Anyaporn Sawasdichai y Nazefah Abdul Hamid muy agradecidos con el Gobierno Real de Tailandia y el Gobierno de Malasia, respectivamente, para doctorado Becas. Este trabajo fue financiado por el Wellcome Trust proyecto de subvención concedida a PSJ y KG. También estamos agradecidos a la Universidad de Bristol Fondo Bioimagen Wolfson.

I. Preparación para el fraccionamiento En esta sección se describe la preparación de poli-L-lisina cubreobjetos recubiertos microscopio y la unión de las células antes de su fraccionamiento. Si es necesario que las células pueden ser transfectadas transitoriamente con los vectores de expresión de la proteína, ya sea antes o después de la unión. A. Preparación de poli-L-lisina cubreobjetos recubiertos <ol><li> Prepare una solución de 1mg/ml de poli-L-lisina en agua destilada. </li><li> Capa limpia cubreobjetos con poli-L-lisina, mediante su incubación en la solución de al menos 1 hora en una plataforma oscilante a 22 ° C. </li><li> Lave el cubreobjetos recubiertos con agua destilada estéril dos veces y seguir con un solo lavado con etanol al 96%. </li><li> Seque al aire los cubreobjetos recubiertos en un pedazo de papel de filtro y guardar en un recipiente seco para uso en el futuro (una vez seca se pueden apilar). </li></ol> B. Fijación de las células a cubreobjetos Las células no adherentes (aquí usamos células K562) <ol><li> Coloque un cubreobjetos recubiertos con poli-L-lisina en un pozo en una placa de 6 pocillos con la superficie recubierta hacia arriba. </li><li> Semillas de las células K562 a una densidad de 7x10 5 células / pocillo en Dulbecco modificado Eagles Medium (DMEM) suplementado con fetal al 10% de suero bovino (FBS) y PS (penicilina 100units/ml, 100mg/ml estreptomicina). En el caso de las células K562 de transfección transitoria se puede realizar utilizando electroporación (0.4cm cubetas de electroporación con 1x10 7 células en 200μl de los medios de comunicación a 250 / 975μF) antes de la siembra de las células. </li><li> Se incuban las células durante 24 horas a 37 ° C en 5% CO 2. </li><li> Se retira el medio y se lavan las células dos veces con fosfato helada (PBS). </li><li> Seguir con el protocolo de fraccionamiento subcelular. </li></ol> Células adherentes (aquí usamos células COS-7) <ol><li> Coloque un cubreobjetos sin revestimiento en un pozo en una placa de 6 pocillos. </li><li> Lavar dos veces en PBS precalentado a 37 ° C. </li><li> Separar las células por digestión con tripsina (0,03% de EDTA, 0,25% de tripsina) en PBS a 37 ° C durante 2 a 5 minutos. Es importante no pasar de digerir las células y para detener la digestión tan pronto como las células flotantes predominantemente individual están presentes. </li><li> Detener la digestión por la siembra de las células a una densidad de 3x10 5 células / pocillo en una placa de 6 pocillos en DMEM suplementado con FBS al 10% y el PSG (penicilina 100 unidades / ml, estreptomicina y 100ug/ml 292ug/ml L-glutamina) , en cubreobjetos normal. Células adherentes en general, un buen agarre en cubreobjetos microscopio normal, sin embargo, el poli-L-lisina cubreobjetos recubiertos puede ser utilizado en caso necesario. </li><li> Se incuban las células durante 24 horas a 37 º C y 5% CO 2. En el caso de Cos-7 en células de transfección transitoria se puede realizar utilizando Fugene 6 (Roche), si es necesario y las células de la izquierda para recuperar por un período de 24 horas a 37 ° C y 5% CO 2. </li><li> Se retira el medio y se lavan las células dos veces con PBS enfriado con hielo. </li><li> Seguir con el protocolo de fraccionamiento subcelular. </li></ol> II. Fraccionamiento subcelular En la fracción de terreno se lleva a cabo de acuerdo con el esquema que se muestra en la Figura 1 y se basa en un método descrito anteriormente 5 con las