Nos gustaría dar las gracias al doctor Claycomb para proporcionar células HL-1 y el protocolo detallado de mantenimiento. También nos gustaría dar las gracias a la señora Elena Carrillo y el Dr. Seth Robia por su ayuda en la generación de la película de células y el Sr. Pete Caron por su ayuda con la fabricación de algunos accesorios para nuestro sistema de mapeo óptico de doble canal. Este trabajo fue apoyado por la American Heart Association (10GRNT3770030 y 12GRNT12050478 a XA), los Institutos Nacionales de Salud (HL113640 a XA), y el Fondo de la Universidad Loyola de Investigaciones para el Desarrollo (a XA).

1. Preparación de la solución <ol><li> La norepinefrina solución (NP) <ol><li> Disolver 40 mg de NP en 25 ml de 30 mM de ácido ascórbico (0,1475 g de ácido ascórbico en 25 ml de Milli-Q (MQ) agua purificada. Evitar exposición a la luz directa durante esta preparación de la solución debido a la norepinefrina (NP) es sensible a la luz. </li><li> Filtrar la solución NP con un filtro de jeringa de 0,22 micras. Alícuota de la solución en 1 ml microtubos estériles y se almacena a -20 ° C. Una vez congelado, NP es estable durante un mes. </li></ol></li><li> Suplementado Lave y Final Claycomb <ol><li> Iniciar con 43,5 ml de medio de Claycomb y complementar con 5 ml de suero bovino fetal (FBS), 0,5 ml de penicilina / estreptomicina, 0,5 ml de NP y 1: 100 diluido L-Glutamina para hacer 50 ml de medio complementado Claycomb final. Evitar exposición a la luz directa durante esta preparación solución porque medio Claycomb es sensible a la luz. </li><li> Preparar el medio de lavado Claycomb usando la misma receta que el supmedio complementado Claycomb excepto agregar 5% de SFB y omitir el NP y los componentes de L-glutamina. NOTA: Los medios de comunicación Final y Wash Claycomb complementado son buenos para dos semanas cuando se almacena a 4 ° C, sin exposición a la luz directa. </li></ol></li><li> Soja solución inhibidora de tripsina <ol><li> Disolver 25 mg de inhibidor de tripsina de soja en 100 ml de Ca-Mg-libre y libre de salina tamponada con fosfato de Dulbecco (PBS). </li><li> Filtrar la solución usando un filtro de jeringa de 0,22 micras. NOTA: La solución esterilizada es estable durante un mes si se almacena a 4 ° C. </li></ol></li><li> Solución de revestimiento plato de gelatina / fibronectina <ol><li> Disolver 0,1 g de gelatina en 500 ml de agua MQ para hacer 0,02% de gelatina. </li><li> Autoclave, luego diluir 1 ml de fibronectina en 199 ml de 0,02% de gelatina y mezclar suavemente. </li><li> Alícuota 6 ml de solución de gelatina en tubos separados y almacenar a -20 ° C. </li></ol></li><li> La solución salina de Hanks <ol><li> Usando un gran revueloplaca, disolver las siguientes sales en agua MQ (mmol / L), NaCl (136,9), KCl (5.366), KH 2 PO 4 (0,4409), NaH 2 PO 4 (0,4000), MgSO4 (0,8142), D-Glc (5.051), NaHCO3 (4.162), HEPES (5.042), CaCl 2 (1.261). </li></ol></li><li> Tinte sensible al calcio solución (Rhod2) <ol><li> Diluir 1 mg de polvo seco Rhod2 en 400 l de 20% de Pluronic F-127 en DMSO para hacer una Rhod2 tinte solución madre y se almacena a -20 ° C. </li><li> Diluir esta solución madre 1: 1000 con una solución salina de Hanks para hacer una solución de trabajo final antes de iniciar el experimento. </li></ol></li><li> Voltaje colorante sensible solución (Rh237) <ol><li> Diluir 5 mg de Rh237 con 2 ml de DMSO para hacer la solución madre y se almacena a -20 ° C. Diluir la solución madre de 1: 1000 con una solución salina de Hanks para hacer una solución de trabajo final antes de iniciar el experimento. </li></ol></li></ol> &amp; #160; NOTA: 1-4 soluciones se basan en el HL-1 protocolo de cultivo celular Claycomb con modificación menor. 2. pases y cultivo HL-1 auricular miocitos en cubreobjetos NOTA: Las células HL-1 se pueden obtener del Dr. Claycomb (Louisiana State University). <ol><li> Se cultivan las células HL-1 en frascos T25 y alimentar con 5 ml de complementado Claycomb Medio diarias. Passage las células en matraces T25 cada cinco días de acuerdo con la HL-1 protocolo de cultivo celular Claycomb (descrito a continuación) 10. </li><li> Cubreobjetos redondos de lugares (20 mm de diámetro) en placas de cultivo de 12 pocillos 24 h antes de las células en el matraz T25 son completamente confluente y listo para ser passaged, y luego cubrir los cubreobjetos con gelatina / fibronectina durante la noche a 37 ° C. </li><li> Al día