Danilo Maddalo es un beneficiario de una beca de investigación como el Grupo de Jóvenes Investigadores (YIG) del Instituto de Tecnología de Karlsruhe.

1. Sembrar las células <ol><li> Antes de empezar, calentar el medio de cultivo, el PBS 1X y tripsina en un baño de agua a 37 ˚ C </li><li> Tomar las células de la incubadora y se aspira el medio. </li><li> Aplique suavemente 5 ml de PBS 1X en las células para lavarlos. </li><li> Aspirar el PBS 1X y aplicar 1 ml de tripsina que contiene 0,025% de EDTA. </li><li> Gire suavemente la placa para tener una distribución uniforme de la tripsina. </li><li> Coloque las células de la incubadora a 37 ˚ C durante 5-10 minutos (dependiendo del tipo de célula). De vez en vez, compruebe si las células están separadas por agitación de la placa. </li><li> Resuspender las células en 10-20 ml de medio y recoger en un tubo Falcon de 50 ml. </li><li> Contar las células mediante un contador Beckman. El número de células debería ser suficiente para garantizar una confluencia de 60-70%. </li><li> Placa de las células y les permiten a las semillas durante 6-8 horas. </li></ol> 2. Transinfección <ol><li> Antes de la transfección, se caliente una alícuota de medio libre de suero en un baño de agua a 37 ˚ C. </li><li> Lleva el gen X-treme 9 a temperatura ambiente. </li><li> Pipetear el medio libre de suero en dos tubos eppendorf (1 tubo = 1 transfección). </li><li> Pipeta de la Gene X-treme 9 en la atención de medio libre de suero que paga la punta no toque la pared de plástico del eppendorf y se incuban durante 5 minutos. Mientras tanto preparar dos mezclas de ADN: en un tubo se mezclan los plásmidos de renilla, luciérnagas y un control de vector vacío, en otro tubo de renilla, luciérnaga y la chaperona molecular. </li><li> Añadir la mezcla de ADN a cada eppendorf conteniendo medio libre de suero + X-treme Gene 9, vortex e incubar a temperatura ambiente durante 20 minutos. </li><li> Añadir gota a gota cada mezcla en cada plato y se incuba durante 24 horas a 37 ° C y 5% de CO 2 </li></ol> 3. La división de las células <ol><li> 24 horas después de la transfección, trypsinize las células como se describe en 1,4 a 1,6 y se diluye a una confluencia final de 60-70% en una placa de 6 pocillos. </li><li> Preparar un plato para el control no sorprendió y tantas placas como los puntos de tiempo de recuperación que tienen que ser analizados, como se muestra en la fig. 2. </li></ol> 4. De choque térmico <ol><li> De choque térmico, pre-caliente el suero que contienen medio de un baño de agua a 37 ˚ C. </li><li> &quot;Amortiguador de choque térmico&quot; preparar diluyendo en medio que contenía suero cicloheximida a una concentración final de 20 mg / ml y MOPS a 20 mm. </li><li> Ahora, corta las placas de la incubadora y aspirar el medio. </li><li> Añadir a cada pocillo 1 ml de &quot;amortiguador de choque térmico, y se incuba durante 30 minutos a 37 ˚ C y 5% de CO 2 (incluye también la placa de referencia en este tratamiento). Esto evitará que la síntesis de novo de proteínas. </li><li> Mientras tanto en el interruptor en un baño de agua y establecer el temperatura a 45 ˚ C. </li><li> Después de la incubación, el sello de la tapa de las placas con Parafilm previamente cortado en tiras. La placa de referencia (100% de refolfed luciérnaga) se deja en la incubadora. </li><li> Las placas se incuban a 45 ˚ C durante 30 minutos. </li><li> Retire las placas del baño de agua, quitar la parafina y poner las placas de nuevo a la incubadora para permitir la recuperación. </li><li> En cada punto de tiempo de la cosecha de las células y los lava en PBS 1X por