Financial support for the experiments presented in the study was provided by the Marie Heim-V&#246;gtlin program of the Swiss National Science Foundation.
We thank Ms. Johanna Nyffeler for developing a set of modified MATLAB programs for screening for statistically significant differences in real-time cell analyzer&#8217;s data and Dr. Yama Abassi for helpful discussion.

1. Preparación <ol><li> Coloque la estación del analizador de células en tiempo real en un incubador de cultivo de tejido fijado en 37 ° C y con 5% de CO 2. Llevar a cabo todas las manipulaciones de cultivo de células y tratamientos farmacológicos en una campana de cultivo de tejidos en condiciones estériles. </li><li> NOTA: Neuronal líneas celulares que requieren diferentes ajustes de temperatura en comparación con las condiciones estándar para la cultura, deben ajustarse en consecuencia. Para las líneas de células débilmente adherentes, utilizar agentes de revestimiento para facilitar la interacción de las células con los microelectrodos de oro en el fondo de los pocillos de la placa de E-96. </li><li> Preparar soluciones 1,000x de stock de los compuestos farmacológicos para el tratamiento del cultivo celular (agentes neuroprotectores o inhibidores de la segunda vías mensajeras en el disolvente apropiado - dimetilsulfóxido o agua estéril) y almacenarlos a -20 ° C. Utilice el disolvente como vehículo en los siguientes tratamientos. </li><li> Células de cultivo de neuroblastoma humano SK-N-SH en DMEM / F12 medio suplementado con 10% de suero bovino fetal (FBS) (medio de proliferación) en matraces de cultivo celular con 175 cm 2 de superficie a una densidad de aproximadamente 75%. Mantenga el número de pases celulares dentro de un rango (de aproximadamente 10 pasajes) para determinar la coherencia entre los experimentos en términos de tasa de proliferación celular y la respuesta a la citotoxicidad. Se prefieren los números de pases bajos. </li><li> Se lava la capa de células SK-N-SH adherente con PBS, y trypsinize con solución de tripsina-EDTA al 0,05% durante 5 min a 37 ° C. Centrifugar las células a 170 × g durante 5 min para eliminar la tripsina. Resuspender las células en 10 ml de medio de proliferación. Contar las células con contador de células Cetro y ajustar el número de células a 300.000 células / ml diluyendo las células con medio de proliferación. </li></ol> 2. Plating y proliferación de las células SK-N-SH <ol><li> Añadir medio de proliferación 100 l a cada pocillo de la placa de E-96 y se deja durante 30 minutos en la campana de cultivo de tejidos a RT se equilibre. Inserte el E-PlaTE 96 en el tiempo real estación analizador de células en el incubador de CO2 a 37 ° C. </li><li> Iniciar el software analizador de células en tiempo real. En la página Layout seleccionar los pozos incluidos en el experimento y entran en los cuadros de edición de la información sobre el tipo de células, el número de células, los nombres y las concentraciones de los compuestos químicos utilizados para el tratamiento de células. NOTA: Para los fines de este protocolo se recomiendan al menos 4 réplicas para cada tratamiento. </li><li> En la página de software Horario determinar &quot;Pasos&quot; incluida en el experimento, seleccionando el número de barridos de medición Índice de Células y el intervalo entre barridos para cada paso. Desde el paso 1 está predeterminado para la medida de fondo, seleccione &quot;Agregar un paso&quot; y establecer el paso 2 para medir el Índice de Células cada 15 minutos durante 96 horas. NOTA: Para los tratamientos, en los que se espera que los efectos con cinética rápida, establecen barridos a intervalos más cortos. </li><li> Haga clic en Iniciar para iniciar el paso predeterminado de forma automática 1y medir la impedancia de fondo de los medios de comunicación. Este valor se resta automáticamente por el software en cada punto de datos una vez que se añadieron las células a los pocillos. </li><li> Utilice el previamente preparado en el paso 1.5) suspensión celular de 300.000 células / ml en medio de proliferación. 