Este trabajo fue financiado por subvenciones del NIH y ACS, incluyendo T32CA009135, CA133027, CA070723, y CA022443. Bill Sugden es una Sociedad Americana del Cáncer Profesor de Investigación.

1. Ingeniería de células con plásmidos visibles <ol><li> Utilice la digestión de restricción estándar o técnicas de recombinación homóloga para introducir un fragmento de ADN que contiene aproximadamente 250 copias de la secuencia operador lactosa (LACO) en el plásmido para ser visualizados. Nuestra plásmido era un bácmido de aproximadamente 170 kpb y contenía el genoma completo de Sarcoma de Kaposi asociado a herpesvirus (KSHV) y la resistencia a la higromicina codificada 12. </li><li> Introducir Laco que contienen ADN plásmido en células de mamífero por transfección con Lipofectamine 2000. Estamos transfectadas las células HeLa y SLK en placas de 60 mm durante 4 horas con 10 g ADN de plásmido purificado con 25 l Lipofectamine 2000, 0,5 OptiMEM ml, y 3,5 ml de DMEM. </li><li> Cambiar el medio de cultivo a 5 ml de DMEM con 10% de células fetales de suero bovino y se incuban durante 48 hr. </li><li> Las células en placas de baja densidad en platos grandes y células de cultivo en un medio que contiene una selección de antibióticos para la presentación plasmid; se seleccionaron las células en DMEM con suero fetal bovino al 10% con 300 mg / ml de higromicina durante 3 semanas. </li><li> Utilice pedazos de estériles, la tripsina papel de filtro empapado de transferir higromicina colonias resistentes a los platos de la nueva cultura. </li><li> Cuando los clones transfectados han crecido hasta llenar el recipiente, para aislar el ADN de la digestión de restricción y transferencia de Southern para garantizar que el polímero de la plásmido Laco es homogéneo y del tamaño esperado. </li><li> Si el plásmido no se ha modificado genéticamente para expresar una fusión de LacI, infectar clones adecuados con un retrovirus que codifica el represor de lactosa (LacI) fusionada a una proteína fluorescente de color rojo, tal como LacI-tdTomato. Utilizamos tdTomato en lugar de proteínas que presentan fluorescencia verde cerca de minimizar photoxicity que ocurrir incluso a dosis múltiples durante largos tiempos de 13. </li><li> Una vez que la proteína de fusión LacI se introduce, cultivar las células en presencia de 200 mg / ml isopropil-bD-tiogalactósido (IPTG) para bloquear las proteínas de fusión de uniónDNA. </li><li> Cultivar las células durante al menos 3 días para permitir la expresión de la proteína de LacI-tdTomato. </li><li> Clones de pantalla (por lavar el IPTG y visualización de las células como se describe más adelante) para aquellos con niveles óptimos de LacI-tdTomato que revelan distintas señales con niveles de referencia mínimos de proteína no unida fusión. </li></ol> 2. Desarrollo del sistema de imágenes Nuestro sistema de formación de imágenes consiste en un Zeiss Axiovert 200M equipado con una platina motorizada y Plan-Aochromat 63x/1.4 objetivo aceite. Luz de excitación de fluorescencia es proporcionado por el &quot;blanco neutral&quot; LED de un sistema de Colibri. Emisión es detectada por un CascadeII: 1024 cámara EMCCD. Esta es una cámara enfriada, back-diluido con un registro de ganancia para amplificar las señales, la corriente oscura es 0,061 electrones por píxel por segundo, y la eficiencia cuántica es mayor que 0,90. El sistema está controlado por software Axiovision 4,8, incluyendo el módulo SmartExperiments. Para la detección de tdTomaa que utilizamos un 75HE Zeiss Juego de filtros para los que el 85% de la luz que pasa puede excitar el flúor y el 95% de la luz emitida pasará el filtro de emisión. <ol><li> Montar un sistema de imágenes con un microscopio invertido y una cámara EMCCD para la detección sensible de señales, una fuente de luz LED de excitación específica y el cierre rápido, una platina motorizada y brazo de enfoque, y el control de software para la adquisición automática de imágenes en intervalos de tiempo largos con múltiples z-pilas. El sistema debe sentarse en una mesa de aire a ser aislado de las vibraciones. </li><li> Montar