centrifugación a 2000Xg durante 1 minuto a 4 ˚ C. </li></ol> 5. Lisis celular y ensayo luciferasa <ol><li> Para una lisis celular eficiente, snap congelar el sedimento de células en nitrógeno líquido. </li><li> Añadir 200 ul de buffer de lisis pasiva diluida 1:5 con agua destilada en un tubo de vidrio. </li><li> Añadir 30 l de lisado de células en una placa de 96 pocillos. Cada muestra está contaminada por triplicado. </li><li> Sólo el &quot;no sorprendió&quot; muestras de referencia será utilizado paraleer la actividad de la luciferasa Renilla, ya que este paso se utiliza para comprobar la eficacia de transfección de igualdad. </li><li> Preparar las soluciones para el ensayo. Para la solución de luciferina diluir luciferina a una concentración final de 0,2 mM de Gly-Gly buffer. Para el tampón de reacción diluir la TDT y la concentración de ATP 1 mM final en Gly-Gly buffer. Para el buffer de coelenterazine reacción, diluir coelenterazine a una concentración final de 0,2 M en tampón coelenterazine. Conservar la solución de luciferina y la reacción coelenterazine luz buffer protegida. </li><li> Ahora coloque la placa de 96 pozos en el luminómetro e iniciar el programa &quot;Wallac 1420 estaciones de trabajo&quot;. </li><li> Introducir el Pump1 en el tubo Falcon que contiene la solución coelenterazine. Cierre la tapa de la luz por lo que no puede entrar en contacto con el tubo. </li><li> Seleccione en el menú de mantenimiento de dispensador de &#39;la opción y marque Pump1. </li><li> Seleccione la opción &#39;Fill&#39;. </li><li> Para modificar el diseño de la placa de elegir la opción &quot;Explorar el protocolo y los resultados&quot; en el menú. Seleccione el protocolo y haga clic dos veces en la misma. A continuación, seleccione los pozos para ser leído. </li><li> Volver a &#39;Wallac Manager&#39; y haga clic en &#39;Inicio&#39;. La reacción entre la luciferasa Renilla y su sustrato coelenterazine emite luz con una longitud de onda máxima de 482 nm. </li><li> Después de la lectura, ir a &quot;mantenimiento del dispensador&quot; y seleccione &quot;Vaciar y Pump1 a liberar a todos de nuevo la solución residual al tubo. </li><li> Ahora mueva el tubo a un tubo Falcon que contiene agua y marca la casilla &quot;Flush&quot;. </li><li> Tras la etapa de lavado, vaciar el tubo mediante la selección de &quot;vacío&quot;. </li><li> Coloque el tubo de Pump1 en el buffer de tubo de reacción de halcón y el de Pump2 en el tubo de la luciferina halcón. </li><li> Cambiar el diseño de la placa. </li><li> Seleccione &#39;Fill&#39; para Pump1 y Pump2 de la Di-Spenser menú de mantenimiento. </li><li> Seleccione el protocolo y empezar la lectura. La reacción entre la luciferasa de luciérnaga y su sustrato luciferina emite luz con una longitud de onda máxima de 560 nm. </li><li> Al final de la lectura, repita el procedimiento vacía-Flush-vacío para Pump1 y Pump2. </li><li> Los resultados ya están disponibles en el &#39;Wallac 1420 Explorer&#39;. </li><li> Cada experimento se asocia a un número de prueba, nombre de usuario, fecha de inicio y fin. </li><li> Los resultados ya se pueden exportar como un archivo de Excel. </li><li> Para los cálculos, las células se dividen en tres grupos: 1. no sorprendió las muestras de referencia (fijado en un 100% de replegarse luciferasa de luciérnaga), 2. células sin chaperón molecular y sometidos a desconchado térmico, 3. células con chaperón molecular y el calor sorprendió. </li></ol> 6. Resultados representante Plegamiento de proteínas se relaciona directamente con el tiempo de recuperación por lo tanto, para probar si el sistema está trabajando a