Este trabajo fue financiado por los Institutos Nacionales de Salud de subvención R01DK080971 y la Fundación de Investigación de Diabetes Juvenil subvención 37-2009-44. También agradecemos el apoyo recibido del Centro de Investigación de Diabetes Pediátrica UCSD (PDRC).

1. Transfección de células HEK293 Capa <ol><li> Para preparar el medio celular HEK293 mediante la adición a las botellas de 500 ml de DMEM (con 4,5 g / ml de glucosa y rojo de fenol y sin glutamina): 50 ml de FBS, 5 ml de 100X penicilina / estreptomicina solución, 5 ml 100x solución de L-glutamina y 500 l anfotericina B. </li><li> Placa cabo HEK293 células en una placa de 24 pocillos con 0,5 ml de HEK293 media por pozo. Asegúrese de que las células se distribuyen uniformemente a través de la parte inferior de la placa. </li><li> Cuando las células HEK293 alcanzan el 100% de confluencia, transfectar con 0,8 g de plásmido que codifica la proteína de interés (insertado en un vector de expresión de mamífero) y 2 l de Lipofectamina 2000 de acuerdo con el protocolo de Lipofectamina. Hemos optado por utilizar el pcDNA 3.3 backbone diseñado para la expresión en células de mamífero. </li><li> Opcionalmente, después de 6 horas de incubación, el intercambio de los medios de transfección se describe en el protocolo Lipofectamine HEK293 con medios normales. </li><li> Para evaluar la expresión de la prot transfectadasen, un subconjunto de los pocillos de cultivo de tejidos transfectadas se puede utilizar para la detección de inmunofluorescencia de la proteína expresada o para el análisis de transferencia de Western de expresión de la proteína usando técnicas estándar. </li></ol> 2. Fijación opcional de células HEK293 que expresan la proteína Transfectados Células HEK293 transfectadas se pueden fijar suavemente con el fin de facilitar el co-cultivo en medios que podrían ser perjudiciales para la vida (por ejemplo, células HEK293 paso 3.9 a continuación) o para permitir la eficiente preparación de antemano de las placas de varios experimentos a la vez (véase también la discusión). <ol><li> Llevar a cabo estudios piloto para determinar el curso temporal de la expresión de la proteína en las células HEK293. </li><li> Después de la transfección de la capa de células HEK293, aspirar los medios de comunicación de las células en el punto de tiempo después de la transfección asociado con más alto nivel de expresión. Si esto ocurre dentro de las primeras 24 horas, no aspirar hasta 24 h después de la transfección. </li><li> Lave HEK293 suavemente las células con D-PBS a continuación, añadir 500 l 4% de paraformaldehído (PFA) y se incuban a temperatura ambiente durante 30 min. (Para obtener la solución PFA 4%, comience con una solución de 16% en PBS 1X y se diluye 1:4 con 1X PBS.) </li><li> Usando PBS estéril, lavar suavemente las células 3 veces con PBS y se incuban O / N a 4 ° C. </li><li> Lavar las células 3 veces con PBS estéril y añadiendo PBS y dejar a 4 ° C hasta que se necesite (tiempo máximo de almacenamiento puede variar en función de la proteína expresada. Almacenamos células HEK293 transfectadas para expresar isoformas neuroligin hasta 2 semanas sin cambios significativos en los efectos observados sobre la secreción de la insulina). </li></ol> 3. Co-cultivo de INS-1 las células beta con células HEK293 <ol><li> Preparar INS-1 mediante la adición de medios de comunicación a 500 ml de RPMI 1640 (con glucosa y rojo de fenol y sin glutamina): 50 ml de FBS, 5 ml de 100X penicilina / estreptomicina, 5 ml de 100X solución de L-glutamina, 5 ml de piruvato de sodio, 500 l anfotericina B, 500 l de beta-mercaptoetanol (esto es casi idéntica a la medicinaum usado típicamente para el cultivo de islote primaria a excepción de la adición de beta-mercaptoetanol). </li><li> Uso de la célula-separador (u otro removedor de células no enzimático), INS-1 cosecha de células en aproximadamente el 70-80% de confluencia de la parte inferior de un matraz de cultivo T75. Cada matraz proporcionará