropipetas conducción. El uso de micropipetas afilados túbulos dividida abiertos y fije sus extremos a la copa para que la superficie apical accesible (Figura 2D). </li></ol> Electrofisiología 2. Single Channel Patch-clamp <ol><li> Llenar la cámara de patch-clamp con solución de baño y transferir el chip de vidrio cubiertacon tejidos aislados a la cámara. Asegúrese de que las micropipetas patch-clamp tienen una resistencia de 7.10 mO para mediciones (células adjunta) fiables sobre las células. </li><li> Para las mediciones de células adjunta, establecer la relación de ganancia del amplificador de 20x y de paso bajo las corrientes de 300 Hz por un filtro de ocho polos de Bessel. Para el monitoreo canales CENa, utilice una solución de baño, en mm: 150 NaCl, CaCl2 1, 2 MgCl2, 10 HEPES (pH 7,4); pipeta: 140 LiCl, 2 de MgCl 2 y 10 HEPES (pH 7,4). </li><li> Llevar a cabo un experimento de patch-clamp convencional en un modo celular conectado a 10. Seleccione una celda en la monocapa epitelial y acercarse a la pipeta a la membrana apical. Forma un sello de alta resistencia entre una pipeta y la membrana celular mediante la aplicación de una suave succión. Una vez que se forma un sello de alta GigaOhm resistentes, un protocolo sin huecos en un potencial de mantenimiento se debe iniciar la actividad de seguimiento del canal de interés. </li><li> Datos actuales tienda brecha gratis solo canal de gigaSellos ohmios para su posterior análisis. Analizar los eventos de canal utilizando un paquete de software en general, se suministra con configuraciones de patch-clamp 18. Calcular la actividad del canal como NPO donde N se refiere al número de canales activos en el parche y Po es probabilidad de apertura promedio de los canales. NOTA: Utilice quistes o túbulos aislados en experimentos de patch-clamp para no más de 30 min. </li></ol> 3. Ratiometric epifluorescencia Las mediciones de concentración de calcio intracelular en las células epiteliales <ol><li> Utilice 5 mM Fura-2AM disuelto en DMSO. Solución Alícuota caldo en tubos cónicos de 500 l individuales (aproximadamente 10 l). Proteger de la luz y guardar en el congelador a -20 ° C durante un máximo de seis meses. </li><li> Incubar quistes aislados y túbulos reventados por 30-40 min en PSS (en mM: 145 NaCl, 4,5 KCl, MgCl2 2, 2 CaCl2 y 10 HEPES a pH 7,35) que contiene 5 mM Fura-02 a.m. colorante y 0,05% de ácido plurónico para ayudar a dispersar los ésteres de acetoximetilo. Para eso, lugar tejidos en el plato de 3,5 cm que contenía cóctel de carga, protegerlo de la luz y se incuba en un agitador lento a temperatura ambiente. </li><li> Cambie Fura-02 a.m. contiene medios para limpiar PSS después de la incubación y colocar el tejido bajo un microscopio para realizar más imágenes de epifluorescencia. </li><li> A su vez en la cámara CCD y la fuente de luz estable (sistema monocromador) equipado con rueda de filtros (filtro de cada posición puede asociarse con su propio nivel de atenuación, seleccionado cada vez que el filtro se llama). </li><li> Encuentra el tejido en campo claro. Cambie a la detección de la señal de fluorescencia y ajustar la intensidad de la fuente de luz utilizando filtros de densidad neutra y potencia de la lámpara para evitar la saturación de la señal. </li><li> Monitorear Fura-2 AM fluorescencia en la muestra de tejido con excitación radiométrica a 340/380 nm con una frecuencia de 0.125 Hz o superior. Use un 40 × / NC 1.3 o similar lente objetivo para resolu adecuadala imagen. </li><li> Para el procesamiento de imágenes y los cálculos importar la secuencia de imágenes. Asegúrese de dividir los canales y utilizar un modo de escala de grises HyperStack. Seleccione varias regiones de interés (células quiste único o áreas seleccionadas de tejido quística) y calcular valores de intensidad para cada canal (340 y 380 nm) en el software de análisis de datos preferido; valores de intensidad fondo restar de cada punto de datos. </li><li> Para cada punto de tiempo de calcular la relación de intensidades de las Fura-2 AM 340 a 380 canales. Gráfico de dispersión / línea cambios en tiempo punto de Ca 2+ transitorio para cada región y calcular los valores medios / SE. </li></ol>

The authors have nothing to disclose.

Describimos aquí las aplicaciones de la técnica de patch-clamp convencional y imágenes de calcio epifluorescencia a monocapas epiteliales quísticas derivadas de un modelo genético murino de ARPKD. El protocolo consiste en tres pasos, de los cuales se debe prestar la mayor atención al aislamiento de los quistes (paso 1.5 de la sección de protocolos) y para los estudios electrofisiológicos. Estos procedimientos clave requieren una amplia formación y la paciencia, y el lector no debe ser frustrada a la vez. <p...