favor<html> Perly, un ensayo se debe realizar la recogida de las células en diferentes momentos. Existe una correlación directa tiempo / porcentaje de replegamiento indica que el experimento ha sido realizado correctamente y por lo tanto representa un factor limitante. Sin embargo debe ser considerado siempre que el repliegue tras el choque térmico es un evento de saturación y por lo tanto, un análisis de tiempo de curso apropiado se debe realizar antes de comenzar cualquier experimento. <img src="/files/ftp_upload/3540/3540fig1.jpg" alt="Figura 1"/> Figura 1. Descripción general del experimento. En el ensayo in vivo replegado requiere de 3 a 4 días para ser completado. El día 1 se siembran las células y transfectadas. El día siguiente, las células están divididos en un formato más pequeño y el día 3 que son sometidos a desconchado térmico. En este punto es posible realizar el ensayo de luciferasa después de recoger las células o para guardar el pellet de células a -80 ° C y realizar el ensayo en otro momento _content &quot;&gt; <img src="/files/ftp_upload/3540/3540fig2.jpg" alt="Figura 2"/> Figura 2. División celular. El día 2 las células se dividen en una placa de 6 pocillos. Cada transfección se diluye en un pozo con el fin de tener toda la transfección en la misma placa. Cada placa se corresponderá con un tiempo de recuperación concreto, más una placa de referencia no sorprendió. <img src="/files/ftp_upload/3540/3540fig3.jpg" alt="Figura 3"/> Figura 3. Representante buen resultado. Porcentaje de A. replegado medido directamente por la actividad luciferasa de luciérnaga. Cada gráfico de barras representa el porcentaje de replegado en ausencia (barras de color negro) y en presencia (barras rojas) de una chaperona molecular en diferentes puntos de tiempo de recuperación. B. La correlación entre el tiempo y replegado en ausencia (línea en negro) y en presencia (línea roja) de acompañante. Los valores de r cuadrado indican una buena correlación. En Fig.3A se muestra un resultado representativo, donde las células se recogieron 15, 30, 45, 60 y 120 después del choque térmico en ausencia (barras de color negro) y en presencia (barras rojas) de chaperona molecular. Porcentaje de aumento de replegarse luciferasa con el tiempo de recuperación prolongado y con la transfección de la chaperona molecular. La correlación entre el repliegue y el tiempo para el control (Fig. 3B, la línea de negro) y el acompañante molecular transfectadas las células (Fig. 3B, línea roja) se muestra. <img src="/files/ftp_upload/3540/3540fig4.jpg" alt="Figura 4"/> Figura 4. Representante mal resultado. Porcentaje de A. replegado mide directamente la actividad de luciferasa. Cada gráfico de barras representa el porcentaje de replegado en ausencia (barras de color negro) y en presencia (barras rojas) de una chaperona molecular en diferentes puntos de tiempo de recuperación. B. La correlación entre el tiempo y replegado en ausencia y en presencia (rojo lINE) de acompañante. Los valores de r cuadrado indican correlación mal. Figura 4A. Muestra un resultado representativo de un experimento no se realiza correctamente. Tanto el control (barras de color negro) y el acompañante molecular transfectadas las células (barras rojas) no mostraron un aumento de la proteína replegado con el tiempo. Además, la transfección de la chaperona molecular no se tradujo en un aumento de replegamiento. Esta mala correlación se confirma en la figura. 4B muestra un valor de R cuadrado de 0,6619 y 0,1882, respectivamente, para el control (línea en negro) y el acompañante molecular transfectadas las células (línea roja).