siguiente, aspirado y desechar la gelatina / fibronectina de las placas de cultivo y reemplazar con 1,5 ml de medio final Claycomb complementado en cada pocillo. </li><li> Dividir celdas de una cultur T25e frasco después de alcanzar plena confluencia en el día 5. Aspirar el medio del matraz T25 que contiene el cultivo confluente y enjuague la cultura brevemente con PBS a 37 ° C. </li><li> Aspirar PBS y añadir 3 ml de 0,05% de tripsina / EDTA a 37 ° C pipeteando la tripsina / EDTA en el fondo del matraz T25 para evitar el contacto directo con las células. Incubar a 37 ° C durante 1 min. </li><li> Eliminar tripsina / EDTA por aspiración y añadir otro 1,3 ml de tripsina / EDTA por matraz T25. Incubar a temperatura ambiente durante 1 min. Examinar al microscopio y si las células todavía se atan al matraz, toque para desalojar completamente las células restantes. </li><li> Añadir 1,3 ml de inhibidor de tripsina de soja a las células y las células de transferencia desde el matraz a un tubo de centrífuga de 15 ml. Lavar el recipiente vacío con 5 ml de medio de lavado Claycomb. Añadir el enjuague a las células en el tubo de centrífuga de 15 ml. Centrifugar a 500 xg durante 5 min. </li><li> Después de centrifugar, aspirar el sobrenadante y suavemente volver a suspender el sedimento celularen 6 ml de medio final Claycomb suplementado. </li><li> Semillas cada pocillo de una placa de 12 pocillos con 0,5 ml de suspensión de células de los pases de células. Alimentar a las células con medio suplementado fresco final Claycomb diaria. NOTA: Vigilar de cerca el crecimiento de las células, por lo general las monocapas de células alcanzan el estado de confluencia en el día 4 y las actividades espontáneas siguen siendo visibles. Entonces, la monocapa se cultivan estará listo para el mapeo óptico en días 4-5. </li></ol> 3. Carga de calcio y de tensión tintes sensibles a las células <ol><li> Para los estudios de mapeo óptico, retire suavemente el cubreobjetos de la placa de cultivo celular de 12 pocillos y enjuague con una solución de sal de Hanks. </li><li> Para teñir las células con colorante sensible al Ca Rhod2, incubar la monocapa celular en el cubreobjetos con 3 ml de la oxigenada (burbujeo con 5% de CO 2/95% O 2 gas) solución de trabajo Rhod2 a 37 ° C en un baño de agua durante 30 min . </li><li> Para la tinción de las células con V m colorante sensibleRh237, sumerja la monocapa celular cargado Rhod2 en 3 ml de solución de trabajo Rh237 oxigenada a 37 ° C en un baño de agua durante otros 15 minutos. </li></ol> Mapeo 4. Canal de Óptica Doble <ol><li> Coloque la monocapa celular, que es de doble teñido con colorantes V m y Ca, en una cámara de circulación en un microscopio de fluorescencia invertida. Utilice un sistema de circulación se calienta para mantener la temperatura de la cámara de la celda a 35 ± 1 ° C. Controlar cuidadosamente la temperatura del líquido de perfusión mediante el ajuste de la velocidad del flujo que circula a través de la cámara para evitar un gradiente de más de 0,3 ° C a través de la cámara. <ol><li> Antes de grabar V m / señales de Ca, lavar la monocapa de células con solución de Hanks fresco durante aproximadamente 5 minutos para eliminar el colorante restante de los pasos de tinción anteriores. </li></ol></li><li> Pace monocapas de células saludables utilizando un electrodo bipolar integrado por una pipeta de vidrio lleno de solución de sal de Hanks y silver alambre enrollado alrededor de la punta de la pipeta a lo descrito previamente 12,17. </li><li> Pace monocapas de células sanas en una longitud de ciclo de 200 a 500 ms y ancho de pulso de 2 ms, contar con un umbral de estimulación en aproximadamente 1 mV. NOTA: Para cada lote de monocapas cultivadas, al menos dos monocapas no tratadas deben ser utilizados para probar la condición del lote celular. Monocapas de control sanos deben ser capaces de ser capturado por la estimulación eléctrica en la duración de los ciclos de 200 a 500 ms. Si las monocapas control fallan a ritmo eléctricamente, esto sugiere que todo el lote de células puede no ser adecuado para los experimentos de mapeo óptico. </li><li> Para excitar tanto V m y Ca colorantes sensibles, utilizar una fuente de luz LED (520 nm longitud de onda) junto con un filtro de excitación (510 nm / 40) y un espejo dicroico (561 nm). <ol><li> Recoger las señales fluorescentes emitidas por las células a través de un objetivo de 40X y se dividió con un