Los canales iónicos juegan un papel importante en muchas funciones fisiológicas, incluyendo el crecimiento y la diferenciación celular. Enfermedades renales autosómicos dominantes y recesivos poliquísticos (PQRAD y ARPKD, respectivamente) son trastornos genéticos caracterizados por el desarrollo de quistes llenos de líquido renales de origen de las células epiteliales tubulares. PQRAD está causada por mutaciones de los genes PKD1 o PKD2 codifican poliquistinas 1 y 2, las proteínas de membrana que participan en la regulación de la proliferación y diferenciación celular. PKD2 por sí mismo o como un complejo con PKD1 también funcionan como un canal catiónico -permeable Ca 2 + 1. Las mutaciones del gen que codifica PKHD1 fibrocystin (una proteína similar al receptor de los cilios asociada a involucrados en el tubulogenesis y / o mantenimiento de la polaridad del epitelio) son el impulso genético de ARPKD 2. Crecimiento quiste es un fenómeno complejo acompañada con la proliferación perturbado 3,4, la angiogénesis 5, la desdiferenciación y pérdida de polardad de las células tubulares 6-8. Reabsorción defectuosa y secreción aumentada en el epitelio quístico contribuyen a la acumulación de líquido en el lumen y el quiste de expansión 9,10. Flujo dependiente Deterioro [Ca 2 +] i de señalización también se ha vinculado a la quistogénesis durante PKD 11-15. Aquí se describe un método adecuado para mediciones de patch-clamp de la actividad de un solo canal e intracelulares de Ca 2+ niveles en monocapas epiteliales quísticas aisladas de ratas PCK. Este método fue aplicado con éxito por nosotros para caracterizar la actividad del canal epitelial de Na + (ENAC) 10 y la [Ca2 +] i dependiente de procesos inducidos por Ca2 + TRPV4 -permeable y la cascada de señalización purinérgico 13. En estos estudios se utilizaron ratas PCK, un modelo de ARPKD causada por una mutación espontánea en el gen PKHD1. La cepa PCK fue originally derivados de las ratas 16 ratas de ese modo SD se utilizan como un control apropiado para la comparación con la cepa Sprague-Dawley PCK (SD). Como resultado, ambos segmentos de la nefrona de ratas SD y conductos no dilatado recogida aisladas de ratas PCK mismos pueden servir como dos grupos de comparación diferentes para experimentos en el epitelio quística.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fura-2 AM</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:183px;">F-14185</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Flou-8</td>
			<td style="width:219px;">AAT Bioquest</td>
			<td style="width:183px;">21091</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Poly-L-lysine</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:183px;">P4707</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Pluronic acid</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:183px;">F-68&#160;</td>
			<td style="width:333px;">solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Shaker</td>
			<td style="width:219px;">Boekel Scientific</td>
			<td style="width:183px;">260350</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Light source</td>
			<td style="width:219px;">Sutter Instrument Co</td>
			<td style="width:183px;">Lambda XL</td>
			<td style="width:333px;">with integrated shutter/filter wheel driver</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Neutral density filters</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;"></td>
			<td style="width:333px;">ND4, ND8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Objective</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;">SFluo&#160;</td>
			<td style="width:333px;">40/1.3 DIC WD 0.22&#160;&#160; oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Camera</td>
			<td style="width:219px;">Andor Technologies</td>
			<td style="width:183px;">Zyla sCMOS</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:283px;">Nikon&#160; microscope (inverted)</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;">Nikon Eclipse TE2000-S</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cover Glass</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:183px;">6661B52</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Diamond pencil</td>
			<td style="width:219px;">Fisher Scientific</td>
			<td style="width:183px;">22268912</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Image acquisition software</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;">Nikon NIS-Elements&#160;</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Image analysis software</td>
			<td style="width:219px;">ImageJ</td>
			<td style="width:183px;">http://imagej.nih.gov/</td>
			<td style="width:333px;">ND Utility plugin allows to import images in the native Nikon Instruments .nd2 format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Recording/perfusion chamber</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:183px;">RC-26</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;"></td>
			<td style="width:219px;"></td>
			<td style="width:183px;"></td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Patch Clamp amplifier</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:183px;">MultiClamp 700B</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Data Acquisition System</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:183px;">Digidata 1440A</td>
			<td style="width:333px;">Axon Digidata&#174; System</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Low Pass Filter</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:183px;">LPF-8</td>
			<td style="width:333px;">8 pole Bessel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Borosilicate glass capillaries</td>
			<td style="width:219px;">World Precision Instruments</td>
			<td style="width:183px;">1B150F-4</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:283px;">Micropipette Puller</td>
			<td style="width:219px;">Sutter Instrument Co</td>
			<td style="width:183px;">P-97</td>
			<td style="width:333px;">Flaming/Brown type micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Microforge</td>
			<td style="width:219px;">Narishige</td>
			<td style="width:183px;">MF-830</td>
			<td style="width:333px;">Japan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Motorized Micromanipulator</td>
			<td style="width:219px;">Sutter Instrument Co</td>
			<td style="width:183px;">MP-225</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Inverted microscope</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;">Eclipse Ti</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Microvibration isolation table</td>
			<td style="width:219px;">TMC</td>
			<td style="width:183px;"></td>
			<td style="width:333px;">equipped with Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Multichannel valve perfusion system</td>
			<td style="width:219px;">AutoMake Scientific</td>
			<td style="width:183px;">Valve Bank II</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Recording/perfusion chamber</td>
			<td style="width:219px;">Warner Instruments</td>
			<td style="width:183px;">RC-26</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Software</td>
			<td style="width:219px;">Molecular Devices</td>
			<td style="width:183px;">pClamp 10 . 2</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;"></td>
			<td style="width:219px;"></td>
			<td style="width:183px;"></td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Temperature controlled surgical table&#160;</td>
			<td style="width:219px;">MCW core</td>
			<td style="width:183px;"></td>
			<td style="width:333px;">for rodents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Binocular stereomicroscope</td>
			<td style="width:219px;">Nikon</td>
			<td style="width:183px;">SMZ745</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Syringe pump-based perfusion system</td>
			<td style="width:219px;">Harvard Apparatus</td>
			<td style="width:183px;"></td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">polyethylene tubing</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:183px;">PE50</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Isofluorane anesthesia</td>
			<td style="width:219px;"><a href="http://www.vetequip.com/">http://www.vetequip.com/</a></td>
			<td style="width:183px;">911103</td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;"></td>
			<td style="width:219px;"></td>
			<td style="width:183px;"></td>
			<td style="width:333px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Other basic reagents</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:183px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

