Los autores desean agradecer a los Dres. Rick Ryan, Umesh Patel, hasta que Jeff Hsu Mateo, Mistry Jehangir y Michele Hatler de apoyo durante el desarrollo de estos proyectos y los protocolos.

Preparación de células: <ol><li> Antes de la siembra de células de las neuronas ensayo de cultivo, / los astrocitos en medios de cultivo hasta ~ confluencia del 70-80% (menos que la siembra directa de células de deshielo o aislamiento). </li><li> Separar las células de los frascos de cultivo / placas mediante el método adecuado para el tipo de células de interés. Si es necesario, cubra los pozos de la placa de ensayo con la proteína de la matriz poli-D-lisina o extracelular para mejorar la adhesión celular. Co-cultivos de astrocitos y neuronas se pueden sembrar de forma simultánea o secuencial. De cabezas de serie secuencial, placas de astrocitos por primera vez como &quot;basal&quot; de la capa se recomienda, seguido por la chapa y la diferenciación neuronal, si es necesario. Ajustar la densidad de células según sea apropiado para el tipo de células, el tiempo de la cultura posterior, los parámetros de interés, etc Dependiendo de la edad la cultura, fuente de células y la densidad de siembra, los cultivos primarios pueden variar considerablemente en la tasa de proliferación, la expresión de GFAP o crecimiento de las neuritas - es importante caracterizar y optimizar su sistema de células para proporcionar mayor relevancia biológica para su modelo experimental, así como para proporcionar imágenes de efectivos, la segmentación y el análisis de uso de HCS (ver Figura 1). Después de añadir las células a la placa (las células pueden ser sembradas en los medios de comunicación 90 l, para facilitar el tratamiento con la toxina como se describe en el Paso 3 a continuación), permiten que la placa de sentarse en una superficie plana a temperatura ambiente durante 15-30 minutos, lo que permite una distribución uniforme de la célula. Después de este período, las células se incuban en un medio de cultivo (37 ° C / 5% CO 2) por lo menos 24 horas, luego cambiar a la diferenciación de las condiciones de cultivo (por ejemplo, niveles séricos bajos / NGF de las células PC12), según corresponda. Al cultivo hasta que las células alcanzan el nivel deseado de confluencia o la diferenciación, el cambio de los medios de comunicación a intervalos apropiados para el tipo de célula. </li><li> Los tratamientos con células (compuestos de control, los compuestos de ensayo, etc) se puede introducir en cualquier momento durante este período de la cultura, en su caso por el tiempo-curso de tratamiento de los intereses. Acrilamida, peróxido de hidrógeno y K-252a se proporcionan en forma de compuestos de control neurotóxicos. Suficientes reactivos se proporcionan para los duplicados de 12 puntos curvas de dosis-respuesta (incluyendo una dH 2 O o conjunto DMSO-control dentro de la relación dosis-respuesta) para los cinco placas de 96 pozos. Los compuestos se proporcionan en la concentración de 250X (para K-252a, en el supuesto de un tratamiento máximo de 1 mM) o 10 veces (de la acrilamida y el peróxido de hidrógeno, suponiendo que los tratamientos máximo de 100 mm y 10 mm, respectivamente). La preparación de tratamiento recomendado consiste en media-log (1: √ 10) series de dilución del compuesto 250X en DMSO, seguido de dilución en tampón de dilución del compuesto 10 veces (10 veces más compuestos de control de origen en dH 2 O puede ser diluido en serie directamente en estériles dH 2 O o tampón de dilución del compuesto). 