espejo dicroico (594 nm) en un componente V m y un componente de Ca.Se filtra la luz emitida en el canal V m con un filtro de pase largo (700 nm) y la luz emitida en el canal de Ca con un filtro de paso de banda (585/40 nm). </li><li> Recoge la luz fluorescente filtrada utilizando dos cámaras CCD (80 x 80 píxeles, 1.000 fps; redshirt Imaging). </li><li> Para evitar colorante photobleaching, hacer grabaciones durante menos de 2 segundos en cada campo de visión para limitar el tiempo de exposición a la luz. Aquí, típicamente imagen cuatro áreas diferentes para cada monocapa. </li></ol></li></ol> 5. Proceso de Datos de V mo Ca Grabaciones <ol><li> Por tanto V señales de Ca (80 x 80 píxeles) m, y el promedio de los valores de intensidad de veinticinco (5 x 5) píxeles vecinos en un valor de intensidad en diferentes puntos de tiempo de grabación para reducir el ruido de fondo. NOTA: Después de que el promedio de datos, cada punto de tiempo de grabación de un potencial de acción individual o Ca transitoria produce una matriz de datos que contiene 16 x 16 promediaron los valores de intensidad (promediadocanales de datos) con una resolución espacial de 50 m. </li><li> Definir el tiempo de activación (AT) de las señales de Ca registrado V mo. <ol><li> Para definir un tiempo de activación (AT) de un potencial de acción individual o Ca transitoria, determinar el tiempo de 50% de amplitud de pico de las señales de CA mediante un algoritmo basado en MATLAB a medida V m o como se describe anteriormente. 12 </li><li> Corregir manualmente el AT analizados para eliminar cualquier error que pueda ocurrir durante el análisis del programa. </li><li> La Web de los definidos en la MATRIZ (16 x 16 promediaron canales de datos) del potencial de acción grabado y transitorios de calcio, respectivamente, en el software utilizado. </li></ol></li><li> Calcule el potencial de acción o transitorios de velocidad de conducción (CV) vectores de calcio en la matriz AT utilizando el método de análisis de campo de vectores como se ha descrito anteriormente, con modificaciones menores 12,18. <ol><li> Calcular el vector de CV de un canal de datos en relación con su entorno en los puntos de datos (5 ×; 5 vecina promedió canales de datos) para simular una superficie lisa polinomio de tiempos de activación (en la superficie) utilizando un algoritmo de mínimos cuadrados dentro del programa MATLAB 12,18. </li><li> Calcular la conducción vector usando las siguientes fórmulas Fórmula 1: <img alt="Ecuación 1" fo:content-width="1.5in" src="/files/ftp_upload/52542/52542eq1.jpg" /> Fórmula 2: θ = arctan [(dy / dt) / (dx / dt)] NOTA: Ve es un vector CV proyectado en xey ejes; dx / dt es la proyección del vector CV en el eje x; dy / dt es la proyección del vector CV en el eje y. T es la superficie AT; x, y se refieren a la 2-D coordenadas; T x = ∂T / ∂x es la derivada parcial de T con respecto a x; T y = ∂T / ∂y es la derivada parcial de T con respecto a Y; θ es la dirección final de un vector de CV. </li><li> Grupo todos los vectores de la matriz AT en 36 vías, cada una de las cuales cubre un sector de diez grados en la brújula. &lt;/ Li&gt; <li> Seleccione la ruta que contiene la mayor población de los vectores de CV entre las 36 vías de conducción. Calcular el promedio de CV de esta vía para representar el CV de la monocapa. </li></ol></li></ol> 6. Visualización de la Propagación de la onda frontal <ol><li> Normalizar la intensidad de señal de un potencial de acción individual o Ca transitoria en diferentes puntos de tiempo de grabación con respecto a su valor de pico utilizando el software MATLAB. </li><li> Almacenar temporalmente los datos de intensidad normalizados de cada punto de tiempo grabado en una matriz de intensidad de 16 x 16 en el software MATLAB utilizado. </li><li> Construir un mapa de intensidad con código de color de la matriz de intensidad almacenada de cada punto de tiempo de grabación utilizando la función surf.m. NOTA: En el mapa de intensidad, el rojo representa alta intensidad de las señales de Ca V mo, y azul refleja baja intensidad de las señales de fluorescencia. </li><li> Crear un archivo .avi mediante la función avifile.m. </li><li> Guarde toda la imapas ntensity en diferentes puntos de tiempo de grabación del potencial de acción o Ca transitoria mediante la función addframe.m. NOTA: En este punto, se generarán dos archivos avi separadas de V m y propagación Ca. </li></ol>