una incubadora de etapa-top para mantener las células a 37 º C, 5-10% de CO2 en una cámara humidificada. Transcurrido el tiempo suficiente para que el sistema llega a una temperatura estable para minimizar los artefactos de movimiento durante el experimento. </li><li> Usar un collar caliente para el objetivo de prevenir el objetivo de sifón de calor de la muestra. </li></ol> 3. Visualización de ADN marcadas <ol><li> Células en placas de 35 mmplato de fondo de vidrio a una densidad de menos de 10% de confluencia, lo que permitirá pares de células que se dividan en las colonias de 8-16 células sin superposición. ¿Es esta hr al menos 24-48 antes de formación de imágenes de modo que las células viables tienen tiempo para dividir. </li><li> Uno a tres horas antes del inicio de formación de imágenes, enjuagar la placa 3 veces con medio de cultivo que carece de ambos fármacos selectivos e IPTG. Este procedimiento lava las células no viables y permite que las proteínas de fusión LacI para enlazar sitios Laco en los plásmidos. </li><li> Sustituir el medio de cultivo con 2 ml de DMEM con suero fetal bovino al 10% y HEPES 25 mM. </li><li> Reemplazar la parte superior de la placa de cultivo con CultFoil estira en un anillo de montaje para un plato de 35 mm. Esta tapa inhibir la evaporación del medio de cultivo, lo que aumentaría la concentración de sal en el tiempo y matar a las células. </li><li> El uso de contraste de interferencia (DIC) de imágenes para ver las células y determinar el plano focal de la parte superior e inferior de las células en múltiples campos de visión.En cada campo, se mueven a un plano focal de aproximadamente 1/3 del camino hacia abajo desde la parte superior de la celda. Guarde el x, y, z las coordenadas en la relación de puestos de fase guardado. Nota: Las señales plásmido puede no ser visible a través de los oculares cuando las células son iluminadas por LEDs de la longitud de onda apropiada para la proteína de fusión LacI. Las señales débiles sólo pueden ser visibles con el uso de la cámara EMCCD, que permite la integración de la señal en el tiempo y por lo tanto amplifica eficazmente señales débiles. </li><li> En el software de control del microscopio, definir un z-pila de imágenes que se deben realizar en cada posición de fase guardado. Elija el tamaño de az paso de 0.35-0.50 m, que le permitirá a la aproximación de muestreo de Nyquist en la dimensión z 14. Incluir &quot;extra&quot; rebanadas por encima y por debajo de los planos en los que se encuentran las células; estas rebanadas de permitir la detección siguiendo movimientos de la célula durante el ciclo celular, y también se utilizan en el procesamiento posterior a la adquisición (deconvolución) de las pilas de imagen. Esta inclusión seresultar en una pila de 40-60 imágenes, dependiendo del grosor de las células. </li><li> En el software de control del microscopio, definir un experimento de series de tiempo para recoger una imagen de campo brillante de la z-pilas a intervalos de 30 min (para el seguimiento de las divisiones celulares y las migraciones más largos tiempos) y las imágenes de fluorescencia a intervalos de 10-60 min (para la visualización de plásmidos dentro de las células). No utilice imágenes DIC durante el experimento, ya que requiere más luz que las imágenes de campo claro estándar, que puede exponer a las células innecesariamente a la fototoxicidad en el curso de experimentos de largo. </li><li> Iniciar el experimento controlado por software. Asegúrese de que el sistema no está perturbado (golpeado, expuesto a la luz ambiente en exceso, etc) durante el experimento. </li><li> Si es necesario, reponer el agua en el sistema de humidificación durante el experimento. </li></ol> 4. Post-adquisición de Procesamiento de Imágenes <ol><li> Revisar las imágenes para cada punto z-pila y el tiempo. Recortar cualquier área que no necesite delas imágenes para reducir el tiempo de procesamiento informático en la siguiente etapa. </li><li> Utilizar un algoritmo de deconvolución para volver a asignar a la intensidad de la señal de píxel de