Estamos agradecidos al Dr Stephen Robinson y la Sra. Hania Czerwinska (Universidad de Monash) por la generosa oferta de cultivos de astrocitos.

1. Determinar ascorbato intracelular en células cultivadas <ol><li> Cultivo de células y la cosecha <ol><li> Crecer de suspensión (por ejemplo, la eritroleucemia humana K562) o células adherentes (por ejemplo, los astrocitos primarios) utilizando procedimientos estándar de cultivo 19-21,32,38. Nota: para garantizar las células contienen ascorbato, se cargan las células cultivadas con ascorbato, ya sea como ascorbato o DHA 33,39. </li></ol></li><li> Crear un extracto celular que contiene ascorbato- <ol><li> Incubar un número apropiado de solución salina tamponada con fosfato de suspensión (PBS), se lavó o células adherentes con 450 l de helado celular permeabilización Buffer [CPB; 0.1% (w / v) de saponina en PBS; 4 º C]. Nota: Determinar el número de células requeridas para obtener un valor de absorbancia en el intervalo lineal del ensayo (véase más adelante) empíricamente por el usuario final. Nota: Los extractos de tejidos también pueden utilizarse (véase la discusión para más detalles). </li><li> Agitacélulas TE en hielo durante 10 minutos para asegurar la lisis celular completa. </li><li> Crear un extracto intracelular que contiene ascorbato-aclarado mediante la eliminación de los desechos celulares por centrifugación del lisado crudo a 16.000 × g durante 5 min en una microcentrífuga refrigerada (4 º C). </li><li> Retire cuidadosamente 4 x 100 ml de alícuotas de cada muestra y añadir a una secuencia horizontal de pozos en una placa de fondo plano de 96 pocillos que contiene ya sea 25 l / pocillo de PBS (&quot;-AO&quot;) o sólo 25 l / pocillo de 45.5 U / ml solución madre de L-ascorbato-oxidasa (AO) en PBS (&quot;+ AO&quot;). Nota: esto debe hacerse en paralelo con la preparación de los estándares (ver más abajo). </li></ol></li><li> Construcción estándar de la curva ascorbato (debe ser construido de nuevo para cada ensayo) <ol><li> Preparar una solución madre de 10 mM de ácido ascórbico en helado de PBS. Nota: Verifique la concentración de ascorbato por espectrofotometría utilizando una cubeta de cuarzo con un 1cm de longitud de vía a 265 nm (coeficiente de extinción = 14,5 mM -1 cm -1) 40. </li><li> Prepare cuidadosamente una serie de normas de ascorbato de entre 0 y 20 mM. </li><li> En paralelo con el paso 1.2.4, retirar con cuidado 4 × 100 ml de alícuotas de cada estándar y añadir a una secuencia horizontal de pozos en una placa de fondo plano de 96 pocillos que contiene 25 l / pocillo de PBS (&quot;-AO&quot;) o un 45,5 U / ml solución madre de AO en PBS (&quot;+ AO&quot;). Nota: 100 l de los estándares de ascorbato anteriores contendrán 0-2 nmol de ascorbato. Tenga en cuenta, el objetivo de la AO es para el control de la contribución de reductores no-ascorbato a ferricianuro reducción. </li></ol></li><li> Determinar ascorbato intracelular - Paso 1: eliminar selectivamente ascorbato y cuantitativamente oxidar ascorbato con ferricianuro <ol><li> Mezclar orbitalmente la placa de 96 pocillos a 550 rpm a temperatura ambiente durante 5 minutos en la oscuridad, cubriendo la placa en papel de aluminio. Nota: Tsu paso debe oxidar todo ascorbato en los pozos &quot;+ AO&quot; a DHA. Los pozos &quot;AO&quot; no se verán afectados. </li><li> Añadir 50 l de 3,5 mM ferricianuro de potasio (en lo sucesivo denominadas &quot;ferricianuro&quot;) en PBS a todos los pocillos. La final [ferricianuro] = 1 mM. Nota: Utilice un multipipettor. </li><li> Mezclar orbitalmente la placa a 550 rpm a temperatura ambiente durante otros 5 minutos en la oscuridad. Nota: Este paso debe resultar en la reducción de ferricianuro a ferrocianuro por el ascorbato en una relación estequiométrica de 2 moléculas de ferricianuro reducido por 1 molécula de ascorbato oxidado. </li><li> Inmediatamente después, añadir 25 l de una solución recién