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	<li>Bukau, B., Weissman, J., Horwich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperones+and+protein+quality+control.">Molecular chaperones and protein quality control.</a> Cell. 125, 443-443 (2006).</li><li>Ritossa, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+the+heat+shock+response.">Discovery of the heat shock response.</a> Cell Stress Chaperones. 1, 97-97 (1996).</li><li>Hendrick, J. P., Hartl, F. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+functions+of+heat-shock+proteins.">Molecular chaperone functions of heat-shock proteins.</a> Annu. Rev. Biochem. 62, 349-349 (1993).</li><li>Fuqua, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+proteins+and+drug+resistance.">Heat shock proteins and drug resistance.</a> Breast. Cancer. Res. Treat. 32, 67-67 (1994).</li><li>Guzhova, I., Margulis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperone+as+a+survival+factor+in+cell+pathology.">Hsp70 chaperone as a survival factor in cell pathology.</a> Int. Rev. Cytol. 254, 101-101 (2006).</li><li>Whitesell, L., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+and+the+chaperoning+of+cancer.">HSP90 and the chaperoning of cancer.</a> Nat. Rev. Cancer. 5, 761-761 (2005).</li><li>Konstantinopoulos, P. A., Papavassiliou, 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=17-AAG:+mechanisms+of+antitumour+activity.">17-AAG: mechanisms of antitumour activity.</a> Expert. Opin. Investig. Drugs. 14, 1471-1471 (2005).</li><li>Galluzzi, L., Giordanetto, F., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP70+for+cancer+therapy.">Targeting HSP70 for cancer therapy.</a> Mol. Cell. 36, 176-176 (2009).</li><li>Ozacmak, V. H., Barut, F., Ozacmak, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melatonin+provides+neuroprotection+by+reducing+oxidative+stress+and+HSP70+expression+during+chronic+cerebral+hypoperfusion+in+ovariectomized+rats.">Melatonin provides neuroprotection by reducing oxidative stress and HSP70 expression during chronic cerebral hypoperfusion in ovariectomized rats.</a> J. Pineal. Res. 47, 156-156 (2009).</li><li>Schroder, H., Langer, T., Hartl, F. U., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DnaK,+DnaJ+and+GrpE+form+a+cellular+chaperone+machinery+capable+of+repairing+heat-induced+protein+damage.">DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.</a> EMBO. J. 12, 4137-41 (1993).</li><li>Thulasiraman, V., Matts, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+geldanamycin+on+the+kinetics+of+chaperone-mediated+renaturation+of+firefly+luciferase+in+rabbit+reticulocyte+lysate.">Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.</a> Biochemistry. 35, 13443-13443 (1996).</li><li>Howarth, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp40+molecules+that+target+to+the+ubiquitin-proteasome+system+decrease+inclusion+formation+in+models+of+polyglutamine+disease.">Hsp40 molecules that target to the ubiquitin-proteasome system decrease inclusion formation in models of polyglutamine disease.</a> Mol. Ther. 15, 1110-1110 (2007).</li><li>Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bag1+functions+in+vivo+as+a+negative+regulator+of+Hsp70+chaperone+activity.">Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity.</a> Mol. Cell. Biol. 20, 1083-1083 (2000).</li></ol>