suficientes células más o menos de 48 pozos. </li><li> Después de centrifugar las células en la centrifugadora durante 3 min a 1200 rpm, volver a suspender en una mezcla de medios de comunicación HEK INS-1 y 50/50. En un matraz confluente 70-80% de las células INS-1, volver a suspender en aproximadamente 25 ml de técnica mixta. </li><li> Si el uso de células HEK293 que han sido fijados en paraformaldehído en lugar de células vivas HEK293, volver a suspender las células INS-1 en 100% INS-1 media en lugar de técnica mixta. </li><li> Usando una pipeta de 10 ml, desprender las células de la pastilla para hacer una suspensión celular homogénea. </li><li> Aspire para eliminar los medios de comunicación sobre las células HEK293. </li><li> Usando una pipeta P1000, añadir con cuidado 500 l de SIN-1 suspensión celular en la capa de células HEK a lo largo de los lados of pozo. Después de cada 6 pozos, utilizar una pipeta de 10 ml para resuspender las células INS-1 de nuevo para asegurar una suspensión celular homogénea. El esquema general de la co-cultivo de configuración se muestra en la Figura 1. </li><li> Se incuban las células durante 24-48 horas, dependiendo del protocolo experimental. </li><li> Si se requiere período de co-cultivo de más de 48 horas, utilice los pasos opcionales en la sección 2 anterior para fijar las células HEK293. </li><li> Si el uso de roedor disociadas primaria o islotes humanos [por ejemplo, ver los protocolos anteriores en JoVe 11-15], asegurar que un medio apropiado tal como medio RPMI con 5 ml de penicilina / estreptomicina, 5 ml de L-GLUT, 0,5 ml de anfotericina-B se utilizan en lugar de INS-1 media. Aunque se pueden usar células de los islotes disociados en una placa de 24 pocillos, en la práctica, debido a la gran cantidad de islotes potencialmente necesarios, se recomienda que se considere la posibilidad de ampliación a una placa de 48 (que requiere un menor número de células de los islotes por pocillo). En una placa de 48 pocillos, agregar aproximadamente 100 islotes valor de disociaciónted células por placa para empezar. Dependiendo de la eficacia de la disociación y el método utilizado, puede ser necesario un menor número de islotes. </li></ol> 4. La glucosa secreción de insulina estimulada <ol><li> Preincubación de células en 250 l de 2,5 mM de glucosa en KRB (tampón de bicarbonato de Krebs-Ringer) durante un mínimo de 1 hora. Para preincubar, quite medio de cocultivo y reemplazar inmediatamente con KRB. Este intercambio se debe hacer un pozo a la vez para minimizar la exposición de las células INS-1 a oxígeno ambiental. Aspirar con una mano mientras pipetear la baja (2,5 mM) KRB glucosa con el otro. </li><li> Tipo de KRB preincubación a 250 l de o bien 2,5 mM o 20 mM de glucosa en tampón KRB un pozo a la vez. </li><li> Si es apropiado para el experimento específico, se pueden añadir secretagogos adicionales tales como IBMX 100 mM para producir una respuesta secretora de insulina estimulada más robusto. Las células beta de los islotes principales disociadas en particular, son poco sensibles a un aumento de las concentraciones de glucosa alone, por lo que aquí especialmente adición de IBMX o secretagogos de otra debe ser considerada 16,17. </li><li> Después de 1 h de incubación, la transferencia de los medios de comunicación para tubos de microcentrífuga y giran 1.500 g durante 5 min. </li><li> Retire 200 l del sobrenadante y utilizar este para el análisis de insulina por RIA o ELISA. </li><li> Para preparar el lisado de células, añadir 100 ml de RIPA buffer de lisis celular (150 mM de NaCl, 1,0% IGEPALCA-630 o Nonidet P-40, 0,5% de desoxicolato de sodio, 0,1% de SDS, 50 mM de Tris, pH 8,0) con inhibidores de la proteasa a la placa inmediatamente después de la eliminación de los medios de comunicación y se incuba a 4 ° C durante 20 min. </li><li> Centrifugar lisado durante 15 min a 10.000 xg y eliminar 50 l de sobrenadante para el análisis de la insulina por RIA o ELISA. </li></ol>