construido que contiene 50% (v / v) de ácido acético y 30% (w / v) de ácido tricloroacético (TCA). Nota: Utilice un multipipettor. </li></ol></li><li> Determinar ascorbato intracelular - Paso 2: cuantificar la cantidad de ferrocianuro formado 37 <ol><li> Añadir 100 l de solución de ferrocianuro de determinación del 37 al eada bien. Nota: Una solución de trabajo debe hacerse inmediatamente antes de su uso: 2 ml de 3 M acetato sódico (pH 6,0); 0,5 ml de ácido acético glacial (~ 17,4 M de ácido acético); 2 ml de ácido cítrico 0,2 M; 2 ml de 3,3 mM FeCl3 en ácido acético 0,1 M; 1 ml de 30 mM ferene-S. El volumen final de esta solución de trabajo debe ser 7,5 ml. </li><li> Mezclar orbitalmente la placa en la oscuridad a 550 rpm durante 30 min a temperatura ambiente. </li><li> Leer los valores de absorbancia de los pocillos a 593 nm (es decir, el máximo de absorbancia de la Fe (II) (Metodo-S) 3 complejo). </li><li> Calcular la cantidad de ascorbato intracelular como nmoles ascorbato por millón de células restando inicialmente los valores de A 593 nm para los pozos de la &#39;+ AO&#39; de los correspondientes - pozos &#39;AO&#39; para cada muestra y luego la interpolación de la curva estándar ascorbato (véase el paso 1.3 ). Al construir la curva estándar, trazar este &quot;valor de la diferencia&quot; para cada estándar frente a la cantidad de ascorbcomió por pocillo. </li></ol></li></ol> 2. Determinación de ascorbato-eflujo de células cultivadas <ol><li> Cultivo de células y la cosecha <ol><li> Crecer suspensión o células adherentes que la anterior (véase el paso 1.1). Nota: llevar a cabo el ensayo a continuación en un formato de plataforma de 24 pocillos con la suspensión y las células adherentes para obtener mejores resultados. </li><li> Volver a suspender las células en suspensión, o superponer las células adherentes, con 400 l de solución salina tamponada con HEPES precalentado, con o sin calcio y magnesio, y que contiene 5 mM de D-glucosa (HBS / D, pH 7,3, 37 ° C) en pozos de una placa de 24 pocillos. Añadir el mismo volumen de HBS / D para pozos sin células especificados en cada placa de 24 pocillos para ser examinados. Nota: este último servirá como controles libres de células para la reacción de la línea de base (ver más abajo). </li></ol></li></ol> 3. Determinar la cantidad de ascorbato de lanzamiento <ol><li> Las siguientes soluciones madre deben prepararse de antemano: 120U / ml en HBS AO / D (preparar fresco); 2,4 mM Ferene-S en HBS / D; 120 M de FeCl3 y 600 mM Na-citrato en HBS / D (preparar inmediatamente de soluciones de reserva más concentradas). </li><li> Para iniciar la reacción ferrireduction (volumen final por pocillo debe ser de 600 l), añadir los siguientes volúmenes de los reactivos a los pocillos individuales que ya contienen 400 l de HBS / D: <ol><li> Añadir 50 l de AO (120 U / ml) o HBS / D para emparejado pozos por triplicado. La concentración final AO debe ser de 10 U / ml. Nota: Label emparejado pozos como &quot;- AO&quot; y &quot;+ AO&quot;. </li><li> Mezclar las placas con mezcla orbital suave durante 5 min a 37 ° C. </li><li> Añadir 50 l de 2,4 mM de fereno-S a todos los pocillos. La concentración final ferene-S debe ser de 200 mM. Mezclar las placas como anteriormente durante 5 min a 37 ° C. </li><li> Añadir 50 l de 120 mM recién preparada citrato férrico. El hierro final y concentraciones de citrato de hierro deben ser 10 mM y 50 mM citrato. Mezclar bien. </li></ol&gt; </li><li> En tres pocillos de control por triplicado, aspirar el medio suprayacente y añadir 600 l de HBS / D que contiene 0,1% de saponina. Nota: Estos servirán como &quot;100% de lisis celular&quot; controles de lactato deshidrogenasa (LDH) ensayo de liberación que se realizará en paralelo (véase más adelante). </li><li> Incubar durante 60 min en la oscuridad a 37 ° C. </li><li> Al final del ensayo de ascorbato de flujo de salida, aspirar rápidamente 500 