Este trabajo fue apoyado por la National Science Foundation (subvención no. DBI 0519984) y los Institutos Nacionales de Salud (concesión no. 5R01GM078279) ..

1) Preparación del material vegetal <ol><li> Protoplastos se puede derivar de muchas especies de plantas y tejidos, siempre que la mezcla correcta de la pared celular enzimas digestivas se utiliza 1. Antes de que un experimento a gran escala se lleva a cabo una digestión de pequeña escala de la materia es aconsejable con el fin de evaluar la eficiencia protoplasting de los tejidos, enzimas, etc, y para estimar el porcentaje de células positivas para la clasificación de la célula. Aquí, los protoplastos derivados de las raíces de las plántulas de Arabidopsis thaliana que tipo de célula específica expresan la proteína verde fluorescente (GFP) se utilizan. La endodermis y el centro de reposo se caracterizan por P SCR:: GFP y el centro de reposo por P WOX5:: GFP 2,3 (Figura 1). </li><li> Las plántulas se cultivan en hidroponia phytatrays (Sigma; Figura 2a) en un filtro de nylon (250 micras de malla; Nitex) que permite que las raíces crecen a través de en el medio de crecimiento (0,22% w / v medio de Murashige y Skoog [Sigma], el 1% w / v de sacarosa, 0,05% w / v MES [2 - (N-morfolino) etanosulfónico ácido], pH 5,7 con KOH). Por otra parte, las plantas pueden ser cultivadas en la parte superior de un filtro de nylon (malla 100 micras) en posición vertical placas de agar al 1% (figura 2b). </li><li> El uso de los filtros antes mencionados no sólo ayuda en la cosecha de las raíces, sino que también facilita el tratamiento fácil adicionales de las plantas, si se desea. Los filtros permiten a las plantas para ser transferido en masa a phytatrays nuevas o las placas de agar complementado con un catalizador de interés. Por ejemplo, el conjunto phytatray hasta se ha utilizado para el tipo de células específicas de análisis de la respuesta transcripcional a un tratamiento de nitrógeno en plantas de Arabidopsis 4. </li><li> Asegúrese de que su microscopio marcador fluorescente se expresa correctamente (especialmente cuando se utiliza la opción de tratamiento, como el tipo de células específicas de marcadores se puede estar influida por el tratamiento). En este caso, las plántulas son inspeccionados bajo un microscopio de fluorescencia (Nikon, Figura 1). Tenga en cuenta que tipo de células específicas líneas de marcador fluorescente que se caracteriza inicialmente con un microscopio confocal para determinar exactamente qué tipos de células se marcan y determinan la variabilidad en la expresión. </li></ol> 2) Preparación de la solución protoplasting <ol><li> Disolver 1,25% w / v celulasa (Yakult), un 0,3% w / v Macerozyme (Yakult), 0,4 M D-manitol, 20 mM MES y 20 mM KCl (de una reserva de 1 M) en agua desmineralizada y ajustar el pH a 5.7 con 1 M Tris / HCl pH 7,5. Esta solución será ligeramente turbio. </li><li> Calentar la solución a 55 ° C durante 10 minutos (la solución se volverá claro) y dejar que se enfríe a temperatura ambiente antes de la adición de 0,1% w / v BSA (albúmina de suero bovino), 10 mM CaCl 2, y 5 mM β-mercaptoetanol . </li></ol> 3) La cosecha y protoplasting del material vegetal <ol><li> Las raíces se cosechan por raspado de ellos fuera de la malla de nylon con un bisturí y se deposita en un frasco que contiene la solución protoplasting. En general, 10 ml de solución protoplasting se utiliza por cada 1.500 raíces de las plántulas. </li><li> Agitar los frascos con cuidado (75 rpm) a temperatura ambiente durante una hora. Un tiempo de incubación más largo puede aumentar el rendimiento de protoplastos, pero también se sumará a los efectos de protoplasting sí mismo en la expresión génica. </li><li> Filtrar la solución de protoplastos con un filtro de 40 micras de células (BD Falcon) y dividir la solución en forma cónica tubos de 15 ml (BD Falcon). </li><li> Girar los tubos en una centrifugadora basculante durante 10 minutos a 500 G. Nota que la velocidad de centrifugación dependerán del tipo de protoplastos utilizados, su fragilidad y la cantidad de restos celulares producidos durante el tratamiento enzimático. </li><li> Eliminar la mayor parte del sobrenadante, resuspender los protoplastos en el resto de la solución e inspeccionarlos microscopio (Figura 3). </li><li> Hacer uso de un hemocitómetro para estimar el número y la densidad de protoplastos. La densidad celular se determinará la velocidad de inyección de la muestra, los eventos por segundo y por lo tanto el tiempo total necesario para ordenar las células en la FACS (ver sección 4.3). </li><li> 3.7) Cualquier proceder directamente a FACS o lavar y volver a suspender los protoplastos en una solución de incubación, como W5 (154 mM NaCl, 125 mM CaCl2, KCl 5 mM, 5 mM MES, ajustar el pH a 5,7 con KOH) o una solución protoplasting sin la añadido enzimas. Sobre todo cuando se observan los cambios transcripcional, es de suma importancia para minimizar la exposición de las muestras a las condiciones que pueden influir en la expresión de genes, como el cambio del buffer los protoplastos se mantienen in Por ello se recomienda mantener los protoplastos en una solución de protoplasting y proceder FACS a la mayor brevedad posible. </li></ol> 4) fluorescencia de células activadas por la clasificación de protoplastos <ol><li> Encender y preparar el clasificador de células. Aquí, un FACSAria (BD) se utiliza. </li><li> Establecer una corriente de flujo con un 100 y mu, m de la boquilla y una presión de 20 psi vaina. </li><li> La densidad de las células y la velocidad de inyección de la muestra se puede ajustar a una experiencia particular en función de si un rendimiento mejor o mayor velocidad alcanzable es deseado. Hemos clasificado con éxito con densidades de hasta 10 millones células / ml. </li><li> Use la opción de la agitación de la muestra en la FACS para prevenir la sedimentación de los protoplastos. Si la obstrucción de la FACS es un problema, hay tres pasos para solucionar problemas que: 1. Realizar una inversión de flujo de la muestra-line. 2. Diluir la suspensión de protoplastos para reducir la densidad. 