modificaciones descritas anteriormente, 7 y en el protocolo se detalla a continuación. Se recomienda la transferencia de los cubreobjetos a un nuevo máximo de 6 pocillos después de cada paso de fraccionamiento quitar el cubre antes de retirar el líquido. Aunque sólo cuatro cubreobjetos se requieren para obtener imágenes, cubreobjetos adicionales se requieren para producir extracto de células enteras y fracciones nucleares matriz de Western Blot si esto se va a realizar en paralelo. <ol><li> Preparar y el filtro del citoesqueleto de amortiguación (CSK): TUBOS 10 mM, sacarosa 300mm, 100mm NaCl, 3 mM MgCl2, 1 mM EGTA. CSK de amortiguación debe prepararse en el día de fraccionamiento. </li><li> Si es necesario que las células de un cubreobjetos se puede utilizar para preparar el extracto de células enteras de Western Blot colocando suavemente 200ul tampón TES (SDS al 1%, 2 mM EDTA, 20 mM Tris-HCl pH 7,4) directamente sobre el portaobjetos y el período de incubación de 1 ó 2 minutos a 22 ° C. El extracto de células enteras se puede retirar y se precipitaron las proteínas mediante la adición de 250ul de 1 M (NH 4) 2 SO 4 e incubando en hielo hasta que las muestras occidentales están listos. </li><li> Lavar los cubreobjetos restantes con 1 ml de hielo frío PBS dos veces a 22 ° C por la inclinación del 6 y la placa dos o tres veces. </li><li> Quitar el cubre representan células enteras y la transferencia a un nuevo máximo de 6 pocillos. Siga con la fijación de células y el protocolo de inmunofluorescencia. </li><li> Retire con cuidado la PBS de los pozos restantes. </li><li> Extracto de las proteínas nucleares y citoplasmáticas en forma no estructurada colocando suavemente 200ul buffer CSK + 0,1% (V / V) de Triton X-100 directamente en cada cubreobjetos restantes e incubando enhielo durante 1 minuto. </li><li> Retire el extracto nuclear citoplasmática y en forma no estructurada y precipitar tal como se describe en el paso 2. </li><li> Lavar los cubreobjetos con 1 ml de PBS frío hielo dos veces a 22 ° C por la inclinación del 6 y la placa. </li><li> Retire el material de cubreobjetos representa firmemente sostiene, nuclear y seguir con la fijación de células y el protocolo de inmunofluorescencia. </li><li> Retire con cuidado la PBS de los pozos restantes a continuación, extraer las proteínas firmemente sostiene, colocando suavemente 200ul buffer CSK + 0,5% (V / V) de Triton X-100 directamente en cada cubreobjetos restantes e incubar en hielo durante 20 minutos. </li><li> Quitar el extracto firmemente sostiene, y se precipitan como se describe en el paso 2. </li><li> Lavar los cubreobjetos con 1 ml de PBS frío hielo dos veces a 22 ° C por la inclinación del 6 y la placa. </li><li> Quitar el cubre representa la fracción de la cromatina y seguir con la fijación de células y el protocolo de inmunofluorescencia. </li><li> Retire con cuidado la PBS de los pozos restantes coloque 200ul CSK de amortiguación + 100ug/ml de DNasa I en el resto de cubreobjetos y se incuba durante 30 minutos a 37 ° C. </li><li> Quitar el extracto de la cromatina y se precipitan como se describe en el paso 2. </li><li> Lavar los cubreobjetos con PBS enfriado con hielo dos veces a 22 ° C por la inclinación del 6 y la placa. </li><li> Quitar los cubreobjetos que representa la fracción de la matriz nuclear y seguir con la fijación de células y el protocolo de inmunofluorescencia. </li><li> Si es necesario que las proteínas de la matriz se puede quitar de un cubreobjetos matriz adicional para el Western Blot con 200ul de amortiguación de los TES como se describe en el paso 2. </li><li> Las muestras para Western Blot se centrifuga a 13000 rpm en una microcentrífuga de sobremesa a 4 ° C y los pellets se resuspendieron en tampón de carga SDS 40ul (62,5 mm Tris-HCl pH 6,8, SDS al 2% (w / v), 50 mM DTT, 10% de glicerol, 0,01% azul de bromofenol (w / v)) antes del análisis por SDS-PAGE. <img src="/files/ftp_upload/1958/1958fig1.jpg" alt="Figura 1" /> Figura 1. Una representación esquemática del fraccionamiento subcelular en situ. Citoesqueleto de amortiguación (CSK) consiste en tubos 10 mM, sacarosa 300mm, 100mm NaCl, 3 mM MgCl2, 1 mM EGTA. TES buffer consiste en SDS al 1%, 2 mM EDTA, 20 mM Tris-HCl pH 7,4. </li></ol> III. Celular y la fijación de inmuno fluorescencia <ol><li> Suavemente agregar 1 ml de formaldehído al 4% / PBS a cada cubreobjetos y se incuba a 4 ° C durante 30 minutos. </li><li> Lave cada cubreobjetos suavemente 2-3 veces con 1 ml de PBS a 22 ° C. </li><li> Añadir 400ul de X-100/PBS Triton 0.1% a cada cubreobjetos y se incuba a 22 ° C durante 10 minutos. </li><li> Lave cada cubreobjetos suavemente 2-3 veces con 1 ml de PBS a 22 ° C. </li><li> Añadir 400ul de bovino al 3% de albúmina sérica (BSA) / PBS y se incuba a 22 ° C durante 20 minutos para reducir el potencial de tinción de fondo. </li><li> Lave cada cubreobjetos suavemente 2-3 veces con 1 ml de PBS a 22 ° C. </li><li> 100 ul lugar del anticuerpo primario en PBS directamente sobre el portaobjetos y se incuba a 22 ° C durante 1 hora. Anticuerpos primarios y secundarios deben ser diluidas en PBS a la concentración requerida. Esto puede tener que ser optimizado para cada anticuerpo utilizado. </li><li> Lave cada cubreobjetos suavemente 2-3 veces con 1 ml de PBS a 22 ° C. </li><li> Lugar 200ul de secundaria de anticuerpos en PBS y se incuba a 22 ° C, protegido de la luz durante 1 hora. </li><li> Lave cada cubreobjetos suavemente 2-3 veces con 1 ml de PBS a 22 ° C. </li><li> Monte cada cubreobjetos (celdas hacia abajo) sobre un portaobjetos de vidrio con medio Vectashield montaje DAPI (4 &#39;,6-diamidino-2-phenylindole clorhidrato). </li><li> Limpie el exceso de medio de montaje de todo el cubreobjetos con una toalla de papel y se incuba el portaobjetos a 22 ° C durante al menos 30 minutos de luz. </li><li> Fijar el cubreobjetos al portaobjetos de vidrio con esmalte transparente de uñas de acrílico. </li><li> Mantenga las diapositivas de la luz antes de la visualización por microscopía de fluorescencia. </li></ol> IV. Los resultados representativos <img src="/files/ftp_upload/1958/1958fig2.jpg" alt="Figura 2" /> Figura 2. Fraccionamiento subcelular de no transfectadas células COS-7. Fraccionado Cos-7 células fueron fijadas con formaldehído y immunostained utilizando α-tubulina, α-histona H1 o α-Lamin A anticuerpos / C conjugada con TRITC seguido por el tratamiento con medio de montaje DAPI . Las imágenes fueron adquiridas con lente ocular de 10x y lente objetivo de 63x utilizando un microscopio confocal Leica. Los núcleos celulares se visualizaron utilizando un conjunto DAPI filtro, mientras que en el mismo campo de las células teñidas con anticuerpos marcador se visualizaron utilizando un conjunto de filtros TRITC. <img src="/files/ftp_upload/1958/1958fig3.jpg" alt="Figura 3" /> Figura 3. Una proteína de fusión GFP-6E2 está asociada a la matriz nuclear. Fraccionamiento subcelular de Cos-7 células que expresan una proteína de fusión que consta de una proteína fluorescente verde (GFP) fusionada con el virus del papiloma humano (VPH) tipos 6Proteína E2 (GFP-6E2). Los núcleos celulares se visualizaron mediante tinción con DAPI, mientras que la localización de GFP-6E2 en las células transfectadas se visualizan directamente a través de la fluorescencia de GFP. Lamin A / C se visualizan mediante α-anticuerpos Lamin A / C conjugada con TRITC y un conjunto de filtros TRITC. Las imágenes revelan que algunos fusión de la proteína GFP-6E2 está asociada a la matriz nuclear (panel inferior derecho). Las imágenes se obtuvieron como se describe en la figura 2.