<ol>
	<li>Kelly, C., McClenaghan, N. H., Flatt, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+islet+structure+and+cellular+interactions+in+the+control+of+insulin+secretion.">Role of islet structure and cellular interactions in the control of insulin secretion.</a> Islets. 3, 41-47 (2011).</li><li>Konstantinova, I., 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=EphA-Ephrin-A-mediated+beta+cell+communication+regulates+insulin+secretion+from+pancreatic+islets.">EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets.</a> Cell. 129, 359-370 (2007).</li><li>Jain, R., Lammert, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+the+endocrine+pancreas.">Cell-cell interactions in the endocrine pancreas.</a> Diabetes Obes. Metab. 11, 159-167 (2009).</li><li>Poitout, V., Olson, L. K., Robertson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-secreting+cell+lines:+classification,+characteristics+and+potential+applications.">Insulin-secreting cell lines: classification, characteristics and potential applications.</a> Diabetes Metab. 22, 7-14 (1996).</li><li>Craig, A. M., Graf, E. R., Linhoff, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+build+a+central+synapse:+clues+from+cell+culture.">How to build a central synapse: clues from cell culture.</a> Trends in Neurosciences. 29, 8-20 (2006).</li><li>Scheiffele, P., Fan, J., Choih, J., Fetter, R., Serafini, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligin+expressed+in+nonneuronal+cells+triggers+presynaptic+development+in+contacting+axons.">Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons.</a> Cell. 101, 657-669 (2000).</li><li>Abderrahmani, A., 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=Neuronal+traits+are+required+for+glucose-induced+insulin+secretion.">Neuronal traits are required for glucose-induced insulin secretion.</a> FEBS Lett. 565, 133-138 (2004).</li><li>Lowe, A. W., Madeddu, L., Kelly, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+secretory+granules+and+neuronal+synaptic+vesicles+have+three+integral+membrane+proteins+in+common.">Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common.</a> The Journal of Cell Biology. 106, 51-59 (1988).</li><li>Suckow, A. T., 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=Expression+of+neurexin,+neuroligin,+and+their+cytoplasmic+binding+partners+in+the+pancreatic+beta-cells+and+the+involvement+of+neuroligin+in+insulin+secretion.">Expression of neurexin, neuroligin, and their cytoplasmic binding partners in the pancreatic beta-cells and the involvement of neuroligin in insulin secretion.</a> Endocrinology. 149, 6006-6017 (2008).</li><li>Suckow, A. T., 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=Transcellular+neuroligin-2+interactions+enhance+insulin+secretion+and+are+integral+to+pancreatic+beta+cell+function.">Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic beta cell function.</a> The Journal of Biological Chemistry. 287, 19816-19826 (2012).</li><li>Kohnert, K. D., Hehmke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+suspensions+of+pancreatic+islet+cells:+a+comparison+of+methods.">Preparation of suspensions of pancreatic islet cells: a comparison of methods.</a> J. Biochem. Biophys. Methods. 12, 81-88 (1986).</li><li>Qi, 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=Human+pancreatic+islet+isolation:+Part+I:+digestion+and+collection+of+pancreatic+tissue.">Human pancreatic islet isolation: Part I: digestion and collection of pancreatic tissue.</a> J. Vis. Exp. (27), e1125 (2009).</li><li>Qi, 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=Human+pancreatic+islet+isolation:+Part+II:+purification+and+culture+of+human+islets.">Human pancreatic islet isolation: Part II: purification and culture of human islets.</a> J. Vis. Exp. (27), e1343 (2009).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> J. Vis. Exp. (7), e255 (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J. Vis. Exp. (50), e2096 (2011).</li><li>Hauge-Evans, A. C., Squires, P. E., Persaud, S. J., Jones, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cell-to-beta-cell+interactions+are+required+for+integrated+responses+to+nutrient+stimuli:+enhanced+Ca2++and+insulin+secretory+responses+of+MIN6+pseudoislets.">Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets.</a> Diabetes. 48, 1402-1408 (1999).</li><li>Spiess, Y., Smith, M. A., Vale, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superfusion+of+dissociated+pancreatic+islet+cells+attached+to+Cytodex+beads.">Superfusion of dissociated pancreatic islet cells attached to Cytodex beads.</a> Diabetes. 31, 189-193 (1982).</li><li>Pham, P. 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=Transient+gene+expression+in+HEK293+cells:+peptone+addition+posttransfection+improves+recombinant+protein+synthesis.">Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.</a> Biotechnol. Bioeng. 90, 332-344 (2005).</li><li>Thomas, P., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEK293+cell+line:+a+vehicle+for+the+expression+of+recombinant+proteins.">HEK293 cell line: a vehicle for the expression of recombinant proteins.</a> J. Pharmacol. Toxicol. Methods. 51, 187-200 (2005).</li><li>Maurisse, R., 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=Comparative+transfection+of+DNA+into+primary+and+transformed+mammalian+cells+from+different+lineages.">Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.</a> BMC Biotechnol. 10, 9 (2010).</li></ol>