l de cada pocillo y añadir a etiquetado adecuadamente pozos en una placa de 24 pocillos. Nota: para células en suspensión, eliminar inicialmente las células por centrifugación a 4 ° C. </li><li> Añadir 300 ml de alícuotas del sobrenadante a una placa de 96 pocillos y luego leer a 593 nm. </li></ol> 4. Determinación de ascorbato extracelular <ol><li> Lea los valores de absorbancia de los pocillos a 593 nm. </li><li> Calcular la cantidad de ascorbato extracelular como nmoles de ascorbato por mg de proteína (o por millón de células) como se describe para la determinación de ascorbato intracelularen el Paso 1.5.4. Nota: el usuario final debe optimizar las condiciones de modo que la liberación de ascorbato no está limitada por el ascorbato intracelular. </li></ol> 5. Determinación de la LDH de lanzamiento <ol><li> Con los 200 l restantes de solución extracelular de cada muestra de llevar a cabo un ensayo de liberación de LDH 32,41 para determinar el grado de lisis celular. Nota: el cálculo en% del total de LDH liberable. Determinar la LDH liberable de las muestras que se trataron con 0,1% de saponina. </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+sensitive+assay+for+ascorbate+using+a+plate+reader.">A simple and sensitive assay for ascorbate using a plate reader.</a> Anal. Biochem. 365, 31-39 (2007).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+colorimetric+microplate+ferrocyanide+assay+applied+to+ascorbate-stimulated+transplasma+membrane+ferricyanide+reduction+and+mitochondrial+succinate+oxidation.">A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation.</a> Anal. Biochem. 373, 287-295 (2008).</li><li>Lane, D. J. R., Robinson, S. R., Czerwinska, H., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Na+/H++exchangers+and+intracellular+pH+in+regulating+vitamin+C-driven+electron+transport+across+the+plasma+membrane.">A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane.</a> Biochem. J. 428, 191-200 (2010).</li><li>Corti, A., Casini, A. F., Pompella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+for+transport+and+efflux+of+ascorbate+and+dehydroascorbate.">Cellular pathways for transport and efflux of ascorbate and dehydroascorbate.</a> Arch. Biochem. Biophys. 500, 107-115 (2010).</li><li>Laroff, G. P., Fessenden, R. W., Schuler, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electron+spin+resonance+spectra+of+radical+intermediates+in+the+oxidation+of+ascorbic+acid+and+related+substances.">The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances.</a> J. Am. Chem. Soc. 94, 9062-9073 (1972).</li><li>Dringen, R., Kussmaul, L., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detoxification+of+exogenous+hydrogen+peroxide+and+organic+hydroperoxides+by+cultured+astroglial+cells+assessed+by+microtiter+plate+assay.">Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.</a> Brain Res. Brain Res. Protoc. 2, 223-228 (1998).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplasma+membrane+electron+transport+comes+in+two+flavors.">Transplasma membrane electron transport comes in two flavors.</a> Biofactors. 34, 191-200 (2009).</li><li>Lin, S., Lin, D. C., Flanagan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+effects+of+cytochalasin+B+on+transport+and+motile+processes.">Specificity of the effects of cytochalasin B on transport and motile processes.</a> Proc. Natl. Acad. Sci. USA. 75, 329-333 (1978).</li><li>May, J. M., Qu, Z. C., Juliao, S., Cobb, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+decreases+oxidant+stress+in+endothelial+cells+caused+by+the+nitroxide+tempol.">Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.</a> Free Radic. Res. 39, 195-202 (2005).</li><li>Avron, M., Shavit, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+simple+method+for+determination+of+ferrocyanide.">A sensitive and simple method for determination of ferrocyanide.</a> Anal. Biochem. 6, 549-554 (1963).</li></ol>