origen en cada 15 z-pila. Si bien este deconvolución se puede basar en una función de dispersión de punto teórico para su sistema de imagen, es preferible medir la función de dispersión de punto de su sistema en particular por formación de imágenes microesferas fluorescentes, y el uso de esta medida en el algoritmo de deconvolución. Se midió la función de dispersión puntual de nuestro sistema de formación de imágenes y aplica a. Limitada algoritmo iterativo de máxima verosimilitud en AxioVision software (descripción AxioVision software, Zeiss) </li><li> Examine las imágenes para el seguimiento de los cambios de intensidad y los movimientos de los plásmidos durante el ciclo celular y la división de los plásmidos a las células hijas. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology 2 volume set. , (2006).</li><li>Adams, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+latent+Epstein-Barr+virus+genomes+in+Raji+cells.">Replication of latent Epstein-Barr virus genomes in Raji cells.</a> J. Virol. 61, 1743-1746 (1987).</li><li>Humme, 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=The+EBV+nuclear+antigen+1+(EBNA1)+enhances+B+cell+immortalization+several+thousandfold.">The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 10989-10994 (1073).</li><li>Verma, S. C., Choudhuri, T., Robertson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+minimal+replicator+element+of+the+Kaposi's+sarcoma-associated+herpesvirus+terminal+repeat+supports+replication+in+a+semiconservative+and+cell-cycle-dependent+manner.">The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner.</a> J. Virol. 81, 3402-3413 (2007).</li><li>Yates, J. L., Guan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epstein-Barr+virus-derived+plasmids+replicate+only+once+per+cell+cycle+and+are+not+amplified+after+entry+into+cells.">Epstein-Barr virus-derived plasmids replicate only once per cell cycle and are not amplified after entry into cells.</a> J. Virol. 65, 483-488 (1991).</li><li>Ye, F. C., 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=Disruption+of+Kaposi's+sarcoma-associated+herpesvirus+latent+nuclear+antigen+leads+to+abortive+episome+persistence.">Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence.</a> J. Virol. 78, 11121-11129 (2004).</li><li>Robinett, C. C., 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=In+vivo+localization+of+DNA+sequences+and+visualization+of+large-scale+chromatin+organization+using+lac+operator/repressor+recognition.">In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.</a> J. Cell Biol. 135, 1685-1700 (1996).</li><li>Nanbo, A., Sugden, A., Sugden, 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+coupling+of+synthesis+and+partitioning+of+EBV's+plasmid+replicon+is+revealed+in+live+cells.">The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells.</a> Embo. J. 26, 4252-4262 (2007).</li><li>Herndier, B. G., 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=Characterization+of+a+human+Kaposi's+sarcoma+cell+line+that+induces+angiogenic+tumors+in+animals.">Characterization of a human Kaposi's sarcoma cell line that induces angiogenic tumors in animals.</a> Aids. 8, 575-581 (1994).</li><li>Zhou, F. C., 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=Efficient+infection+by+a+recombinant+Kaposi's+sarcoma-associated+herpesvirus+cloned+in+a+bacterial+artificial+chromosome:+application+for+genetic+analysis.">Efficient infection by a recombinant Kaposi's sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis.</a> J. Virol. 76, 6185-6196 (2002).</li><li>Shaner, N. C., 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=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Points,+pixels,+and+gray+levels:+digitizing+image+data.">Points, pixels, and gray levels: digitizing image data.</a> Handbook of Biological Confocal Microscopy. , 59-79 (2006).</li><li>Cannell, M. B., McMorland, A., Soeller, C., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+enhancement+by+deconvolution.">Image enhancement by deconvolution.</a> Handbook of biological confocal microscopy. , 488-500 (2006).</li></ol>