No tenemos nada que revelar.

El método de cocultivo descrito aquí proporciona una forma eficaz para determinar la importancia fisiológica de las proteínas de las células beta-superficie, transmembrana específicos, y específicamente de sus dominios extracelulares. Por el cultivo de las células beta o células de insulinoma (tales como las células INS-1 empleadas aquí) en contacto con las células HEK293 que presentan una proteína de interés en la superficie celular, se pueden diseñar experimentos para determinar los efectos extracelular...

Se describe aquí un método para facilitar las investigaciones de cómo los dominios extracelulares de proteínas de transmembrana específicos afectan a la secreción de insulina. Las sondas método de los efectos de las interacciones de la proteína de interés con las proteínas (o, posiblemente, otras moléculas) en la superficie de las células beta pancreáticas. El método permite la investigación de cómo las proteínas de la superficie celular expresadas por las células beta o por otras células vecinas (por ejemplo, células endoteliales, neuronas, células alfa pancreáticas) afectan a la función de las células beta a través de interacciones transcelular (es decir, a través de interacciones con los compañeros de interacción sobre la superficie adyacente de células beta). La membrana plasmática celular contiene una serie compleja de proteínas estructurales y funcionales que sirven como puentes para el medio ambiente extracelular. Por formación de conexiones transcelulares o inicio de eventos de señalización de plástico, las interacciones entre las proteínas de la superficie celular puede ayudar a coordinar lala función de las células vecinas. Las células beta pancreáticas están agrupados juntos dentro de los islotes pancreáticos y actúan de forma coordinada para mantener la homeostasis de la glucosa 1. Como se reveló, por ejemplo, por la importancia de la extracelular EphA-ephrinA y neuroligina-2 interacciones en la regulación de la glucosa estimulada por la secreción de insulina, que se está convirtiendo cada vez más claro que el aumento de los conocimientos de las interacciones entre las proteínas extracelulares que se producen en las superficies adyacentes de células beta serán de gran importancia para obtener una comprensión completa de la secreción de insulina, beta maduración funcional celular y el mantenimiento de la homeostasis de la glucosa 1-3. El objetivo del método descrito aquí es permitir a las investigaciones de los efectos sobre la función de las células beta de las interacciones que implican transcelular transmembrana específico o proteínas de otra manera-de la superficie celular asociadas. Por células beta el co-cultivo con células HEK293 transfectadas con diferentes construcciones de expresión, los efectos sobre el bfunción de las células eta de diferentes proteínas de la superficie celular o variantes mutadas de los mismos puede ser eficiente sondeó. Esto se consigue sin tener que transfectar las células beta a sí mismos. La elucidación de los papeles de las interacciones particulares transcelular por estudios de caída, knockout o la sobreexpresión en células beta en cultivo o in vivo requiere perturbación directa de ARNm de las células beta y la expresión de la proteína, afectando potencialmente salud de las células beta y / o función en formas que podrían confundir los análisis de los efectos de las interacciones extracelulares específicas. Estos enfoques también altera los niveles de los dominios intracelulares de las proteínas específicas y, además, no permiten que los efectos debidos a las interacciones entre las proteínas sobre o dentro de la misma célula debe distinguirse de los efectos de las interacciones transcelular. Aquí, un método para determinar el efecto de las interacciones específicas transcelular en la capacidad de secreción de insulina y la capacidad de respuesta de las células beta es descrIbed. Este método es aplicable a líneas secretoras de insulina de las células beta, tales como las células INS-1, 4 y para primarias de roedor disociadas o células beta humanos. Se basa en modelos desarrollados por cocultivo neurobiólogos, que encontraron que la exposición de las neuronas cultivadas a proteínas neuronales específicas expresadas en células HEK293 (o COS) capas de células podría identificar las proteínas que la unidad de formación de sinapsis 5,6. Dadas las similitudes entre el mecanismo de secreción de las sinapsis neuronales y de las células beta, que razonó que la función de las células beta y la maduración funcional podrían ser impulsados ​​por las interacciones transcelulares similares 7-9. Con el fin de sondear estas interacciones, hemos desarrollado el sistema descrito en este documento en el que las células beta se co-cultivaron sobre una capa de células HEK293 que expresan una proteína de interés 10. Este sistema permite que el citoplasma de las células beta de permanecer intacta mientras extracelulares interacciones proteína-proteína son manipulados.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