30.000 células / pocillo para la toxicidad celular / estudios de neuroprotección y 15.000 células / pocillo para estudios de proliferación celular. Saque el E-Plate y añadir 100 l de suspensión celular por pocillo durante toxicidad celular / estudios de neuroprotección y añadir 50 l de suspensión celular y medio de proliferación de 50 l por pocillo de estudios de proliferación celular. El número de células sembradas por pocillo debe ser optimizado para cada línea celular empíricamente. </li><li> Remover con suavidad la E-Plate 96, incluso para chapado de las células. Deje el E-Plate 96 durante 30 min en la campana de cultivo de tejido a temperatura ambiente para permitir que las células se asientan de manera uniforme a la parte inferior de los pocillos. </li><li> Inserte el E-Plate 96 en la estación de analizador de células en tiempo real y comenzar el paso 2 de la Lista pla edad. Monitorear los valores del Índice Cell largo del experimento al ver las curvas de células en la página Terreno y los datos del índice de la célula primas en la página Índice de Células del software. </li></ol> 3. Suero privación <ol><li> 24 horas después de la siembra de las células, una pausa en el experimento, y quitar la E-96 Placa de la estación de analizador de células en tiempo real. Diluir inhibidores de segundo mensajero de las existencias 1,000x con medio libre de suero / DMEM F12 - a 200 veces la concentración final. Tratar las células con 1 mu l inhibidores de las vías de segundos mensajeros que se realizarán las pruebas de su participación en la neurotoxicidad / neuroprotección 30 minutos antes de iniciar la citotoxicidad, de inhibir las respectivas vías. Devuelva el E-Placa 96 a la estación y reanudar mediciones experimentales Índice Cell. </li><li> Después de 30 minutos en pausa el experimento de nuevo. Retire con cuidado medio de proliferación plenamente de la E-Placa 96 pocillos con una pipeta (o una pipeta multicanal), teniendo cuidado de no perturbar la capa de células adherentesdirigiendo el borde de la punta de la pipeta a la esquina del pozo. Añadir 200 l / pocillo de DMEM libre de suero / F12 (medio de privación de suero). Asegúrese de intercambio rápido de medio de cultivo celular para reducir al mínimo la agitación mecánica de las células. </li><li> Diluir compuestos a ensayar por sus efectos neuroprotectores de 1.000X de stock con medio DMEM / F12 libre de suero a 200 veces la concentración final. Añadir 1 mu l compuestos a ensayar por sus efectos neuroprotectores y 1 mu l inhibidores de las vías de segundos mensajeros en los pocillos individuales de acuerdo con el plan experimental. </li><li> En las células para estudios de proliferación no cambie medio. Diluir compuestos a ensayar desde 1,000X de stock a 200 veces la concentración final en el medio de proliferación, el tratamiento con 1 l por pocillo y continuar a la cultura en medio de proliferación. </li><li> Reanudar el experimento para continuar con Cell mediciones de índice cada 15 minutos durante 96 horas según lo establecido previamente. </li></ol> 4. datos Análisis <ol><li> Para neurotoxicidad / datos neuroprotección normalizan Índice de Células hasta el último punto de tiempo antes del tratamiento farmacológico o cambio de medio para reducir la variación entre los experimentos seleccionando &quot;normalizado Índice de Células&quot; y &quot;normalizar la hora&quot; en la página Mapa. </li><li> Destacar los pozos en la página Parcela en las condiciones experimentales de interés y haga clic en &quot;Añadir&quot; para trazar las curvas de índice de células normalizadas. Observar la cinética del efecto neurotóxico / neuroprotector, o la inhibición de segundo mensajeros como curvas de índice de células normalizado como una función del tiempo. </li><li> Tenga en cuenta las curvas de índice de la célula para los tratamientos con fármacos ensayados en medio de proliferación como una función de tiempo para evaluar si un efecto sobre la proliferación está implicado en el efecto neuroprotector / neurotóxico. </li><li> Exportar la información experimental para todos los puntos de la célula de tiempo Índice de la página de software Índice de Células en un archivo de Excel para iniciar la evaluación estadística de los resultados <br/&gt; NOTA: Como primer paso en el análisis estadístico de un conjunto de programas de MATLAB modificados que utilizan la prueba t de Welch, con el valor p representa semilogarithmically, utilizar eje invertida contra la escala de tiempo (incluidos como archivos de código suplementarios). Estos programas permiten la detección de los valores de p en el tiempo-luego de todo un experimento en tiempo real analizador de células, y por lo tanto ayudan a la selección de los puntos de tiempo para el análisis estadístico más detallado. Ver el material complementario para obtener información detallada sobre los programas. </li><li> Para el análisis estadístico de las diferencias en los valores de índice de células en diferentes condiciones de tratamiento en puntos específicos de tiempo, seleccione los valores de índice de la célula en respectivos puntos de tiempo desde el archivo exportado Excel. Analizar los valores del índice de la célula de los puntos de tiempo seleccionados con una sola vía o de dos vías de análisis de varianza (ANOVA) seguido de una prueba post-hoc utilizando el software estadístico. </li></ol>