10 l de cada tratamiento y luego se pueden añadir a los 90 l de medios de cultivo ya está presente en cada pozo, para una concentración final 1X (0,4% DMSO o el 10% dH 2 O). Datos de la muestra se proporciona por 24 o 96 horas de tratamiento compuesto a 37 ° C antes de la fijación. </li></ol> Celular fijación y la tinción de inmunofluorescencia: Nota: El tiempo de tinción es de ~ 2,5 horas posteriores a la fijación. No permita que los pozos se sequen entre los pasos de tinción. Aspiración y dispensación de los reactivos deben llevarse a cabo en las tasas de flujo bajo para disminuir la pérdida de células debido al corte del fluido. Todos los recomendados &#39;por así los volúmenes se refieren a un solo pozo de una microplaca de 96 pozos. Todos los recomendados &#39;por placa de 96 pocillos de los volúmenes de incluir un 25% superior para la pérdida de volumen de líquido de manejo. Todos los pasos de tinción se realiza a temperatura ambiente (TA). Todos los tampones y soluciones de anticuerpos se mantienen estables durante al menos 24 horas a temperatura ambiente. <ol start="4"><li> Al final del período de cultivo, pre-caliente solución de fijación HCS (2X) a temperatura ambiente (RT) o 37 º C si se desea (12 mL/96-well placa). En una campana para vapores químicos, añadir 100 ml / directamente bien a los medios de cultivo y permiten fijar durante 30 min a temperatura ambiente. Retire el fijador / que contiene la toxina y disponer de los medios de comunicación en cumplimiento de la normativa de residuos peligrosos (ver MSDS). Si procediendo de inmediato a las manchas, lavar cada pocillo dos veces con 200 uL de buffer HCS inmunofluorescencia. Por otra parte, si las placas están a teñir en un momento posterior, lavar dos veces con 200 l de tampón de lavado, deje segundo enjuague de volumen en los pozos y las placas de almacén cerrado herméticamente a 4 º C hasta que las manchas. </li><li> Si las muestras de uso, han sido almacenadas a 4 º C antes de la tinción, lavar dos veces con 200 l de búfer HCS inmunofluorescencia antes de proceder con el protocolo de tinción. </li><li> Prepare la solución de conejo anti-tubulina βIII / ratón anti-GFAP anticuerpos HCS Primaria (6 mL/96-well placa) de la siguiente manera: Añadir 60 l de cada anticuerpo primario descongelado a 5,88 ml de Buffer HCS inmunofluorescencia. Mezclar bien. Quitar Buffer inmunofluorescencia anterior enjuague. Añadir 50 l de solución de anticuerpo primario a cada pocillo e incubar durante 1 hora a temperatura ambiente. </li><li> Eliminar el anticuerpo primariosolución. Enjuáguese la boca tres veces con 200 l de búfer HCS inmunofluorescencia. </li><li> Prepare una solución de trabajo de anticuerpos HCS Secundaria / HCS Hoechst tinción nuclear (6 mL/96-well placa) de la siguiente manera: Añadir 30 l de cada anticuerpo descongelado secundaria y 30 L de descongelado nuclear Hoechst HCS mancha a 5,91 ml de Buffer HCS inmunofluorescencia. Mezclar bien, la protección de solución de la luz. Quitar anterior HCS Buffer inmunofluorescencia enjuague. Añadir 50 l de anticuerpo secundario HCS / Hoechst HCS manchas solución nuclear y se incuba durante 1 hora a temperatura ambiente, protegido de la luz. </li><li> Quitar HCS anticuerpo secundario / Hoechst HCS manchas solución nuclear. Enjuague dos veces con 200 l de búfer HCS inmunofluorescencia. </li><li> Quitar anterior HCS Buffer inmunofluorescencia enjuague. Enjuague dos veces con 200 l de tampón de lavado HCS, dejando segundo enjuague de volumen en los pozos. </li><li> Placa de placa de sello y la imagen de inmediato, o se almacenan a 4 º C