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En este trabajo se presentan dos ensayos de microplaca colorimétricos rápidos, concretos y relativamente sensibles para la determinación de ascorbato derivado de los compartimentos intra y extracelulares en las células cultivadas. Los ensayos pueden ser completados con el acceso a equipos de laboratorio estándar y los reactivos. El reactivo sólo moderadamente costosa requerida para el ensayo es AO, que es esencial, ya que imparte un alto grado de especificidad analito hacia L-ascorbato.</sp...

El descubrimiento de la naturaleza química del ácido ascórbico (vitamina C), y su identificación como el largamente buscado &quot;factor anti-escorbuto&quot;, de Albert Szent-Györgyi y otros en los artículos publicados desde 1928 hasta 1934 1 han sido hitos importantes en la historia de la bioquímica. De hecho, estos descubrimientos han contribuido a Szent-Györgyi siendo galardonado con el Premio Nobel de Fisiología o Medicina en 1937. La suite en constante expansión de los roles de ascorbato en la fisiología animal y vegetal, así como la salud humana, siguen siendo los temas de activo científica investigación y controversia. L-ascorbato es una abundante reductor fisiológica y la enzima cofactor en sistemas de mamíferos, y contribuye a numerosas reacciones enzimáticas bien definidas que implican la hidroxilación del colágeno, carnitina y la biosíntesis de norepinefrina, metabolismo de la tirosina y de la hormona péptido amidación 2. Curiosamente, evide montajena vez que sugiere que el ascorbato juega un papel en la estimulación de otras dioxigenasas dependientes de hierro, tales como las prolil hidroxilasas y asparaginilo implicados en la hidroxilación y la orientación de los factores inducibles por hipoxia (HIF) 1α y 2α 3. Un informe reciente sugiere que el ascorbato juega un papel en la maduración de células T a través de desmetilación que afecta a la cromatina a través de su actividad en la estimulación de las hidroxilasas nucleares, Jumonji C (JmjC) dominio de las proteínas; el último de los cuales parecen requerir ascorbato para la plena actividad 4. De hecho, la estimulación de tales enzimas por el ascorbato parece ocurrir por un mecanismo similar a la estimulación por el ascorbato de las hidroxilasas de HIF y de colágeno. Entre otros efectos clásicos, ascorbato contribuye significativamente a la antioxidación celular como una cadena-romper eliminador de radicales soluble en agua 5 y para el reciclaje de membrana plasmática α-tocoferol (vitamina E) a través de la reducción de la α-tocoferoxilo radical 6, Which es importante en la protección contra la peroxidación lipídica de la membrana 7. Es importante destacar que, aunque la mayoría de los mamíferos son capaces de la síntesis hepática de novo de ascorbato a partir de D-glucosa, primates superiores, conejillos de indias y algunos murciélagos dependen de fuentes dietéticas de la vitamina 8. Esto es debido a la inactivación del gen GULO, los ortólogos de mamíferos que en no afectados codifican la enzima, γ-gulono-lactona oxidasa 9-13. Esta enzima es necesaria para la reacción final en la biosíntesis de ascorbato a partir de glucosa 13. Después de la absorción mediada por transportador desde la luz intestinal en los seres humanos, ascorbato se distribuye por todo el cuerpo por el sistema circulatorio. La vitamina se encuentra típicamente en su forma reducida en concentraciones milimolares intracelularmente (con la notable excepción de los eritrocitos en la que las concentraciones son típicamente similar a la concentración de plasma que prevalece), y en HCl conc micromolarentrations (por ejemplo 50-200 micras) en la mayoría de los fluidos extracelulares 14,15. En condiciones fisiológicas, ascorbato típicamente se somete a una oxidación de un electrón reversibles a la libre radical ascorbilo (AFR; también conocido como monodehydroascorbate o semidehydroascorbate). Mientras que la AFR es un radical relativamente estable 16, en ausencia de su rápida reducción enzimática de un electrón de nuevo a ascorbato, dos AFR pueden más dismutan a uno ascorbato y uno deshidroascorbato (DHA) 9,13,17. En el interior de la célula, el producto de oxidación de dos electrones de ascorbato, el DHA, se puede reducir rápidamente de nuevo a ascorbato por el glutatión-y NAD (P) H-enzimática dependiente y reacciones no enzimáticas 13. Si bien se acepta que clásicamente único papel importante de ascorbato en el metabolismo del hierro es el de estimular la absorción dietética de hierro no hemo 18, nosotros y otros han aportado pruebas de strongly lo que sugiere que el ascorbato juega un papel ampliado en gran medida en el metabolismo de este metal. En primer lugar, ascorbato que se libera por las células-ascorbato repleta parece jugar un papel importante en la modulación de la absorción de hierro no unido a transferrina por las células 19,20, y la evidencia reciente indica que el ascorbato también modula la absorción de hierro unido a transferrina por las células 21, el último de los cuales corresponde a una ruta principal de hierro-captación fisiológica 22. Ascorbato es esencial para la función normal del sistema nervioso central de los mamíferos 23,24. Junto con la corteza suprarrenal, glándula pituitaria, el timo, la retina y el cuerpo lúteo, el cerebro contiene altas concentraciones de ascorbato en relación con otros tejidos del cuerpo 23,25-27. Además, la exposición de ambos astrocitos 28,29 y neuronas como las células 30 al glutamato se sabe para accionar la liberación de ascorbato en el espacio extracelular, donde el ascorbate se cree que ayuda a proteger a las neuronas contra el glutamato inducida por la disfunción neuronal 31. Aunque el mecanismo exacto de glutamato inducida por la liberación de ascorbato de astrocitos se desconoce, se ha proporcionado recientemente pruebas que indican la implicación de células inflamación causada por la captación de glutamato por el glutamato de los astrocitos y transportador de aspartato (GLAST, también conocida aminoácido excitador transportador isoforma 1 [EAAT1 ] en los seres humanos) y la consiguiente activación de osmolíticas y aniones canales sensibles al volumen (VSOACs) que son permeables a las pequeñas aniones orgánicos tales como ascorbato 32. Las identidades moleculares de los conductos de membrana de plasma implicados en la formación VSOAC quedan aún por identificar 33,34. Aunque muchos ensayos se han desarrollado para la determinación de ascorbato en muestras biológicas, que incluyen ensayos espectrofotométricos, fluorométricos y cromatográficas 35,36, existe mucha variabilidad en la especificidad, la sensibilidad, interference por contaminantes químicos, rango lineal efectiva y la estabilidad del analito punto final. Además, otros factores importantes que influyen en la elección de ensayo son la rapidez, la facilidad de uso y acceso a equipos relativamente especializada, como la cromatografía líquida de alta resolución (HPLC) aparato. Aquí presentamos un ensayo simple y altamente específico de microplacas colorimétrico para la determinación de ascorbato intracelular en células cultivadas, así como un ensayo separado para la determinación de ascorbato-flujo de salida a partir de células cultivadas. El último ensayo tiene como objetivo evitar el problema de la subestimación de la liberación de ascorbato a partir de células debido a la rápida re-absorción de ascorbato liberado por los transportadores de ascorbato de sodio-dependiente (SVCTs). Aunque ambos métodos han aparecido en algunas de nuestras publicaciones anteriores 19,20,32,37,38, este manuscrito proporciona un conjunto explícito de instrucciones y directrices para su ejecución efectiva.