<ol>
	<li>Cavallucci, V., D'Amelio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matter+of+life+and+death:+the+pharmacological+approaches+targeting+apoptosis+in+brain+diseases.">Matter of life and death: the pharmacological approaches targeting apoptosis in brain diseases.</a> Curr. Pharm. Des. 17 (3), 215-229 (2011).</li><li>Bull, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPA+CHaPD+001:+Review+of+Environmental+Chemicals+and+Neurotoxicity.">HPA CHaPD 001: Review of Environmental Chemicals and Neurotoxicity.</a> Focus on Neurological Diseases. , (2007).</li><li>Ke, N., Wang, X., Xu, X., Abassi, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+xCELLigence+system+for+real-time+and+label-free+monitoring+of+cell+viability.">The xCELLigence system for real-time and label-free monitoring of cell viability.</a> Methods Mol. Biol. 740, 33-43 (2011).</li><li>Limame, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+dynamic+cell+viability,+migration+and+invasion+assessments+by+novel+real-time+technology+and+classic+endpoint+assays.">Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.</a> PLoS One. 7 (10), e46536 (2012).</li><li>Diemert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impedance+measurement+for+real-time+detection+of+neuronal+cell+death.">Impedance measurement for real-time detection of neuronal cell death.</a> J. Neurosci. Methods. 203 (1), 69-77 (2012).</li><li>Kustermann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+label-free,+impedance-based+real+time+assay+to+identify+drug-induced+toxicities+and+differentiate+cytostatic+from+cytotoxic+effects.">A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects.</a> Toxicol In Vitro. 27 (5), 1589-1595 (2013).</li><li>Marinova, Z., Walitza, S., Gr&#252;nblatt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-HT2A+serotonin+receptor+agonist+DOI+alleviates+cytotoxicity+in+neuroblastoma+cells:+role+of+the+ERK+pathway.">5-HT2A serotonin receptor agonist DOI alleviates cytotoxicity in neuroblastoma cells: role of the ERK pathway.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 44, 64-72 (2013).</li><li>Gonz&#225;lez-Maeso, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens+recruit+specific+cortical+5-HT(2A)+receptor-mediated+signaling+pathways+to+affect+behavior.">Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Tsuchioka, M., Takebayashi, M., Hisaoka, K., Maeda, N., Nakata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+(5-HT)+induces+glial+cell+line-derived+neurotrophic+factor+(GDNF)+mRNA+expression+via+the+transactivation+of+fibroblast+growth+factor+receptor+2+(FGFR2)+in+rat+C6+glioma+cells.">Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells.</a> J. Neurochem. 106 (1), 244-257 (2008).</li><li>Galante, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+toxicity,+conformation+and+morphology+of+typical+initial+aggregation+states+of+A&#946;1-42+and+A&#946;py3-42+beta-amyloids.">Differential toxicity, conformation and morphology of typical initial aggregation states of A&#946;1-42 and A&#946;py3-42 beta-amyloids.</a> Int. J. Biochem. Cell. Biol. 44 (11), 2085-2093 (2012).</li><li>Sheline, C. T., Cai, A. L., Zhu, J., Shi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+or+target+deprivation-induced+neuronal+death+causes+oxidative+neuronal+accumulation+of+Zn2+and+loss+of+NAD.">Serum or target deprivation-induced neuronal death causes oxidative neuronal accumulation of Zn2+and loss of NAD.</a> Eur. J. Neurosci. 32, 894-904 (2010).</li></ol>