3. Limpieza de la solución de protoplastos mediante la repetición de la etapa de filtración (3.3) después de la centrifugación y resuspensión. </li><li> Prepare el aparato para medir la dispersión frontal (FSC), dispersión lateral (SSC) y la emisión de 530/30 nm para GFP y 610/20 nm para el espectro de color rojo con autofluorescencia (RSA) después de la excitación por un láser de 488 nm. Estos son, en esencia, los únicos parámetros que utilizan para aislar la GFP positivos protoplastos. En este caso, los valores de voltaje son los siguientes: FSC - 60 V, 250 V SSC, 350V y 335V GFP RSA. Tenga en cuenta que los valores de voltaje óptimo será diferente para cada FACS e incluso tendrá que ser ajustado a lo largo de la vida del clasificador de células. </li><li> Comience por la creación de un diagrama de puntos de dispersión frontal versus dispersión lateral. Aplicar los valores de voltaje de modo que los eventos medidos se centran en la parcela. </li><li> A continuación, cree un gráfico de puntos de señales de fluorescencia verde frente a roja. Aplicar los valores de voltaje de modo que los acontecimientos se cuantificó el rendimiento de una población centrada en diagonal en la trama cuando se mira en una suspensión de protoplastos de tipo salvaje (no-GFP). Una suspensión de protoplastos derivados de una línea de marcador GFP se produce una población libre de color verde fluorescente eventos nunca antes vista en las muestras de tipo salvaje. </li><li> Establecer restricciones para ajustar la compensación de solapamiento espectral entre las buenas prácticas agrarias y la RSA. Correcta configuración de restricción de la compensación permitirá una mejor separación de los protoplastos GFP-positivas de los protoplastos no las buenas prácticas agrarias y los escombros. Las restricciones se utiliza aquí es el siguiente: RSA, menos 17,91% GFP. </li><li> Configurar una puerta para identificar las buenas prácticas agrarias-positivas eventos, un control negativo de la no-GFP protoplastos se debe utilizar para ayudar a definir los límites de la puerta (Figura 4). </li><li> Implementar un corte de dispersión hacia adelante para dejar pequeños restos fuera del análisis. Visualizar los eventos de GFP-positivas en el FSC frente a SSC complot para ayudar a determinar la ubicación de la corte. Aquí, el punto de corte se estableció en 5000. Tenga en cuenta que la FACS contará escombros ordenar eventos y una muestra con altos niveles de residuos puede tener una serie de eventos por ciento diferente GFP positivos de lo esperado. Esto no es necesariamente un problema. Sin embargo, los restos más en la muestra, mayor será el tipo va a tomar. </li><li> Dependiendo del experimento y la abundancia del tipo de células a analizar, establecer el modo de precisión, FACS, ya sea para un rendimiento óptimo o la pureza óptima de las células ordenados. </li><li> Para la extracción de RNA, preparar los tubos de recogida (1,5 ml tubos de microcentrífuga) con la cantidad adecuada de tampón de extracción de RNA. Con esta configuración, 20.000 eventos de clasificación dará un volumen total de aproximadamente 100 l, que se clasificaron en 350 l de tampón de extracción (micro RNeasy kit, QIAGEN). Mezcla de las muestras después de la finalización de la especie como la suspensión de células pueden reunir en la parte superior. </li><li> Almacenar las muestras o proceder directamente a la extracción de RNA. Análisis de microarrays exitosos se han preformado con el ARN extraído de tan poco como 500 eventos ordenados. En este caso, se utilizó un kit de extracción RNeasy micro (QIAGEN), el WT-Ovation Pico Sistema de amplificación de ARN y FL-Ovation cDNA biotina módulo V2 (NuGEN). </li></ol> Resultados representante Una phytatray de aproximadamente 1.500 SCR de una semana de edad, P:: GFP plántulas produjo unos 60.000 protoplastos (medida por el hemocitómetro). 2,6% de 65.000 eventos FACS-procesado se define como GFP-positivas y fueron ordenados (Figura 4b). Ocho placas de aproximadamente 1500 de cuatro días de edad, P WOX5:: GFP plantas cada uno (12.000 en total) producido por 30.000.000 protoplastos (medida por el hemocitómetro). 0,063% de los 16.000.000 eventos FACS-procesado se define como GFP-positivas y fueron ordenados (Figura 4c). 10.000 eventos ordenados se utilizan normalmente para la extracción de ARN y puede producir 20 a 140 ng de ARN total (Figura 5). <img src="/files/ftp_upload/1673/1673fig1.jpg" alt="Figura 1" /> Figura 1. Tipo de células específicas líneas marcador GFP en la raíz de Arabidopsis. Imágenes de microscopía de fluorescencia fueron tomadas con contraste de interferencia diferencial (DIC) y un filtro de buenas prácticas agrarias en un microscopio Eclipse 90i (Nikon) que se ejecutan en el software Metamorph (Molecular Devices). Las imágenes DIC y GFP se superpusieron para fines de visualización. Las dos líneas de marcador utilizado en este experimento visual se muestran;a) P SCR:: GFP y b) P WOX5:: GFP. <img src="/files/ftp_upload/1673/1673fig2.jpg" alt="Figura 2" /> Figura 2. Plántulas condiciones de crecimiento. Crecieron en un controlador del medio ambiente hidropónico en phytatrays (a) o en placas de agar en posición vertical (b). <img src="/files/ftp_upload/1673/1673fig3.jpg" alt="Figura 3" /> Figura 3. Protoplastos de plantas que expresan GFP. Imágenes de microscopía de fluorescencia fueron tomadas con contraste de interferencia diferencial (DIC) y un filtro de buenas prácticas agrarias en un microscopio Eclipse 90i (Nikon) que se ejecutan en el software Metamorph (Molecular Devices). Las imágenes DIC y GFP se superpusieron para fines de visualización. Las flechas indican el estallido de una célula, restos celulares y protoplastos GFP-positivas. La distancia entre dos líneas blancas es de 50 micras. <img src="/files/ftp_upload/1673/1673fig4.jpg" alt="Figura 4" /> Figura 4. Fluorescencia de células activadas por la clasificación de la GFP-positivas protoplastos protoplastos derivados de tipo salvaje (a) P SCR.:: GFP (b) o P WOX5:: GFP (c) líneas de marcadores fueron analizados y ordenados con una FACSAria (BD) con puertas definido en una gráfica de puntos verdes (530/30 nm; eje x) frente roja (610/20 nm; eje y) de fluorescencia. 100.000 eventos se presentan en cada parcela. Los eventos incluidos en la puerta de la clasificación se destacan las buenas prácticas agrarias verde. <img src="/files/ftp_upload/1673/1673fig5.jpg" alt="Figura 5" /> Figura 5. Representante de las extracciones de ARN de 10.000 células seleccionadas. Las células fueron ordenados directamente en la extracción del ARN de amortiguación (QIAGEN), el ARN fue purificado y se analiza la concentración, la pureza y la integridad en un Bioanalyzer 2100 (Agilent). Tres réplicas de las dos líneas marcador se muestran.