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			<td height="60" style="height:60px;width:284px;">Nunc 96-well flat-bottom plates</td>
			<td style="width:135px;">Thermo</td>
			<td style="width:127px;">269620</td>
			<td style="width:132px;">Any flat-bottom 96-well plate can be used</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:284px;">Refrigerated benchtop microcentrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5415D</td>
			<td style="width:132px;">A non-refrigerated microcentrifuge that has been equilibrated to temperature in a cold room can also be used</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:74px;width:284px;">Refrigerated bench-top centrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5810R</td>
			<td style="width:132px;">Swing-bucket</td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:284px;">Bio-Rad Benchmark Plus Microplate Spectrophotometer</td>
			<td style="width:135px;">Bio-Rad</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Any microplate spectrophotometer capable of reading at 593 nm can be used and is recommended. If a filter-based plate reader is used, choose the closest wavelength possible and use the standard-curve method.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:284px;">Ependorf MixMate (microplate orbital mixer)</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">This is a very versatile and reliable microplate mixer and works very well for these assays</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;"></td>
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			<td></td>
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			<td height="20" style="height:20px;width:284px;">General-purpose buffers</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Phosphate-buffered saline (PBS), pH 7.4</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:284px;">MOPS-buffered saline (MBS); 137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">MBS + 5 mM D-glucose (MBS/D)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:284px;">HEPES-buffered saline + 5 mM D-glucose (HBS/D); 137 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2&#8226;2 H2O, 0.8 mM MgSO4&#8226;7 H2O, 5 mM D-glucose, 20 mM HEPES-Na+, pH 7.3)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Cell permeabilisation buffer (CPB; 0.1% saponin in PBS)</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">General chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">L-ascorbic acid or sodium L-ascorbate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Highest purity preparations should be obtained</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Dehydro-L-ascorbic acid (DHA) dimer</td>
			<td>Sigma-Aldrich</td>
			<td>30790</td>
			<td style="width:132px;">Aqueous solutions theoretically yield 2 moles of DHA monomer per mole of DHA dimer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Cytochalasin B</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">C6762</td>
			<td style="width:132px;">Stock solutions prepared in DMSO or ethanol</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Ascorbate oxidase (AO)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">A0157</td>
			<td style="width:132px;">Stock solutions (120 U/ml) can be prepared in PBS or MBS and then frozen in aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Potassium ferricyanide (FIC)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">455989</td>
			<td style="width:132px;">Trihydrate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Ferene-S (3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5&#8242;,5&#8242;&#8242;-disulfonic acid disodium salt)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">92940</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Sodium L-glutamate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">L-glutamine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Saponin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">47036</td>
			<td style="width:132px;">Prepare a 0.1% stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Stock solutions for intracellular ascorbate determination assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">3 M sodium acetate (pH 6.0)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Glacial acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.2 M citric acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">3.3 mM FeCl3 in 0.1 M acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">30 mM ferene-S</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">50% (v/v) acetic acid + 30% (w/v) trichloroacetic acid (TCA)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stock solutions for ascorbate-efflux assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">AO (120 U/ml)</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">2.4 mM ferene-S</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.12 mM FeCl3 in 0.6 mM sodium-citrate</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
	</tbody>
</table>