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En este trabajo un protocolo para medir la actividad intracelular de replegado de las chaperonas moleculares se presenta. Todo el ensayo se puede realizar en 3 ó 4 días como lo demuestra la visión de la figura. 1. La robustez y la linealidad de la señal de luz producida por la luciérnaga y la luciferasa Renilla representan una base sólida para la reproducibilidad del protocolo. El paso crítico de la prueba es la elección de un reactivo de...

Para este experimento &quot;Luz Vector &#39;una forma luminómetro Perkin Elmer &#39;1420 contador de luminiscencia se utilizó Programas para el luminómetro: Renilla: la bomba 1, 100 l de inyección Tampón de reacción: Pump1, inyectar 70 l Luciferina: Pump2, inyección 30μl Para los buffers de preparar estas soluciones madre con agua destilada: <ul style="list-style:none"><li> - 1M MgSO 4: 246,48 g MgSO 4 * 7H 2 O en 1 litro </li><li> - 0,5 M EGTA: 190,18 g EGTA en 1 litro </li><li> - 1M KH 2 PO 4: 136,09 g KH 2 PO 4 en 1 litro </li><li> - 1M K2 HPO 4: 228,23 g K 2 HPO 4 * 3 H 2 O en 1 litro </li><li> - 5 M NaCl: 292,2 g de NaCl en 1 litro </li><li> - 0,5 M EDTA: 186,12 g EDTA * 2H 2 O en 1 litro &nbsp;</li></ul> Gly-Gly-buffer: <ul style="list-style:none"><li> - 25mm Glycylglycin </li><li> - 15 mM MgSO 4 </li><li> - 4 mm EGTA </li></ul> Para un uso litro a 3,3 g de Glycylglycin, 15 ml de una solución 1M MgSO4 y 8 ml de una solución 0,5 M EGTA. Resolver en el agua, ajustar el pH a 7,8 y llevar a un volumen total de 1 litro. Tampón de reacción para Renilla / Coelenterazine búfer: <ul style="list-style:none"><li> - &amp; Nbsp; 13,4 mM KH 2 PO 4 </li><li> - 86,6 mM KH 2 PO 4 </li><li> - 0,5 M NaCl </li><li> - 1 mM EDTA </li></ul> Para un uso litros 13,4 ml de 1M KH 2 PO 4 solución, 86,6 ml de 1M K 2 HPO 4 solución, 100 ml de una solución de NaCl 5M y 2 ml de una solución 0,5 M EDTA. <table border="1"><tr><td></td><td> Empresa </td><td> Catálogo / Número de producto: </td></tr><tr><td> DMEM (Dulbecco modificado de Eagle Medium 1X) </td><td> GIBCO </td><td> 41966029 </td></tr><tr><td> Oro suero bovino fetal </td><td> PAA </td><td> A15-151 </td></tr><tr><td> Gene X-treme 9 ADN reactivo de transfección </td><td> Roche </td><td&gt; 06365809001 </td></tr><tr><td> PBS Fosfato de Dulbecco solución salina tamponada 1X </td><td> GIBCO </td><td> 14190-094 </td></tr><tr><td> MOPS 3 - (N-morfolino) propanosulfónico </td><td> SIGMA-ALDRICH </td><td> M1254 </td></tr><tr><td> Cicloheximida </td><td> SIGMA-ALDRICH </td><td> C7698 </td></tr><tr><td> Tampón de lisis pasiva 5X </td><td> Promega </td><td> E194A </td></tr><tr><td> Glycylglycin EGTA MgSO 4 (MgSO 4 * 7H 2 O) </td><td> SIGMA-ALDRICH Roth Roth </td><td> G7278 3054.2 P027.1 </td></tr><tr><td> KH 2 PO 4 K 2 HPO 4 (K 2 HPO 4 * 3 H 2 O) NaCl EDTA </td><td> Roth Roth Roth Roth </td><td> 3904.1 6878.2 3957.1 8043.1 </td></tr><tr><td> Luciferina Firefly </td><td> Biosynth </td><td> L8200 </td></tr><tr><td> Coelenterazine </td><td> Biosynth </td><td> C7000 </td></tr><tr><td> DTT (ditiotreitol) </td><td> Roth </td><td> 6908.2 </td></tr><tr><td> ATP (trifosfato de adenosina) </td><td> Roche </td><td> 10519987001 </td></tr></table>