<ol>
	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Salama, G., Hwang, S. M., 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=Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts.">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]. 12 (Unit 12 17), (2009).</li><li>Laurita, K. R., Singal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+action+potentials+and+calcium+transients+simultaneously+from+the+intact+heart.">Mapping action potentials and calcium transients simultaneously from the intact heart.</a> Am J Physiol Heart Circ Physiol. 280 (5), H2053-H2060 (2001).</li><li>Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+optical+imaging+of+membrane+potential+and+intracellular+calcium.">Simultaneous optical imaging of membrane potential and intracellular calcium.</a> J Electrocardiol. 38 (4 Suppl), 107-112 (2005).</li><li>Sowell, B., Fast, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+mechanism+of+shock-induced+arrhythmias:+role+of+intracellular+calcium.">Ionic mechanism of shock-induced arrhythmias: role of intracellular calcium.</a> Heart Rhythm. 9 (1), 96-104 (2012).</li><li>Fast, V. G., Kleber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+conduction+in+cultured+strands+of+neonatal+rat+heart+cells+measured+with+voltage-sensitive+dyes.">Microscopic conduction in cultured strands of neonatal rat heart cells measured with voltage-sensitive dyes.</a> Circ Res. 73 (5), 914-925 (1993).</li><li>Hou, L., 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=A+major+role+for+HERG+in+determining+frequency+of+reentry+in+neonatal+rat+ventricular+myocyte+monolayer.">A major role for HERG in determining frequency of reentry in neonatal rat ventricular myocyte monolayer.</a> Circ Res. 107 (12), 1503-1511 (2010).</li><li>Beauchamp, P., 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=Relative+contributions+of+connexins+40+and+43+to+atrial+impulse+propagation+in+synthetic+strands+of+neonatal+and+fetal+murine+cardiomyocytes.">Relative contributions of connexins 40 and 43 to atrial impulse propagation in synthetic strands of neonatal and fetal murine cardiomyocytes.</a> Circ Res. 99 (11), 1216-1224 (2006).</li><li>Xia, M., 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=Functional+expression+of+L-+and+T-type+Ca2++channels+in+murine+HL-1+cells.">Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.</a> J Mol Cell Cardiol. 36 (1), 111-119 (2004).</li><li>Claycomb, W. C., 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=HL-1+cells:+a+cardiac+muscle+cell+line+that+contracts+and+retains+phenotypic+characteristics+of+the+adult+cardiomyocyte.">HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte.</a> Proc Natl Acad Sci U S A. 95 (6), 2979-2984 (1998).</li><li>Fahrenbach, J. P., Ai, X., Banach, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+intercellular+coupling+improves+the+function+of+cardiac+pacemakers+derived+from+mouse+embryonic+stem+cells.">Decreased intercellular coupling improves the function of cardiac pacemakers derived from mouse embryonic stem cells.</a> J Mol Cell Cardiol. 45 (5), 642-649 (2008).</li><li>Yan, J., 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=c-Jun+N-terminal+kinase+activation+contributes+to+reduced+connexin43+and+development+of+atrial+arrhythmias.">c-Jun N-terminal kinase activation contributes to reduced connexin43 and development of atrial arrhythmias.</a> Cardiovasc Res. 97 (3), 589-597 (2013).</li><li>White, S. M., Constantin, P. E., Claycomb, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+physiology+at+the+cellular+level:+use+of+cultured+HL-1+cardiomyocytes+for+studies+of+cardiac+muscle+cell+structure+and+function.">Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function.</a> Am J Physiol Heart Circ Physiol. 286 (3), H823-H829 (2004).</li><li>Yang, Z., Shen, W., Rottman, J. N., Wikswo, J. P., Murray, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+stimulation+causes+electrical+remodeling+in+cultured+atrial+myocytes.">Rapid stimulation causes electrical remodeling in cultured atrial myocytes.</a> J Mol Cell Cardiol. 38 (2), 299-308 (2005).</li><li>Brundel, B. J., Kampinga, H. H., Henning, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain+inhibition+prevents+pacing-induced+cellular+remodeling+in+a+HL-1+myocyte+model+for+atrial+fibrillation.">Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation.</a> Cardiovasc Res. 62 (3), 521-528 (2004).</li><li>Umapathy, K., 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=Electrogram+fractionation+in+murine+HL-1+atrial+monolayer+model.">Electrogram fractionation in murine HL-1 atrial monolayer model.</a> Heart Rhythm. 5 (7), 1029-1035 (2008).</li><li>Fast, V. G., Cheek, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+arrhythmias+induced+by+strong+electrical+shocks+in+myocyte+cultures.">Optical mapping of arrhythmias induced by strong electrical shocks in myocyte cultures.</a> Circ Res. 90 (6), 664-670 (2002).</li><li>Bayly, P. V., 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=Estimation+of+conduction+velocity+vector+fields+from+epicardial+mapping+data.">Estimation of conduction velocity vector fields from epicardial mapping data.</a> IEEE Trans Biomed Eng. 45 (5), 563-571 (1998).</li></ol>