protegido de la luz hasta que esté listo para la imagen. </li></ol> Adquisición de imágenes y análisis: <ol start="12"><li> Imágenes y análisis de placas teñidas se puede realizar en una variedad de plataformas disponibles HCS, incluyendo el IN Cell Analyzer (GE Healthcare), ArrayScan (ThermoFisher Científico) u Opera (Perkin Elmer). Algunas pautas para la imagen y el análisis se presentan en la Tabla 1: </li></ol><table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4"> HCS222 Directrices de adquisición de imágenes </td></tr><tr><td> Detección de los reactivos </td><td> Lente del objetivo </td><td> Rango de excitación del filtro [pico / ancho de banda (nm)] </td><td> Rango de emisión de filtro [pico / ancho de banda (nm)] </td></tr><tr><td> Hoechst tinción nuclear HCS </td><td> 20X </td><td> 360/40 </td><td> 460/40 (535/50 o si es necesario) </td></tr><tr><td> HCS secundaria de anticuerpos, FITC burro IgG anti-conejo </td><td> 20X </td><td> 480/40 </td><td> 535/50 </td></tr><tr><td> HCS secundaria de anticuerpos, Cy3-burro IgG anti-ratón </td><td> 20X </td><td> 535/50 </td><td> 600/50 </td></tr></tbody></table> <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="background:#000000; color:#FFFFFF;" colspan="4" width="545"> HCS222 Directrices de Análisis de Imagen </td></tr><tr><td> Celda Parámetro </td><td> Detección </td><td> Segmentación / Medida </td><td> Razón fundamental </td></tr><tr><td> Número de celular, características nucleares </td><td> Hoechst tinción nuclear HCS </td><td> Región nuclear (460 nm de emisión del canal). Contar el número de núcleos. Contenido de ADN (intensidad nuclear) o análisis de la zona nuclear también son posibles. </td><td> Utilice el número de células, las características nucleares para determinar la pérdida de células, los fenotipos toxicidad, etc pueden &quot;filtrar&quot; los núcleos de los asociados con βIII-tubulina o la expresión de GFAP para obtener por separado los recuentos de células neuronales y los astrocitos / caracterizaciones (muy recomendable). </td></tr><tr><td> βIII-tubulina expresión, fruto de neuritas </td><td> HCS anticuerpo secundario, conjugado con FITC </td><td> Región citoplasmática (535 nm de emisión del canal). Señal de FITC puede ser utilizado para distinguir los cuerpos de células neuronales de las neuritas (por ejemplo, a través de las áreas del cuerpo mínimo / promedio de células, las longitudes de las neuritas mínimo / máximo y ancho). Determinar parámetros como la longitud de las neuritas total, cuenta con las neuritas / celulares, etc </td><td> Mediciones de las neuritas consecuencia puede ser modulada durante la diferenciación neuronal o como consecuencia de una lesión química, los estados de enfermedad, etc pueden &quot;filtrar&quot; los cuerpos celulares de las asociadas con la expresión βIII-tubulina para obtener la caracterización distinta neuronal (muy recomendable). </td></tr><tr><td> La expresión de GFAP, Zona de astrocitos </td><td> HCS anticuerpo secundario, conjugado con Cy3 </td><td> Región citoplasmática (600 nm de emisión del canal). Cy3 señal puede ser utilizada para definir la segmentación del citoplasma astrocíticos. Determinar parámetros como la intensidad media de la señal citoplásmica, área de la celda, etc </td><td> Expresión de GFAP y el área celular de astrocitos puede ser modulada durante el desarrollo de los astrocitos o como consecuencia de una lesión química, los estados de enfermedad, etc pueden &quot;filtrar&quot; los cuerpos celulares de las relacionadas con la expresión de GFAP para obtener la