<ol>
	<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts.">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127, 1466-1475 (2001).</li><li>Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E., Benfey, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+SCARECROW+function+reveals+a+radial+patterning+mechanism+common+to+root+and+shoot.">Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot.</a> Development. 127, 595-603 (2000).</li><li>Blilou, I., Xu, J., Wildwater, M., Willemsen, V., Paponov, I., Friml, J., Heidstra, R., Aida, M., Palme, K., Scheres, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PIN+auxin+efflux+facilitator+network+controls+growth+and+patterning+in+Arabidopsis+roots.">The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots.</a> Nature. 433, 39-44 (2005).</li><li>Gifford, M. L., Dean, A., Gutierrez, R. A., Coruzzi, G. M., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+nitrogen+responses+mediate+developmental+plasticity.">Cell-specific nitrogen responses mediate developmental plasticity.</a> Proc Natl Acad Sci U S A. 105, 803-808 (2008).</li><li>Bargmann, B. O. R., Birnbaum, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+fluorescent+selection+permits+precise,+rapid,+and+in-depth+overexpression+analysis+in+plant+protoplasts.">Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.</a> Plant Physiol. 149, 1231-1239 (2009).</li><li>Petersson, S. V., Johansson, A. I., Kowalczyk, M., Makoveychuk, A., Wang, J. Y., Moritz, T., Grebe, M., Benfey, P. N., Sandberg, G., Ljung, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Auxin+Gradient+and+Maximum+in+the+Arabidopsis+Root+Apex+Shown+by+High-Resolution+Cell-Specific+Analysis+of+IAA+Distribution+and+Synthesis.">An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis.</a> Plant Cell. 21, 1659-1668 (2009).</li></ol>