No conflicts of interest declared.

Este artículo describe los aspectos clave de mapeo óptico en una culta HL-1 miocitos auricular monocapa teñido con calcio y tensión tintes fluorescentes sensibles. Incluye el cultivo de una óptima HL-1 monocapa celular, la configuración de los equipos de la cartografía, la cartografía de una monocapa cultivadas, y el análisis de datos. Para asignar con éxito células cultivadas, la clave es preparar una monocapa de células uniformemente distribuida. Cuando la siembra de las célul...

Una calcio único (Ca) y la tensión (V m) técnica de mapeo óptico de doble canal 5.1 se está convirtiendo en una herramienta eficaz para registrar simultáneamente señales V m y Ca en ambos corazones intactos y monocapas de células cultivadas. Esta técnica hace posible la obtención de información de gran alcance respecto a la relación entre los transitorios de calcio y los potenciales de acción de entender mejor los mecanismos subyacentes electrofisiológicas de las arritmias cardiacas. Monocapas de células cultivadas han demostrado ser un modelo celular útil para estudiar la electrofisiología cardiaca y el mecanismo subyacente de las arritmias. 4,6-8 HL-1 células son una línea de cultivo de miocitos fibrilación bien caracterizado. Células HL-1 también mantienen un genotipo y el fenotipo diferenciado de forma única que incluye morfológica, electrofisiológico, y las características farmacológicas de los miocitos auriculares adultos. Estas células expresan genes y proteínas cardíacas, incluyendo important canales iónicos cardíacos (es decir, L y los canales de calcio tipo T) 9 y isoformas maduras de las proteínas contráctiles del sarcómero se encuentran normalmente en miocitos auriculares adultos como los demás y nos han informado anteriormente. 10-13 Además, las células HL-1 pueden ser cultivan para formar un 2-dimensional (2-D) monocapa de miocitos. Por lo tanto, las ventajas de utilizar cultivadas monocapas HL-1 incluyen: 1) el costo relativamente más bajo y más fácil de mantener una línea de cultivo de los miocitos de aislamiento y cultivo de miocitos neonatales primarios; 2) una monocapa confluente de células reduce las complejidades estructurales que resultan de la estructura 3-D del corazón; 3) una monocapa celular se puede eliminar la interferencia de la fibrosis intersticial que se produce en el corazón intacto. Esto se puede utilizar para diseccionar funciones electrofisiológicas específicas de un grupo de miocitos sin interferencia de los fibroblastos y la matriz intersticial; 4) evaluación de las consecuencias funcionales de la manipulación farmacológica o genética en culmonocapas de células radas se pueden lograr con eficacia. Por lo tanto, las células HL-1 se han convertido en un modelo celular ampliamente utilizado para el estudio de diversos aspectos de la fisiología de los miocitos, así como actividades eléctricas anormales inducidos por estimulación. 13-16 Sin embargo, se requiere un manejo y cuidado especial a la cultura monocapas de células sanas que responden al exterior estimulación eléctrica para los estudios de mapeo óptico. Además, la doble procedimiento de tinción de colorante fluorescente puede dañar fácilmente la integridad de las monocapas de células confluentes cultivadas. Por lo tanto, la realización de V m / CA mapeo óptico de doble canal en cultivos de HL-1 monocapas ha sido un gran reto. El objetivo de este método es proporcionar los pasos clave para llevar a cabo con éxito mapeo óptico de doble canal en cultivos de HL-1 monocapas. Aquí hemos proporcionado amplios detalles sobre un protocolo optimizado para la preparación monocapa-1 HL celular, mapeo óptico de doble canal de una monocapa de células cultivadas, y procesamiento de datos de mapas.