caracterización distinta astrocíticos (muy recomendable). </td></tr></tbody></table> Tabla 1. Adquisición de imágenes y análisis de las Directrices - HCS222 βIII-Tubulin/GFAP (Co-cultivo) Ensayo Los resultados representativos: <img src="/files/ftp_upload/1173/figure31173.png" alt="Figura 3" /> Figura 1. βIII-tubulina y immunofluore GFAPscence de cultivos mixtos de células nerviosas de ratas. Co-cultivos de astrocitos de hipocampo de rata primaria, ya sea con las neuronas del hipocampo de rata primaria o PC12 células de rata se han generado por los astrocitos pre-revestimiento de varios días en la cultura, seguido por la siembra de las neuronas. Neuronas primarias fueron cultivadas por un período adicional de 11 días, mientras que PC12s se cultivaron durante 2 días en condiciones de diferenciación (niveles séricos bajos / NGF). Manipulación de células, la fijación y tinción se realizó de acuerdo a HCS222 protocolos de ensayo. Las células fueron fotografiadas en el GE EN Celular Analizador de 1000 (3.3) a 20X (neuronas primarias) o 10x (PC12) aumento del objetivo y se analizaron con el analizador de GE En la celda de estación de trabajo 1000 (3.4) y el crecimiento de las neuritas múltiples algoritmos de análisis objetivo del panel superior.: imágenes fusionadas de Hoechst HCS manchas Nuclear (azul), βIII-tubulina (verde) y GFAP (rojo) de fluorescencia. Análisis por separado del canal de fluorescencia βIII-tubulina permite la segmentación crecimiento de las neuritas (panel del medio: los cuerpos celulares con un contorno azul (neuronas primarias) o amarillo (PC12), neuritas en verde (neuronas primarias) o azul claro (PC12)). Análisis de los canales de GFAP permite la segmentación de astrocitos (panel inferior: núcleos con un contorno azul, el citoplasma de color verde). Segmentación GFAP para la rata primaria neuronas del hipocampo / co-cultivo de astrocitos demuestra la capacidad de distinguir entre los órganos de núcleos / célula que son GFAP (+) (Verde describe) y los que son GFAP (-) (rojo), lo que cuenta por separado para celulares neuronas y astrocitos en un ambiente de cultivo mixto. <img src="/files/ftp_upload/1173/figure41173.png" alt="Figura 4" /> <img src="/files/ftp_upload/1173/figure4.51173.png" alt="Figura 4 part2" /> Figura 2. Respuestas dosis de rata primaria neuronas del hipocampo / co-cultivos de astrocitos a las tensiones tóxicas. Astrocitos de hipocampo de rata primaria (P6) se sembraron en placas de 96 pozos en los medios de cultivo y se cultivaron durante 6 días. Neuronas primarias de hipocampo de rata (directo de deshielo) se sembraron luego en la parte superior de la pre-plateado astrocitos y cultivo en medios de cultivo durante 11 días. Las células fueron tratadas con diluciones seriadas de peróxido de hidrógeno o de acrilamida (concentraciones máximo = 100 mm y 10 mm, respectivamente) para las últimas 24 horas de la cultura. Manipulación de células, la fijación y tinción se realizó de acuerdo a HCS222 protocolos de ensayo. Las células fueron fotografiadas en el GE EN Celular Analizador de 1000 (3.3) a 20X (10 campos / pocillo) y se analizaron (nuclear / citoplasmática / neuritas segmentación), utilizando el GE EN Celular Analizador de estación de trabajo 1000 (3.4) y el crecimiento de las neuritas múltiples algoritmos de análisis objetivo. Los datos presentados son la media ± SEM, n = 3. Varios parámetros (crecimiento de las neuritas, la intensidad de GFAP, área de los astrocitos y los recuentos de células neuronales o astrocitos) fueron investigados por dosis-dependiente de las tendencias.