intracellular refolding assay

Este protocolo describe un método para medir la actividad enzimática de las chaperonas moleculares en un sistema basado en células y los posibles efectos de los compuestos con actividad inhibitoria / estimulante. Chaperonas moleculares son proteínas implicadas en la regulación de un plegamiento de proteínas y tienen un papel crucial en la promoción de la supervivencia celular al estrés, como los insultos de choque térmico 2, el hambre de nutrientes y la exposición a productos químicos / venenos 3. Por esta razón, los acompañantes se encuentran para participar en eventos como el desarrollo de tumores, chemioresistance de 4 células de cáncer, así como la neurodegeneración 5. El diseño de pequeñas moléculas capaces de inhibir o estimular la actividad de estas enzimas es por lo tanto, una de las estrategias más estudiadas para el tratamiento del cáncer y enfermedades neurodegenerativas 7 9. El ensayo aquí descrito ofrece la posibilidad de medir la actividad de replegamiento de una chaperona molecular en particular y para estudiar el efecto de un borradorHeridas en su actividad. En este método el gen de la chaperona molecular se investiga transfectadas con un vector de codificación de expresión para el gen de la luciferasa de luciérnaga. Ya se ha descrito que desnaturalizan luciferasa de luciérnaga puede ser replegada por chaperonas moleculares 10,11. Como la normalización de control de transfección, un vector de codificación para el gen de la luciferasa Renilla es transfectadas. Todas las transfecciones se describe en este protocolo se realizan con X-treme Gene 11 (Roche) en células HEK-293. En el primer paso, la síntesis de proteínas es inhibida por el tratamiento de las células con cicloheximida. A partir de entonces se desarrolla la proteína inducida por choque térmico a 45 ° C durante 30 minutos. Sobre la recuperación a 37 ° C, las proteínas son re-doblado en su conformación activa y la actividad de la luciferasa de luciérnaga se utiliza como lectura: la luz se producirá más, la proteína más tendrá recuperó la conformación original. No-calor células sorprendido se establecen como referencia (100% de replegarseluciferasa).

Watch this Scientific Journal Video about Intracellular Refolding Assay at JoVE.com

Intracellular Refolding Assay

En este protocolo un método para medir el replegamiento de proteínas intracelulares tras el choque térmico se describe. Este método puede ser usado para estudiar foldases como chaperones moleculares y sus co-factores o componentes capaces de influir en su actividad. La actividad luciferasa de luciérnaga se utiliza como reportero para medir la actividad de las chaperonas replegado.

Ensayo Replegado intracelular

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds ...

Research

JoVE Journal

Biology

14.1K vistas.  Karlsruhe Institute of Technology. En este protocolo un método para medir el replegamiento de proteínas intracelulares tras el choque térmico se describe. Este método puede ser usado para estudiar foldases como chaperones moleculares y sus co-factores o componentes capaces de influir en su actividad. La actividad luciferasa de luciérnaga se utiliza como reportero para medir la actividad de las chaperonas replegado.

Video: Intracellular Refolding Assay

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Cerca de la medición de la membrana plasmática y Global Dynamics calcio intracelular en los astrocitos

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Investigación

Biología

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Un método para ratón islotes pancreáticos Aislamiento y cAMP intracelular Determinación

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Los ensayos acoplados para Monitoreo replegamiento de proteínas en<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by &#946; and &#947;-secretases. Despite the importance of A&#946; to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.
While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the &#947;-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.
We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments.

While the transport of cell surface proteins is relatively easily studied, visualizing the trafficking of intracellular proteins is much more difficult. Here, we use constructs incorporating photoactivatable GFP and demonstrate a method to accurately follow the amyloid precursor protein from the Golgi apparatus to down-stream compartments and follow its clearance.

Imágenes del tráfico intracelular de APP con GFP fotoactivable

Beta-amyloid (A&#946;) is the major constituent of senile plaques found in the brains of Alzheimer&#8217;s disease patients. A&#946; is derived from the ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

La detección de Intracelular expresión génica en células vivas de murino, humano y porcino Origen Utilizando nanopartículas fluorescencia marcado

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purificación y replegamiento de amiloide fibrillas de (Su)<sub&gt; 6</sub&gt;-etiquetados proteína recombinante Shadoo Expresado como cuerpos de inclusión en<em&gt; E. coli</em

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Identificación de eventos de señalización intracelular inducida en células viables por la interacción con las células vecinas sometidos a la célula apoptótica de la muerte

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Óptica pH cuantificación de intracelular en epitelios de túbulos de Malpighi de Drosophila melanogaster con fluorescentes genéticamente codificado pH indicador

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

Lente libre Video microscopia para la dinámica y el análisis cuantitativo del cultivo de células adherentes

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...