Costos de publicación para el video-artículo actual fueron patrocinados por &quot;ACEA Biosciences&quot;.

El protocolo actual presenta la utilidad de un analizador de células en tiempo real para evaluar de manera continua y en condiciones de etiqueta libre de los efectos neuroprotectores / neurotóxicos de compuestos en líneas de células neuronales y para profundizar en los segundos mensajeros vías implicadas en el efecto. A pesar de que la utilidad del analizador de células en tiempo real para estudiar la citotoxicidad y efectos de las drogas sobre la proliferación celular es generalmente...

La muerte celular neuronal desempeña un papel fundamental en la fisiopatología de muchos trastornos relacionados con el cerebro 1. La disponibilidad de información confiable y de alto rendimiento en los ensayos de toxicidad in vitro es fundamental para tener un mejor conocimiento de los mecanismos de neurotoxicidad y para ayudar a seleccionar moléculas neuroprotectoras como candidatos terapéuticos en el desarrollo de fármacos 2. Sin embargo, hay muchas limitaciones a la mayoría ampliamente utilizado en assays.They neurotoxicidad vitro evaluar la neurotoxicidad / neuroprotección en un solo punto de tiempo no permitir la resolución cinética; a menudo utilizan etiquetas o sonda que puede interferir con las vías de señalización y limitar los estudios adicionales en la misma población de células, y son a menudo mano de obra, y en muchos casos no proporcionan una visión mecanicista. En el presente estudio se demuestra la utilidad de un tiempo real basados ​​en la impedancia analizador de células para determinar la neurotoxicidad y neuroprotección en una línea celular neuronal en tiempo real y bajo libre de etiquetascondiciones y para proporcionar información sobre los mecanismos descendentes a través del análisis de las vías de segundos mensajeros involucrados en el efecto. Estudios previos han confirmado la validez del analizador de células en tiempo real para determinar la citotoxicidad así como los efectos sobre la proliferación celular en líneas celulares en comparación con las técnicas estándar 3,4,5,6. Por ejemplo, se observó una buena correlación entre las lecturas de la viabilidad celular WST-1 ensayo estándar y los valores de índice de células en varios puntos de tiempo bajo condiciones de proliferación basal y después de dos paradigmas diferentes tóxicos en células HeLa 3. En las células A549 y MDA-MB-231 proliferación y citotoxicidad provocado con el estabilizador de microtúbulos paclitaxel mostraron valores muy similares cuando evaluada por mediciones del índice de la célula y de la forma estándar utilizado sulforrodamina B (SRB) de ensayo 4. En la línea celular neuronal de las neuronas del hipocampo inmortalizadas HT-22 Cell mediciones de índice fueron validados por su capacidad to detectar la proliferación celular, la citotoxicidad glutamato y citoprotección contra el ampliamente utilizado de 3 (4,5-dimetiltiazol-2-il) 2,5-difeniltetrazolio bromuro (MTT) 5. En el mismo estudio, los resultados del ensayo de MTT y las mediciones de índice de la célula también una buena correlación en la medición de la proliferación de las células progenitoras neuronales, citotoxicidad después de la privación de factores de crecimiento y de rescate de la citotoxicidad por el pan-caspasa inhibidor QVD 5. Inducida en células NIH 3T3 por vandetanib (receptor del factor de crecimiento endotelial vascular y el inhibidor del receptor del factor de crecimiento epidérmico) Citotoxicidad mostró resultados similares medidos con los valores de índice de la célula o ensayo de captación de rojo neutro 6. Recientemente, hemos utilizado el sistema analizador de células en tiempo real para evaluar los efectos neuroprotectores de la serotonina 2A (5-HT 2A) agonista del receptor de clorhidrato de (±) -2,5-dimetoxi-4-iodoamphetamine (DOI) en una línea celular neuronal ( células SK-N-SH) y se seleccionaron para la participación de second vías de mensajería a través de monitorizar el efecto de la inhibición química de la neuroprotección observada 7. Curiosamente, el receptor 5-HT 2A tiene agonistas tanto alucinógenas y no alucinógeno (como DOI y lisurida, respectivamente), que pueden activar las dos vías de segundos mensajeros comunes y distintas 8. Las ventajas de la técnica presentada son que permite recoger información en tiempo real sobre la supervivencia celular en el transcurso de días, para delinear las vías de segundos mensajeros implicados, para evaluar la posible contribución de los efectos de proliferación a la neuroprotección, y para seleccionar un tiempo óptimo para estudios de punto final adicionales en la misma población de células. Un diagrama esquemático del flujo de trabajo en el protocolo actual se presenta en la Figura 1.