<ol>
	<li>Donaldson, M. M., Boner, W., Morgan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopBP1+regulates+human+papillomavirus+type+16+E2+interaction+with+chromatin.">TopBP1 regulates human papillomavirus type 16 E2 interaction with chromatin.</a> J Virol. 81, 4338-4342 (2007).</li><li>Javed, A., Guo, B., Hiebert, S., Choi, J. Y., Green, J., Zhao, S. C., Osborne, M. A., Stifani, S., Stein, J. L., Lian, J. B., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Groucho/TLE/R-esp+proteins+associate+with+the+nuclear+matrix+and+repress+RUNX+(CBF(alpha)/AML/PEBP2(alpha))+dependent+activation+of+tissue-specific+gene+transcription.">Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBF(alpha)/AML/PEBP2(alpha)) dependent activation of tissue-specific gene transcription.</a> J Cell Sci. 113, 2221-2231 (2000).</li><li>Kowalczyk, A. M., Roeder, G. E., Green, K., Stephens, D. J., Gaston, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+induction+or+inhibition+of+apoptosis+by+HPV+proteins.">Measuring the induction or inhibition of apoptosis by HPV proteins.</a> Methods Mol. Med. 119, 419-432 (2005).</li><li>McLarren, K. W., Theriault, F. M., Stifani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+with+the+nuclear+matrix+and+interaction+with+Groucho+and+RUNX+proteins+regulate+the+transcription+repression+activity+of+the+basic+helix+loop+helix+factor+Hes1.">Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.</a> J Biol. Chem. 276, 1578-1584 (2001).</li><li>McNeil, S., Guo, B., Stein, J. L., Lian, J. B., Bushmeyer, S., Seto, E., Atchison, M. L., Penman, S., van Wijnen, A. J., Stein, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+the+YY1+transcription+factor+to+the+nucleolus+and+the+nuclear+matrix+in+situ:+the+C-terminus+is+a+principal+determinant+for+nuclear+trafficking.">Targeting of the YY1 transcription factor to the nucleolus and the nuclear matrix in situ: the C-terminus is a principal determinant for nuclear trafficking.</a> J Cell Biochem. 68, 500-510 (1998).</li><li>Melan, M. A., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+and+differential+extraction+of+soluble+proteins+in+permeabilized+cultured+cells.+Implications+for+immunofluorescence+microscopy.">Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Implications for immunofluorescence microscopy.</a> J Cell Sci. 101, 731-743 (1992).</li><li>Zou, N., Lin, B. Y., Duan, F., Lee, K. Y., Jin, G., Guan, R., Yao, G., Lefkowitz, E. J., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hinge+of+the+human+papillomavirus+type+11+E2+protein+contains+major+determinants+for+nuclear+localization+and+nuclear+matrix+association.">The hinge of the human papillomavirus type 11 E2 protein contains major determinants for nuclear localization and nuclear matrix association.</a> J. Virol. 74, 3761-3770 (2000).</li></ol>