a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells

La vitamina C (ascorbato) desempeña numerosos papeles importantes en el metabolismo celular, muchos de los cuales sólo han salido a la luz en los últimos años. Por ejemplo, dentro del cerebro, ascorbato actúa de una manera neuroprotector y neuromodulador que implica el ciclismo ascorbato entre las neuronas y astrocitos vecinales - una relación que parece ser crucial para la homeostasis ascorbato cerebro. Además, la evidencia emergente sugiere fuertemente que el ascorbato tiene un papel ampliado en gran medida en la regulación del metabolismo del hierro celular y sistémica que se reconoce clásicamente. El creciente reconocimiento del papel integral de ascorbato en la fisiología celular y del organismo normal y desregulado exige un rango de mediano rendimiento y alta sensibilidad técnicas analíticas que se pueden ejecutar sin la necesidad de equipo especializado altamente costoso. Aquí proporcionamos instrucciones explícitas para un medio de rendimiento, ensayo específico y relativamente barato microplaca para la determinación de la boª ascorbato intra y extracelular en cultivos de células.

Determinación de ascorbato intracelular en las células cultivadas en suspensión En el primer ensayo (Figura 1), ascorbato intracelular se determina, después de ascorbato-específica (es decir, AO-sensible) reducción de ferricianuro a ferrocianuro, utilizando la determinación altamente sensible de ferrocianuro por un procedimiento publicado previamente 37. La detección de ascorbato se basa en la quelación colorimétrico de hierro ferroso que se genera...

Watch this Scientific Journal Video about A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells at JoVE.com

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

El ascorbato desempeña numerosos papeles importantes en el metabolismo celular, muchos de los cuales sólo han salido a la luz en los últimos años. Aquí se describe un rendimiento medio, ensayo específico y de bajo costo de microplacas para la determinación tanto de ascorbato intra y extracelular en cultivos de células.

Un ensayo rápido y específico de microplacas para la Determinación de ascorbato intra y extracelular en células cultivadas

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

a-rapid-specific-microplate-assay-for-determination-intra

Research

JoVE Journal

Biology

13.4K vistas.  University of Sydney.  El ascorbato desempeña numerosos papeles importantes en el metabolismo celular, muchos de los cuales sólo han salido a la luz en los últimos años. Aquí se describe un rendimiento medio, ensayo específico y de bajo costo de microplacas para la determinación tanto de ascorbato intra y extracelular en cultivos de células. 

Video: A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Contar y determinar la viabilidad de las células cultivadas

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Investigación

Biología

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

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, ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

Un rápido, método escalable para el aislamiento, estudio funcional, y análisis de la matriz extracelular derivado de células

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Una interfaz gráfica de usuario para el seguimiento asistido por software de la concentración de proteínas en protones dinámicos celulares

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Expresión transitoria y localización celular de proteínas recombinantes en células cultivadas de insectos

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Ensayo de co-inmunoprecipitación utilizando proteínas nucleares endógenas de las células cultivadas bajo condiciones hipóxicas

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...