<table border="0" cellpadding="0" cellspacing="0" width="1015">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Name of Material/ Equipment</td>
			<td style="width:161px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:457px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">xCELLigence&#160; RTCA SP system bundle</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 00380601030</td>
			<td>consists of RTCA Analyzer, RTCA SP Station and RTCA Control Unit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">E-plate 96</td>
			<td style="width:161px;">ACEA Biosciences</td>
			<td style="width:119px;">No: 05232368001</td>
			<td>for culturing the cells, inserted in the RTCA SP Station</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">SK-N-SH cells</td>
			<td style="width:161px;">ATCC - (in partnership with LGC Standards)</td>
			<td style="width:119px;">HTB-11</td>
			<td>can be replaced by another adherent neuronal cell line of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DMEM/F12&#160;</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8437</td>
			<td>cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">fetal bovine serum</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">16140-063</td>
			<td>supplements proliferation, but not serum deprivation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">tisue culture flask T175</td>
			<td style="width:161px;">Sarstedt</td>
			<td style="width:119px;">83.1812.302&#160;</td>
			<td>for culturing cells, which will be later plated on the E-Plate 96</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">0.05% Trypsin-EDTA</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">25300-054</td>
			<td>for trypsinization of cells cultured in tissue culture flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Scepter 2.0 cell counter</td>
			<td style="width:161px;">Merck Millipore</td>
			<td style="width:119px;">PHCC20060</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">phosphate-buffered saline</td>
			<td style="width:161px;">Life Technologies</td>
			<td style="width:119px;">10010-015</td>
			<td>for washing the cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">(&#177;)-DOI hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D101</td>
			<td>5-HT2A agonist for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">LY-294,002 hydrochloride</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">L9908</td>
			<td>PI3-K inhibitor for cell culture treatment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">lisuride maleate</td>
			<td style="width:161px;">Tocris Bioscience</td>
			<td align="right" style="width:119px;">4052</td>
			<td>compound with 5-HT2A agonistic activity for cell culture treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">dimethyl sulfoxide</td>
			<td style="width:161px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4540</td>
			<td>for dissolving LY-294,002 and lisuride maleate</td>
		</tr>
	</tbody>
</table>

real-time impedance-based cell analyzer as a tool to delineate molecular pathways involved in neurotoxicity and neuroprotection in a neuronal cell line