Protoplastos puede, en principio, se derivan de una variedad de tejidos de la planta, optimización de las condiciones favorables en gran medida mejorar la calidad y la cantidad de ARN. Tanto la solución protoplasting y el tampón de incubación electiva utilizado influirá en este aspecto. Muchas proteínas fluorescentes se pueden utilizar diferentes, dependiendo de las capacidades de la FACS utiliza, por ejemplo, GFP, RFP, YFP, PPC o sus muchas variantes y derivados. La expresió...

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		<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>250 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-250/50</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;m nylon mesh</td>
<td>&nbsp;</td>
<td>Sefar Filtration</td>
<td>NITEX 03-100/47</td>
<td>&nbsp;</td>
<tr>
<td>Square petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-10k</td>
<td>&nbsp;</td>
<tr>
<td>Phytatrays</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1552</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog Basal Medium (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5519</td>
<td>&nbsp;</td>
<tr>
<td>sucrose</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>S5-3</td>
<td>&nbsp;</td>
<tr>
<td>MES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M2933</td>
<td>&nbsp;</td>
<tr>
<td>KOH</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1767</td>
<td>10 M stock</td>
</tr>
<tr>
<td>Eclipse 90i microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cellulase R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>Yakult Pharmaceutical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9546</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8041</td>
<td>1 M stock</td>
</tr>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3912</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>CALBIOCHEM</td>
<td>444203</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2536</td>
<td> 1 M stock</td>
</tr>
<tr>
<td>orbital shaker</td>
<td>&nbsp;</td>
<td>LAB-LINE</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352340</td>
<td>&nbsp;</td>
<tr>
<td>conical 15 ml tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352196</td>
<td>&nbsp;</td>
<tr>
<td>table centrifuge</td>
<td>&nbsp;</td>
<td>Sorvall</td>
<td>Legend RT</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3014</td>
<td>&nbsp;</td>
<tr>
<td>FACSAria</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microfuge tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20170-38</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy micro kit</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>74004</td>
<td>&nbsp;</td>
<tr>
<td>WT-Ovation Pico RNA Amplification System</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>3300_12</td>
<td>&nbsp;</td>
<tr>
<td>FL-Ovation cDNA Biotin Module V2</td>
<td>&nbsp;</td>
<td>NuGEN</td>
<td>4200_12</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence activated cell sorting of plant protoplasts