Las interacciones entre las proteínas de la superficie celular ayudan a coordinar la función de las células vecinas. Las células beta pancreáticas se agrupan dentro de los islotes pancreáticos y actúan de forma coordinada para mantener la homeostasis de la glucosa. Cada vez es más claro que las interacciones entre las proteínas transmembrana en las superficies de las células beta adyacentes son determinantes importantes de la función de las células beta. La elucidación de los papeles de las interacciones particulares transcelular por estudios de caída, knockout o la sobreexpresión en células beta en cultivo o in vivo requiere perturbación directa de ARNm y la expresión de la proteína, afectando potencialmente a la salud de las células beta y / o función en formas que podrían confundir los análisis de los efectos de interacciones específicas. Estos enfoques también alteran los niveles de los dominios intracelulares de las proteínas específicas y pueden prevenir los efectos debidos a las interacciones entre las proteínas dentro de la misma membrana celular para ser disdistinguirse de los efectos de las interacciones transcelular. Aquí se presenta un método para determinar el efecto de las interacciones específicas transcelular en la capacidad de secreción de insulina y la capacidad de respuesta de las células beta. Este método es aplicable a las líneas de las células beta, como INS-1 en las células, y las células beta primarios disociados. Se basa en modelos desarrollados por cocultivo neurobiólogos, que encontraron que la exposición de las neuronas cultivadas a las proteínas neuronales específicas expresadas en células HEK293 (o COS) capas de células de proteínas identificadas importantes para la conducción de la formación de sinapsis. Dadas las similitudes entre el mecanismo de secreción de las sinapsis neuronales y de las células beta, que razonó que la maduración funcional de las células beta podría ser impulsada por las interacciones transcelulares similares. Hemos desarrollado un sistema en el que las células beta se cultivan sobre una capa de células HEK293 que expresan una proteína de interés. En este modelo, el citoplasma de las células beta está sin tocar mientras extracelular INTERACCIO proteína-proteínans son manipulados. Aunque aquí nos centraremos principalmente en los estudios de la glucosa estimulada por la secreción de insulina, otros procesos pueden ser analizados, por ejemplo, cambios en la expresión génica según lo determinado por inmunotransferencia o qPCR.