implementing patch clamp and live fluorescence microscopy to monitor functional properties of freshly isolated pkd epithelium

Iniciación quiste y expansión durante la enfermedad renal poliquística es un proceso complejo caracterizado por anormalidades en la proliferación celular tubular, acumulación de líquido luminal y la formación de matriz extracelular. La actividad de los canales iónicos y la señalización de calcio intracelular son parámetros fisiológicos clave que determinan las funciones de epitelio tubular. Hemos desarrollado un método adecuado para la observación en tiempo real de la actividad de los canales iónicos con la técnica de patch-clamp y el registro de nivel intracelular de Ca2 + en monocapas epiteliales recién aisladas de quistes renales. PCK ratas, un modelo genético de la enfermedad poliquística autosómica recesiva del riñón (ARPKD), se utilizaron aquí para el análisis ex vivo de los canales iónicos y el flujo de calcio. Descrito aquí es un procedimiento detallado paso a paso diseñado para aislar monocapas quísticas y túbulos no dilatado de PCK o ratas normales Sprague Dawley (SD), y supervisar la actividad de un solo canal e intracelulares de Ca 2+ dinámica.Este método no requiere procesamiento enzimático y permite el análisis en un entorno nativo de recién aisladas monocapa epitelial. Además, esta técnica es muy sensible a los cambios de calcio intracelular y genera imágenes de alta resolución para mediciones precisas. Por último, aislado epitelio quístico se puede utilizar más de tinción con anticuerpos o colorantes, preparación de cultivos primarios y purificación de diversos ensayos bioquímicos.

Participación ENaC potencial en quistogénesis ha sido demostrado por varios estudios que observan factor de crecimiento epidérmico perturbado (FEAG) de señalización en PKD progresión de 22-25 y la reabsorción de sodio anormal en ARPKD modelos murinos y cultivos de tejidos 26-28. Por ejemplo, Veizis et al. Mostraron que la absorción sensible a amilorida Na + se reduce en las células de CD desde el modelo de ratón BPK no ortólogos de ARPKD 29. Recientemente h...

Watch this Scientific Journal Video about Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium at JoVE.com

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Los canales iónicos expresadas en el epitelio tubular renal desempeñan un papel importante en la patología de la enfermedad poliquística del riñón. Aquí se describen los protocolos experimentales utilizados para realizar mediciones de análisis y el nivel de calcio intracelular patch-clamp en el epitelio quística recién aisladas de los riñones de roedores.

La implementación de Patch Clamp y vivo microscopía de fluorescencia para supervisar propiedades funcionales de recién aisladas PKD Epitelio

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

implementing-patch-clamp-live-fluorescence-microscopy-to-monitor

Research

JoVE Journal

Biology

9.7K vistas.  Medical College of Wisconsin.  Los canales iónicos expresadas en el epitelio tubular renal desempeñan un papel importante en la patología de la enfermedad poliquística del riñón. Aquí se describen los protocolos experimentales utilizados para realizar mediciones de análisis y el nivel de calcio intracelular patch-clamp en el epitelio quística recién aisladas de los riñones de roedores. 

Video: Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Métodos para grabaciones de parche Capacidad de la pinza del cáliz

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Investigación

Biología

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pulido a presión para la Mejora de pipetas de patch-clamp grabación

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Cuantitativa de células vivas por fluorescencia microscopia análisis de la levadura de fisión

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Preparación de liposomas gigantes de la Imagen y Patch-Clamp Electrofisiología

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal celular conectado a Patch-clamp grabación

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitución de una proteína transmembrana, el canal iónico dependiente de voltaje, KvAP, en Gigante vesículas unilamelares para Microscopía y Patch Clamp Estudios

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Análisis y calcio monocanal Imaging en los podocitos de los glomérulos recién aisladas

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...