Los problemas más comunes y sugerencias: Todos o la mayoría de las células se pierden durante el lavado. Durante los pasos de líquidos de limpieza debe realizarse lentamente hacia el lado de la placa de 6 pocillos evitando el cubreobjetos. Del mismo modo los líquidos deben ser removidos cuidadosamente inclinando la placa y lentamente pipetear el exceso de líquido. Adhesión celular se puede aumentar el uso de poli-L-lisina cubreobjetos recubiertos. ADN genómi...

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>BSA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>A9647-100G</td>
<td>&nbsp;</td>
<tr>
<td>DNase I</td>
<td>Chemical</td>
<td>Sigma</td>
<td>DN25-1G</td>
<td>&nbsp;</td>
<tr>
<td>poly-L-Lysine</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-8920</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>Chemical</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EGTA</td>
<td>Chemical</td>
<td>Sigma</td>
<td>E4378-25G </td>
<td>&nbsp;</td>
<tr>
<td>Fomaldehyde</td>
<td>Chemical</td>
<td>BDH</td>
<td>284216N</td>
<td>&nbsp;</td>
<tr>
<td>PIPES</td>
<td>Chemical</td>
<td>Sigma</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>Chemical</td>
<td>Fisher Scientific</td>
<td>S/8600/53</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Chemical</td>
<td>Sigma</td>
<td>T-6876</td>
<td>&nbsp;</td>
<tr>
<td>Mounting Medium with DAPI</td>
<td>Chemical</td>
<td>Vector Laboratories</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Fugene 6 Transfection Reagent</td>
<td>Chemical</td>
<td>Roche </td>
<td>11814443001</td>
<td>&nbsp;</td>
<tr>
<td>Histone H1</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-8030</td>
<td>&nbsp;</td>
<tr>
<td>Lamin A/C</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-20681</td>
<td>&nbsp;</td>
<tr>
<td>Tubulin-&alpha;</td>
<td>Antibodies</td>
<td>Santa Cruz</td>
<td>SC-32293</td>
<td>&nbsp;</td>		</tbody>
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in situ subcellular fractionation of adherent and non-adherent mammalian cells

Función de la proteína está íntimamente unido a la localización de la proteína. A pesar de algunas proteínas se limitan a una ubicación específica o compartimento subcelular, muchas proteínas están presentes en una población libre difusión de libre cambio con una sub-población que está estrechamente asociada a un dominio particular o de la estructura subcelular. En fraccionamiento subcelular situ permite la visualización de proteína de la compartimentación y también puede revelar proteína sub-poblaciones que se localizan en las estructuras específicas. Por ejemplo, la eliminación de proteínas solubles citoplásmicos y suelto y sujeto proteínas nucleares puede revelar la asociación estable de algunos factores de transcripción con la cromatina. La digestión posterior de ADN en algunos casos pueden revelar asociación con la red de proteínas y ARN que se denominan colectivamente la nuclear andamio o matriz nuclear. A continuación se describen los pasos necesarios en el fraccionamiento de las células de mamíferos in situ de adherentes y no adherentes en cubreobjetos microscopio. Visualización de la proteína se puede lograr utilizando anticuerpos específicos o proteínas fluorescentes de fusión y microscopía de fluorescencia. Los anticuerpos y / o tintes fluorescentes que actúan como marcadores para los compartimientos o estructuras específicas permiten la localización de proteínas para ser cartografiado con detalle. En el fraccionamiento situ también se puede combinar con el Western Blot para comparar la cantidad de proteínas presentes en cada fracción. Este enfoque bioquímico simple puede revelar asociaciones que de otra manera no se detectan.

Watch this Scientific Journal Video about In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells at JoVE.com

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

En el fraccionamiento subcelular situ de células de mamíferos en cubreobjetos microscopio permite la visualización de la localización de la proteína.

En Fraccionamiento subcelular situ de células mamíferas adherentes y no adherentes-

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

in-situ-subcellular-fractionation-adherent-non-adherent-mammalian

Research

JoVE Journal

Biology

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

15.7K vistas.  University of Bristol. En el fraccionamiento subcelular situ de células de mamíferos en cubreobjetos microscopio permite la visualización de la localización de la proteína.

Video: In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinizing Siembra y células de mamífero

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Investigación

Biología

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

MISIÓN esiRNA sobre el cribado del ARNi en células de mamífero

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

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

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

Ensayo clonigénicas: las células adherentes

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

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Análisis de la posición del ciclo celular en células de mamífero

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

La visualización de los ARNm retículo endoplásmico localizados en células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polisoma Fraccionamiento y análisis de los mamíferos Translatomes en un genoma de gran escala

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medición de niveles hemo síntesis en células de mamífero

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagen Ca Local<sup&gt; 2 +</sup&gt; Las señales en las células de mamífero en cultivo

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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

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

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

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

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