No hay conflictos de interés declarado.

El método descrito aquí se puede utilizar para seguir los plásmidos en células de mamíferos vivos en el tiempo que abarca múltiples generaciones. Al limitar la exposición a la luz de excitación, hemos utilizado estas técnicas para seguir las células de parejas recién divididas a través de las colonias de 16-32 células, lo que representa por lo menos 72 horas y 3 divisiones celulares. Estos experimentos proporcionan información que no puede obtenerse a partir de ensayos estáticos, tales como PCR cuantitati...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 11965 </td><td></td></tr><tr><td> OptiMEM </td><td> GIBCO </td><td> 31985 </td><td></td></tr><tr><td> El suero fetal bovino </td><td> Hyclone </td><td> SH30910.03 </td><td></td></tr><tr><td> Penicilina </td><td> Sigma </td><td> P3032 </td><td></td></tr><tr><td> Sulfato de estreptomicina </td><td> Sigma </td><td> S9137 </td><td></td></tr><tr><td> Higromicina </td><td> Calbiochem </td><td> 400050 </td><td> 400 g / ml para SLK; 300 g / ml para HeLa </td></tr><tr><td> Isopropil-bD-tiogalactósido (IPTG) </td><td> Roche </td><td> 10 724 815 001 </td><td> Concentración final 200 ug / ml </td></tr><tr&gt; <td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-027 </td><td></td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td></td></tr><tr><td> Con fondo de cristal platos </td><td> MatTek </td><td> P35G-1.5-10-C </td><td></td></tr><tr><td> CultFoil </td><td> Pecon </td><td> 000000-1116-08 </td><td></td></tr><tr><td> Axiovert 200M </td><td> Zeiss </td><td></td><td></td></tr><tr><td> Colibri </td><td> Zeiss </td><td> 423052-9500-000 </td><td></td></tr><tr><td> Neutral LED blanco </td><td> Zeiss </td><td> 423052-9120-000 </td><td></td></tr><tr><td> Filtrar Cube 75 HE </td><td> Zeiss </td><td> 489075-0000-000 </td><td></td></tr><tr><td> Plan-Apochromat 63x/1.4 objetivo </td><td> Zeiss </td><td> 440762-9904-000 </td><td></td></tr><tr><td> CascadeII: 1024 EMCCD cámara </td><td> Fotometría </td><td> B10C892007 </td><td></td></tr><tr><td> Tempcontrol mini- </td><td> Zeiss / Pecon </td><td> 000000-1116-070 </td><td></td></tr><tr><td> Tempcontrol 37-2 digitales </td><td> Zeiss / Pecon </td><td> 000000-1052-320 </td><td></td></tr><tr><td> Controlador digital de CTI 3700 </td><td> Zeiss / Pecon </td><td> 411856-9903 </td><td></td></tr><tr><td> Objetivo calentador </td><td> Zeiss / Pecon </td><td> 440760-0000-000 </td><td></td></tr><tr><td> Fase de calentamiento inserto P </td><td> Zeiss / Pecon </td><td> 411861-9901-000 </td><td></td></tr><tr><td> Stage-top Incubadora S </td><td> Zeiss / Pecon </td><td> 411860-9902-000 </td><td></td></tr><tr><td> Humidificador Sistema </td><td> Zeiss / Pecon </td><td> 000000-1116-065 </td><td></td></tr></tbody></table>

monitoring plasmid replication in live mammalian cells over multiple generations by fluorescence microscopy