Muchos trastornos relacionados con el cerebro tienen la muerte celular neuronal implicado en su fisiopatología. Mejora en modelos in vitro para estudiar los efectos neuroprotectores o neurotóxicos de las drogas y las vías descendentes involucradas ayudaría a aumentar el conocimiento sobre los mecanismos moleculares de neuroprotección / neurotoxicidad y potencialmente podría facilitar el desarrollo de fármacos. Sin embargo, muchos ensayos de toxicidad in vitro existentes tienen limitaciones importantes - más evaluar la neurotoxicidad y neuroprotección en un solo punto de tiempo, no permitiendo observar la evolución temporal y la cinética del efecto. Además, la oportunidad de recoger información sobre las vías de señalización corriente abajo implicados en la neuroprotección en tiempo real sería de gran importancia. En el protocolo actual se describe el uso de un tiempo real basados ​​en la impedancia analizador de células para determinar los efectos neuroprotectores de la serotonina 2A (5-HT 2A) agonistas del receptor en una línea celular neuronal menores de etiqueta libre y en tiempo real Conditiones utilizando mediciones de impedancia. Además, se demuestra que los inhibidores de las vías de segundo mensajero se pueden utilizar para delinear las moléculas de aguas abajo implicadas en el efecto neuroprotector. También se describe la utilidad de esta técnica para determinar si un efecto sobre la proliferación celular contribuye a un efecto neuroprotector observado. El sistema utiliza placas microelectrónicos especiales denominados E-planchas que contienen alternando microelectrodos de oro en la superficie inferior de los pozos, que sirven como sensores celulares. La lectura de impedancia se modifica por el número de células adherentes, la viabilidad celular, la morfología y la adhesión. Un parámetro adimensional llamado Índice de Células se deriva de las mediciones de impedancia eléctrica y se utiliza para representar el estado celular. En general, el tiempo real basados ​​en la impedancia analizador de células permite en tiempo real, la evaluación de la etiqueta libre de la neuroprotección y la neurotoxicidad, y la evaluación de la implicación de las rutas segundo mensajero, contribuyendo a másevaluación detallada y de alto rendimiento de potenciales compuestos neuroprotectores in vitro, para la selección de candidatos terapéuticos.

<html> Suero privación conduce a una disminución en los valores de índice de células, que pueden ser monitoreados continuamente con el analizador de células en tiempo real Estímulos neurotóxicos a las células conducen a una disminución en los valores de índice de células, que puede ser monitoreado en tiempo real con la técnica presentada y la dinámica de los cuales depende de los estímulos neurotóxicos específico y el tipo de célula estudiado. Figura ...

Watch this Scientific Journal Video about Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line at JoV

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Cell Analyzer basados ​​en impedancia Real-Time como una herramienta para delinear vías moleculares implicadas en la neurotoxicidad y neuroprotección en una línea celular neuronal

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

real-time-impedance-based-cell-analyzer-as-tool-to-delineate

Research

JoVE Journal

Neuroscience

11.3K vistas.  University of Zürich. Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Video: Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Automatizado de Análisis de la morfología neuronal Sholl digitalizados en múltiples escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Investigación

Neurociencias

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

Derivación eficiente de los Derechos Humanos progenitores neuronales y neuronas de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Electroencefalografía simultánea, en tiempo real Medición de la concentración de lactato y Manipulación optogenético de la actividad neuronal en la corteza cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Microglia como una biosensor sustituto para determinar la neurotoxicidad de nanopartículas

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

La electroporación en el útero métodos para estudiar la excitabilidad de las subpoblaciones neuronales y Conectividad Una sola célula

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Culturas de rebanada Hippocampal Organotypic como un modelo para estudio neuroprotección e invasividad de las células tumorales

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Cell Analyzer basados ​​en impedancia Real-Time como una herramienta para delinear vías moleculares implicadas en la neurotoxicidad y neuroprotección en una línea celular neuronal

Cell Analyzer basados en impedancia Real-Time como una herramienta para delinear vías moleculares implicadas en la neurotoxicidad y neuroprotección en una línea celular neuronal