De alta resolución, el tipo específico de célula de análisis de la expresión génica mejora considerablemente la comprensión de la regulación del desarrollo y las respuestas a los estímulos del medio ambiente en cualquier organismo multicelular. Hibridación in situ y la visualización del gen reportero puede hasta cierto punto se utiliza para este fin, sino para alta resolución cuantitativa RT-PCR o de alto rendimiento en todo el análisis del transcriptoma el aislamiento de ARN a partir de tipos de células es un requisito. Disociación celular de los tejidos que expresan un marcador de la proteína fluorescente en un tipo específico de célula y la posterior clasificación de células activadas fluorescencia (FACS) hace que sea posible reunir una cantidad suficiente de material para la extracción de ARN, el análisis de cDNA síntesis / amplificación y microarrays. Un amplio conjunto de tipo de células específicas líneas reportero fluorescente está disponible para la comunidad de investigación de la planta. En este caso, dos líneas de marcador de la raíz de Arabidopsis thaliana se utilizan: P SCR:: GFP (endodermis y el centro de reposo) y P WOX5:: GFP (centro de reposo). Un gran número (miles) de las plántulas se cultivan hidropónicos o en placas de agar y se cosechan para obtener material suficiente raíz para su posterior análisis. Disociación celular de material vegetal se obtiene por digestión enzimática de la pared celular. Este procedimiento hace uso de alta osmolaridad inducida plasmólisis y celulasas disponibles en el mercado, pectinasas y hemicelulasas para liberar protoplastos en la solución. FACS de las buenas prácticas agrarias de células positivas hace uso de la visualización de los verdes contra los espectros de emisión de color rojo de protoplastos excitado por un láser de 488 nm. GFP-positivas protoplastos se pueden distinguir por su relación cada vez mayor de verde a la emisión de color rojo. Protoplastos son típicamente ordenados directamente en tampón de extracción de ARN y se almacena para su posterior procesamiento en un momento posterior. Esta técnica se revela que es sencillo y práctico. Además, se demuestra que se puede utilizar sin dificultad de aislar un número suficiente de células para el análisis de transcriptoma, incluso para los tipos de células muy escasos (por ejemplo, las células del centro de reposo). Por último, una configuración de crecimiento de plántulas de Arabidopsis se ha demostrado que permite el tratamiento sin complicaciones de las plantas antes de la clasificación de células (por ejemplo, para el tipo específico de célula de análisis de las respuestas al estrés biótico o abiótico). Complementarios usos potenciales de FACS de protoplastos de plantas se discuten.

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting of Plant Protoplasts at JoVE.com

Fluorescence Activated Cell Sorting of Plant Protoplasts

Un método para el aislamiento de determinados tipos de células a partir de material vegetal se demuestra. Esta técnica emplea las líneas transgénicas que expresan proteínas fluorescentes marcador en los tipos de célula en particular, la disociación y la fluorescencia celular selección de células activadas. Además, un crecimiento de configuración se ha establecido aquí que facilita el tratamiento de<em&gt; Arabidopsis thaliana</em&gt; Plantones antes de la clasificación de células.

Fluorescencia de células activadas por la clasificación de los protoplastos de la planta

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

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24.6K vistas.  New York University. Un método para el aislamiento de determinados tipos de células a partir de material vegetal se demuestra. Esta técnica emplea las líneas transgénicas que expresan proteínas fluorescentes marcador en los tipos de célula en particular, la disociación y la fluorescencia celular selección de células activadas. Además, un crecimiento de configuración se ha establecido aquí que facilita el tratamiento de Arabidopsis thaliana Plantones antes de la cla...

Video: Fluorescence Activated Cell Sorting of Plant Protoplasts

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Lípidos vesículas mediada por cromatografía de afinidad utilizando magnética clasificación de células activadas (LIMACS): un nuevo método para analizar la interacción de proteínas y lípidos

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Investigación

Biología

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Bromodeoxyuridine (BrdU) El etiquetado y posterior clasificación de células activadas fluorescencia para la Cultura independiente de la identificación de carbono orgánico disuelto degradación bacterioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Análisis específico de célula de<em&gt; Arabidopsis</em&gt; Hojas usando citometría de flujo

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Aislamiento de fibroblastos normales y el cáncer asociado-de los tejidos frescos por Clasificación células activadas por fluorescencia (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Interacciones proteína-proteína se visualizó por fluorescencia Bimolecular Complementación en protoplastos de tabaco y hojas

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medición de la osmótica de agua Coeficiente de Permeabilidad (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos de plantas aisladas como ejemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Análisis rápido de los fenotipos circadianos en Arabidopsis protoplastos transfectadas con un reloj luminiscentes Reportero

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

La visualización de frecuencia Stromule con microscopía de fluorescencia

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

Microscopía de luz Hoja de fluorescencia de las raíces de plantas que crecen en la superficie de un gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...