Pocos plásmidos de origen natural se mantienen en células de mamífero. Entre éstos están los genomas de gamma-herpesvirus, incluyendo el virus de Epstein-Barr (EBV) y el sarcoma de Kaposi asociado a herpesvirus (KSHV), que provocan múltiples malignidades humanas 1-3. Estos dos genomas se replican de manera autorizada, cada uno usando una sola proteína viral y maquinaria de replicación celular, y se pasan a las células hijas durante la división celular a pesar de que carecen de sus centrómeros tradicionales 4-8. Mucho trabajo se ha realizado para caracterizar las repeticiones de estos genomas plásmido utilizando métodos tales como Southern blot y la hibridación fluorescente in situ (FISH). Estos métodos están limitados, sin embargo. PCR cuantitativa y las transferencias de Southern proporcionar información sobre el número medio de plásmidos por célula en una población de células. La FISH es un ensayo de una sola célula que revela tanto el número medio y la distribución de plásmido<html> s por célula en la población de células, pero es estática, no permitiendo ninguna información sobre el padre o progenie de la célula examinada. Aquí se describe un método para visualizar los plásmidos en células vivas. Este método se basa en la unión de una proteína marcada con fluorescencia lactosa represor a múltiples sitios en el plásmido de interés 9. El DNA de interés está diseñada para incluir aproximadamente 250 repeticiones en tándem del operador lactosa (LACO) secuencia. Laco es unido específicamente por la proteína represora de la lactosa (LacI), que puede ser fusionada con una proteína fluorescente. La proteína de fusión puede expresarse a partir del plásmido de ingeniería o introducida por un vector retroviral. De esta manera, las moléculas de ADN están marcados fluorescentemente y por lo tanto se hacen visibles a través de microscopía de fluorescencia. La proteína de fusión se bloquea la unión de la ADN plasmídico por cultivo de células en presencia de IPTG hasta los plásmidos están listos para ser visto. ONTENIDO &quot;&gt; Este sistema permite que los plásmidos sea controlados en células vivas a través de varias generaciones, revelando propiedades de su síntesis y la partición a las células hijas. células ideales son adherente, fácilmente transfectadas, y tienen núcleos grandes. Esta técnica se ha utilizado para determinar que 84% de EBV derivados de plásmidos se sintetizan cada generación y 88% de la partición recién sintetizado plásmidos fielmente a las células hijas en células HeLa. pares de estos plásmidos EBV se observaron a ser atados o asociado a las cromátidas hermanas después de su síntesis en S- fase hasta que se observaron para separar como las cromátidas hermanas en la anafase separados 10. El método se está utilizando actualmente para estudiar la replicación de los genomas HVSK en células HeLa y células SLK. células HeLa se inmortalizan las células epiteliales humanas, y las células inmortalizadas se SLK endoteliales humanas las células. Aunque las células SLK se derivaron originalmente de una lesión KSHV, ni la línea HeLa ni SLK celular naturalmente harbors HVSK genomas 11. Además de estudiar la replicación viral, esta técnica de visualización se puede utilizar para investigar los efectos de la adición, eliminación o mutación de los diversos elementos de secuencia de ADN en la síntesis, la localización, y la partición de otros ADN de los plásmidos recombinantes.

<html> Proyecciones de máxima intensidad de z-pilas adquiridas en un experimento representativo se muestra en la Figura 3. Típicas señales plásmido están presentes en 3-8 rebanadas de la Z-Stack, dependiendo de si representan única o grupo de plásmidos. En el caso de EBV y KSHV plásmidos derivados, las señales se mueven lentamente dentro de la célula hasta que la célula alcanza la mitosis. Las células se migran durante el transcurso del experimento. También se vuelven esféricos y desprenden...

Watch this Scientific Journal Video about Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy at JoVE.com

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Un método de observación de moléculas individuales de ADN en células vivas se describe. La técnica se basa en la unión de una proteína marcada con fluorescencia represor lac a sitios de unión ingeniería genética en el ADN de interés. Este método puede ser adaptado para seguir muchos ADNs recombinantes en células vivas en el tiempo.

Supervisión de la replicación del plásmido en células de mamíferos en vivo durante varias generaciones por microscopía de fluorescencia

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

monitoring-plasmid-replication-live-mammalian-cells-over-multiple

Research

JoVE Journal

Biology

11.7K vistas.  University of Wisconsin - Madison. Un método de observación de moléculas individuales de ADN en células vivas se describe. La técnica se basa en la unión de una proteína marcada con fluorescencia represor lac a sitios de unión ingeniería genética en el ADN de interés. Este método puede ser adaptado para seguir muchos ADNs recombinantes en células vivas en el tiempo.

Video: Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basado en la fluorescencia de medición de la entrada de calcio de la tienda que funciona en las células vivas: a partir de células cancerosas cultivadas de fibra muscular esquelética

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Cuantitativa de células vivas por fluorescencia microscopia análisis de la levadura de fisión

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET Microscopía de Monitoreo en tiempo real de eventos de señalización en las células vivas Uso de biosensores unimoleculares

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

En Vivo Tracking 4-Dimensional de madre hematopoyéticas y células progenitoras en ratones adultos de calota de Médula Ósea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

La detección de Intracelular expresión génica en células vivas de murino, humano y porcino Origen Utilizando nanopartículas fluorescencia marcado

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...