Usando el método descrito aquí, se ha probado el efecto de diferentes variantes de la proteína neuroligina sobre la secreción de insulina. Esto complementa nuestro trabajo publicado investigar el efecto de neuroligina-2 en la función de las células beta 10. Figura 2, por ejemplo, representa los resultados obtenidos a partir de INS-1 cocultivo de las células beta con células HEK293 transfectadas para expresar una isoforma neuroligina se hace referencia aquí como NL- X. Este experiment...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interacciones proteína transcelular son determinantes importantes de la función de las células beta pancreáticas. Detalladas aquí es un método adaptado de un modelo de co-cultivo de sinaptogénesis-para investigar cómo las proteínas transmembrana específicos influyen en la secreción de insulina. Células HEK293 transfectadas expresan proteínas de interés; células beta no necesitan ser transfectadas o de otro modo perturbado directamente.

Análisis Coculture de interacciones entre proteínas extracelulares que afectan a la secreción de insulina por las células beta del páncreas

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

coculture-analysis-extracellular-protein-interactions-affecting

Research

JoVE Journal

Medicine

12.8K vistas.  University of California, San Diego. Interacciones proteína transcelular son determinantes importantes de la función de las células beta pancreáticas. Detalladas aquí es un método adaptado de un modelo de co-cultivo de sinaptogénesis-para investigar cómo las proteínas transmembrana específicos influyen en la secreción de insulina. Células HEK293 transfectadas expresan proteínas de interés; células beta no necesitan ser transfectadas o de otro modo perturbado directamente.

Video: Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Mejora de la administración IV de la insulina en un hospital de la comunidad

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Investigación

Medicina

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema Coculture con un cerebro rebanada organotípicos y 3D esferoide de células de carcinoma

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Humano Dupuytren<em&gt; Ex Vivo</em&gt; Cultura para el Estudio de miofibroblastos y matriz extracelular Interacciones

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Neurodegeneración en un modelo animal de beta-amiloide crónica Oligómero de infusión es contrarrestado por el anticuerpo Tratamiento Con una infusión de bombas osmóticas

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Application of End-to-end Anastomosis in Robotic Central Pancreatectomy

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of pancreatic body. With technological development, the robotic surgical system has shown its advantage in minimally invasive surgery and been increasingly applied in central pancreatectomy. However, reconstruction of the continuity of pancreas with end-to-end anastomosis after robotic central pancreatectomy has not been applied. In this study, we report surgical techniques for robotic central pancreatectomy with end-to-end anastomosis. The pancreas is reconstructed by duct-to-duct anastomosis of the pancreatic duct with a pancreatic stent inserted in the two stumps of pancreatic duct, and by end-to-end anastomosis of the pancreatic parenchyma. Compared with traditional central pancreatectomy with pancreaticoenteric anastomosis, this approach decreases the operative injury to the patient, and also conserves the integrity and continuity of the digestive duct and pancreatic duct. The robotic surgical system integrated with multiple instruments with flexible and precise movement is particularly suitable for the dissection and reconstruction of the pancreatic duct. We found that robotic central pancreatectomy with end-to-end anastomosis is safe and feasible, and we need more experience to evaluate its best indications and long-term outcomes.

The robotic central pancreatectomy with end-to-end anastomosis is feasible and safe for tumor in the pancreatic neck and proximal portion of pancreatic body. The operative techniques of this operation are presented.

Aplicación de End-to-end Anastomosis en robótica Pancreatectomía Central

Central pancreatectomy is carried out for the treatment of benign or low-malignant potential tumors located in the pancreatic neck or proximal part of ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Homogeneous Time-resolved F&amp;#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Homogénea tiempo resuelto Förster Resonancia energía basada en la transferencia de ensayo para la detección de la secreción de insulina

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Aislamiento de hepatocitos primario de ratón para proteína naciente síntesis análisis por el método de etiquetado no radiactivo L-azidohomoalanine

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Bio-energética investigación de Candida albicans utilizando en tiempo real extracelular flujo análisis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...