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhod2 dry powder</td>
			<td style="width:103px;">&#160;AAT Bioquest</td>
			<td style="width:119px;">21062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pluronic</td>
			<td style="width:103px;">AAT Bioquest</td>
			<td style="width:119px;">20050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">DMSO</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rh237</td>
			<td style="width:103px;">Invitrogen</td>
			<td style="width:119px;">&#160;S-1109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4&#160;</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">P0662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">S6014</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPEs</td>
			<td style="width:103px;">sigma</td>
			<td style="width:119px;">H3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage and calcium dual channel optical mapping of cultured hl-1 atrial myocyte monolayer

Mapeo óptico ha demostrado ser una técnica valiosa para detectar la actividad eléctrica cardiaca tanto intacto ex vivo corazones y en monocapas de miocitos en cultivo. Células HL-1 han sido ampliamente utilizado como un modelo celular 2-Dimensional para el estudio de diversos aspectos de la fisiología cardiaca. Sin embargo, ha sido un gran reto al mapa ópticamente calcio (Ca) los transitorios y los potenciales de acción simultáneamente desde el mismo campo de visión en una culta HL-1 monocapa celular auricular. Esto se debe a que se requiere un manejo y cuidado especial para preparar las células sanas que se pueden capturar eléctricamente y mapeados ópticamente. Por lo tanto, hemos desarrollado un protocolo de trabajo óptimo para el mapeo óptico de doble canal. En este manuscrito, que hemos descrito en detalle cómo realizar el experimento de doble canal mapeo óptico. Este protocolo es una herramienta útil para mejorar la comprensión de la acción potencial de propagación y la cinética de Ca en el desarrollo de arritmias.

Una monocapa confluente culta presenta un ritmo intrínseco regular como se demuestra en la película 1. A continuación realizó V m dual mapeo óptico canal / Ca en una totalmente confluente HL-1 monocapa. Figura 1A muestra un ejemplo de los rastros de las señales V Ca my de un grabado único superar. Mapas isócronos representativos de las señales de Ca utilizando el sistema de mapeo óptico de doble canal propagado uniformemente V m y se muestran e...

Watch this Scientific Journal Video about Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer at JoVE.com

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Tensión y calcio Dual Channel Mapping óptico de Cultivadas HL-1 auricular miocitos monocapa

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

voltage-calcium-dual-channel-optical-mapping-cultured-hl-1-atrial

Research

JoVE Journal

Biology

10.1K vistas.  Loyola University Chicago. This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Video: Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Limpieza y cuidado apropiado del microscopio

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Investigación

Biología

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Contraste de fase y de interferencia diferencial microscopía de contraste (DIC)

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Mapeo óptico de corazones de ratas Langendorff perfundidos

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

Grabación óptica de la actividad eléctrica en el conejillo de Indias Redes entérica con tintes sensibles al voltaje

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Fotoconversión purificada de proteínas fluorescentes y de doble sonda óptica destacar en células vivas

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

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 ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

Aislamiento y grabaciones Kv Canal murinos en miocitos auriculares y ventriculares

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

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 ...

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Mapeo óptico de Intra-retículo sarcoplásmico Ca<sup&gt; 2 +</sup&gt; Y potencial transmembrana en el corazón de conejo perfundidos Langendorff

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...