<ol>
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M., Gass&#243;, S., V&#237;lchez, D., Rodr&#237;guez-Farr&#233;, E., Sanfeliu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+effects+of+trimethyltin+and+triethyltin+on+human+fetal+neuron+and+astrocyte+cultures:+a+comparative+study+with+rat+neuronal+cultures+and+human+cell+lines.">Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.</a> Toxicol Lett. 152, 35-46 (2004).</li></ol>

Los autores son todos los empleados de Millipore Corporation.

Hasta la fecha, los experimentos in vitro diseñado para estudiar la evaluación de la neurotoxicidad de riesgo utilizando cultivos mixtos de neuronas y astrocitos han sido limitados. Es ampliamente aceptado que las células gliales son esenciales en la prestación de apoyo espacial y metabólica de las neuronas y juegan un papel fundamental en la modulación de diversas funciones neuronales, como la migración neuronal, la diferenciación y la actividad sináptica. Astrocitos gliales también liberan citoquinas y otros factores solubles que son capaces de inducir la tolerancia tanto en las respuestas adversas en los alrededores de tejido neuronal, así como la promoción de las neuronas de muchas toxinas. Demostrando la profundidad de interacción neuronal-glial, en los estudios de co-cultivo, los astrocitos se ha demostrado que protegen a las neuronas contra la toxicidad del estrés oxidativo, mientras que el deterioro de las funciones de los astrocitos, como el mantenimiento de la defensa antioxidante y los niveles de energía celular, se ha demostrado que críticamente influir en la supervivencia neuronal. Estudios recientes han sugerido que el uso de los astrocitos en un ensayo in vitro de la neurotoxicidad del sistema puede resultar más relevante para la salud humana la estructura del sistema nervioso central y la función de las células neuronales solo. A pesar de la creciente conciencia de la importancia fisiológica de las neuronas, los astrocitos interacciones, que ya ha sido difícil de realizar análisis cuantitativos de estas interacciones en células intactas. El advenimiento de filtrado de contenido de alta permite el desarrollo de poderosos nuevos ensayos para abordar esta cuestión. En este estudio, hemos demostrado que el análisis cuantitativo de varios fenotipos neuronales y los astrocitos en un único ensayo son posibles gracias a HCA. En las neuronas primarias que han realizado las mediciones cuantitativas de los marcadores de neurotoxicidad como el número de neuronas, y la longitud de las neuritas contar las neuritas. En los astrocitos, gliosis reactiva es un marcador biológico conocido de la neurotoxicidad, caracterizada por alteraciones en la expresión de GFAP, el número de astrocitos y la morfología de los astrocitos. Hemos demostrado que cada uno de estos criterios de valoración puede ser fácilmente evaluada a través de HCA. Estos estudios apoyan el concepto de que el HCA de nervios específicos biomarcadores podría ser utilizado como parte de una batería de pruebas in vitro para detectar rápidamente grandes cantidades de productos químicos para los puntos finales neurotóxicos. HCS222 kit de alta Millipore análisis de contenido de co-cultivo de neuronas y astrocitos se compone de reactivos de detección de alta calidad y protocolos para la creación de perfiles simultáneamente βIII-tubulina y la proteína glial fibrilar ácida (GFAP) en una amplia variedad de modelos celulares de neurotoxicidad. Los reactivos altamente validados suministrado con el kit permiten al usuario estandarizar los ensayos, minimizar el ensayo a ensayo de la variabilidad, y para generar imágenes de forma reproducible con una elevada relación señal-a-fondo de relación.

<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Rabbit Anti-&#x03B2;III-Tubulin HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201672</td>
<td>1 vial containing 300 &micro;L 
Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-rabbit IgG, FITC conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201649</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Mouse Anti-GFAP HCS Primary Antibody, 100X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201671</td>
<td>1 vial containing 300 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Secondary Antibody (donkey anti-mouse IgG, Cy3 conjugate), 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200437</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hoechst HCS Nuclear Stain, 200X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200438</td>
<td>1 vial containing 150 &micro;L Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Fixation Solution with Phenol Red, 2X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200434</td>
<td>1 bottle containing 100 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Immunofluorescence Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200435</td>
<td>1 bottle containing 1000 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>HCS Wash Buffer, 1X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. 2007643</td>
<td>1 bottle containing 500 mL Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>Acrylamide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201679</td>
<td>1 vial containing 500 &micro;L of 1M acrylamide in dH2O Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Hydrogen Peroxide (Control Compound), 10X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201730</td>
<td>1 vial containing 500 &micro;L of 100 mM hydrogen peroxide in dH2O Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>K-252a (Control Compound), 250X</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS201741</td>
<td>1 vial containing 100 &micro;L of 250 &micro;M K-252a in DMSO Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>DMSO for Compound Serial Dilution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200441</td>
<td>1 bottle containing 10 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Compound Dilution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200442</td>
<td>1 bottle containing 25 mL Millipore HCS222 Kit Component</td>
</tr>
<tr>
<td>Plate Sealers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Part No. CS200443</td>
<td>10 each Millipore HCS222 Kit Components</td>
</tr>
<tr>
<td>tissue culture-treated black/clear bottom microplates </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HCS imaging/analysis system</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>

a neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity

Análisis de contenido de alta (HCA) se combinan los ensayos de las células y los reactivos de detección con imágenes automatizada y potentes algoritmos de análisis de imagen, permitiendo la medición de varios fenotipos celulares dentro de un único ensayo. En este estudio, hemos utilizado HCA para desarrollar un ensayo novedoso para la neurotoxicidad. Neurotoxicidad evaluación representa una parte importante de la evaluación de seguridad de los medicamentos, además de ser un importante foco de los esfuerzos de protección del medio ambiente. Además, la neurotoxicidad es también una buena aceptación<em&gt; In vitro</em&gt; Marcador del desarrollo de enfermedades neurodegenerativas como el Alzheimer y el Parkinson. Recientemente, la aplicación de HCA a la selección neuronal ha sido reportado. Mediante el etiquetado, las células neuronales con βIII-tubulina, los ensayos de HCA puede proporcionar alto rendimiento, no subjetivo, la medición cuantitativa de parámetros como el número de neuronas, y la longitud de las neuritas contar neuritas, todo lo cual puede indicar efectos neurotóxicos. Sin embargo, el papel de los astrocitos permanece inexplorado en estos modelos. Los astrocitos tienen un papel fundamental en el mantenimiento del sistema nervioso central (SNC) la homeostasis, y están asociadas con la neuroprotección y neurodegradation cuando se activan en respuesta a sustancias tóxicas o enfermedades. GFAP es una proteína de filamentos intermedios expresa predominantemente en los astrocitos del sistema nervioso central. La activación de los astrocitos (gliosis) lleva a la regulación al alza de GFAP, comúnmente acompañado por la proliferación de astrocitos y la hipertrofia. Este proceso de gliosis reactiva ha sido propuesto como un marcador precoz de daño al sistema nervioso. El método tradicional para la cuantificación de GFAP es por inmunoensayo. Este enfoque es limitado por la incapacidad de proporcionar información sobre la localización celular, la morfología y el número de células. Se determinó que el HCA podría ser utilizado para superar estas limitaciones y medir simultáneamente múltiples funciones relacionadas con gliosis - cambios en la expresión de GFAP, la hipertrofia de los astrocitos, y la proliferación de astrocitos - dentro de un único ensayo. En los estudios de co-cultivo, los astrocitos se ha demostrado que protegen a las neuronas contra varios tipos de insultos tóxicos y de influir en forma crítica la supervivencia neuronal. Estudios recientes han sugerido que el uso de los astrocitos en un<em&gt; In vitro</emNeurotoxicidad&gt; sistema de prueba puede resultar más relevantes para la estructura y función del SNC humano de células neuronales solo. En consecuencia, hemos desarrollado un ensayo HCA para el co-cultivo de neuronas y astrocitos, compuesta por protocolos y validados, el objetivo de reactivos específicos para la detección de perfiles de βIII-tubulina y la proteína ácida glial fibrilar (GFAP). Este ensayo permite el análisis simultáneo de neurotoxicidad, crecimiento de las neuritas, gliosis, la morfología neuronal y los astrocitos y el desarrollo neuronal y de los astrocitos en una amplia variedad de modelos de celulares, lo que representa una novela, no subjetivo, de alto rendimiento de ensayo para la evaluación de la neurotoxicidad. El ensayo tiene un gran potencial para mejorar la detección de neurotoxicidad y mejora de la productividad en la investigación en neurociencias y el descubrimiento de fármacos.

Watch this Scientific Journal Video about A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity at JoVE.com

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Este artículo describe un nuevo protocolo y el reactivo conjunto diseñado para la medición sensible de los efectos neurotóxicos de los compuestos y los tratamientos en co-cultivos de neuronas y astrocitos utilizando el análisis de contenido de alta. Los resultados demuestran que el análisis de contenido de alta tecnología representa una apasionante novela para la evaluación de la neurotoxicidad.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

a-neuronal-astrocyte-co-culture-assay-for-high-content-analysis

Research

JoVE Journal

Biology

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Video: A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Las culturas de las células nerviosas de Aplysia para imágenes de alta resolución de los Conos de Crecimiento

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Investigación

Biología

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un compartimiento de Multi-CNS neuronas-glia Co-cultivo de microfluidos Plataforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidos Co-cultivo de células epiteliales y bacterias para la Investigación de la señal mediada Soluble Interacciones

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

La cuantificación de ADN de doble cadena con el Hitachi F-7000 espectrofotómetro de fluorescencia y Dye PicoGreen

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

Absoluta Quantum medición del rendimiento de las muestras de polvo

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Un alto contenido de imágenes de flujo de trabajo para estudiar Grb2 complejos de señalización por clonación de expresión

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Un ensayo de unión de alto rendimiento MHC II para el análisis cuantitativo del péptido epítopos

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

Análisis alta Content Screening para evaluar los efectos toxicológicos de nocivo Componentes y potencialmente perjudicial (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...