Los autores se apoyan en la investigación sueca del Consejo, la fundación sueca para la investigación estratégica, la Sociedad Sueca del Cáncer, la sociedad del cáncer infantil sueco, Ake Wibergs y la Fundación Jeanssons.

1. La introducción de la sonda a las células <ol><li> Co-transfectar células con una sonda FRET y basado en un plásmido de resistencia que confieren. Elegir un método de transfección que sea eficiente en su celda de tipo de interés. Para las células U2OS, estándar de calcio métodos de transfección de fosfato dan resultados adecuados 15. </li><li> Seleccione las celdas en el antibiótico apropiado para al menos siete días. Esto enriquece la cantidad de células que expresan la sonda y limita la cantidad de células con niveles de expresión de tóxicos, o los niveles de expresión que afectan gravemente el ciclo celular. </li><li> (Opcional) Seleccione de clones estables. </li><li> El uso de células fijas o en vivo, compruebe que la PPC y YFP están presentes en todas las células en aproximadamente la misma proporción. </li><li> En el caso de algunas células contienen sólo la PPC o YFP, la recombinación es probable que haya ocurrido. La recombinación puede ocurrir ya sea durante el crecimiento bacteriano o después de la transfección de la sonda y puede en este último caso dependerá de la calidad del plásmido. Repita el plásmido preparación y linealizar el plásmido antes de la transfección si el problema persiste. </li></ol> 2. Radiométrica de vigilancia FRET <ol><li> Las células de las semillas en placas con fondo de cristal, o para el montaje de la cubierta se desliza en una cámara. Asegúrese que los platos / cubreobjetos hay 1,5 (170 m de espesor). </li><li> Monte las células en medio de cultivo sin rojo fenol en un microscopio de epifluorescencia motorizado con control de temperatura a 37 ° C. Garantizar la regulación del pH de los medios de comunicación, ya sea por el CO 2 o mediante el uso de CO 2-medios de comunicación independientes como Liebowitz-15. </li><li> Centrarse en las células con luz transmitida. No busques en las células con luz fluorescente. </li><li> Para empezar, la adquisición de una imagen de 12 bits o 16 con la excitación YFP (YFPex) y YFP filtros de emisión (YFPem) y un espejo dicroico adecuado. El uso de intervalos máximos disponibles que todavía permite la resolución subcelular necesario. El uso de un binning de alta para reducir el tiempo de exposición o la intensidad es crucial para evitar la fototoxicidad. </li><li> Measure o estimar la intensidad de fondo y el ruido aproximado de medir o estimar el promedio aproximado, valores mínimo y máximo de píxeles en un área desprovista de células. Tiempo de cambio de la exposición y los filtros de densidad neutra para que la media de la señal en la célula es aproximadamente la intensidad de fondo además de cinco a diez veces la diferencia entre la máxima y la intensidad de fondo min. </li><li> Compruebe que las imágenes no están cerca de estar saturado. La intensidad máxima de la imagen debe ser menos de la mitad del rango dinámico (por ejemplo, max 2000, en una imagen de 12 bits) para permitir que las diferencias de intensidad cuando las células entrar en mitosis. </li><li> Repita el paso desde 2,4 hasta 2,6 con excitación PPC y YFP filtros de emisiones y un espejo dicroico adecuado. </li><li> Seleccionar varias regiones, con las células transfectadas, con exclusión de las regiones utilizadas para el punto 2.4 a 2,7, y asegurarse de que la intensidad máxima es de menos de la mitad del rango dinámico en todas las células. </li><li> Iniciar un experimento de lapso de tiempo la adquisición de dos imágenes de cada 45 min con YFPex - YFPem y CFPex - YFPem conjuntos de filtros. </li></ol> 3. Verificación y optimización de las condiciones <ol><li> Abrir las imágenes adquiridas y controlar el tiempo de ciclo celular de la mitosis de la mitosis de las células que expresan la sonda transfectadas. </li><li> Comparar el tiempo del ciclo celular medido con los datos publicados por el tipo de células empleadas. Por otra parte, la película no transfectadas las células mediante la adquisición de una imagen de la luz transmitida solamente (DIC por ejemplo, o de contraste de fase) cada hora para obtener referencia los tiempos de ciclo celular. </li><li> En caso de que el tiempo de la mitosis de la mitosis corresponde a la referencia los tiempos del ciclo celular, vaya al paso 4. Por otra parte, volver a adquirir las imágenes con más exposición, más tiempo o múltiples puntos z-niveles y repita el paso 3. </li><li> En caso de que el tiempo de la mitosis de la mitosis no corresponde hacer referencia a los tiempos del ciclo celular, la película de células transfectadas con la adquisición de una imagen de la luz transmitida sólo (por ejemplo, DIC o de contraste de fase) cada hora, seguido por una imagen al finalde la experiencia para identificar las células transfectadas. </li><li> Si las células transfectadas aún muestran los tiempos de ciclo más largo de células, repita el paso 1, pero menos transfectar sonda o seleccionar clones estables que contienen cantidades muy bajas de la sonda. </li><li> Si las células transfectadas muestran tiempos normales del ciclo celular en ausencia de luz fluorescente, repita el paso 2, pero reducir la exposición mediante la modificación de filtros de hurgar en la basura, de densidad neutra, el tiempo de exposición, y el tiempo entre las imágenes. </li></ol> 4. El análisis de FRET <ol><li> El análisis puede ser realizado por la mayoría de los software dedicados a la microscopía. A continuación se describe cómo realizar el análisis utilizando el software gratuito ImageJ ( <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a> ) utilizando la relación de plug-in Plus ( <a href="http://imagej.nih.gov/ij/plugins/ratio-plus.html" target="_blank">http://imagej.nih.gov/ij/plugins/ratio-plus . html</a> ). </li><li> Abrir las imágenes en ImageJ. En caso de que las imágenes están en el formato de un STAC multicolork (por ejemplo, DeltaVision), separar los canales individuales, en primer lugar a la conversión a un HyperStack: Imagen-HyperStack pila de HyperStack y, posteriormente, dividir a las pilas de un solo canal con: Imagen-HyperStack-reducir la dimensionalidad </li><li> Multiplique el YFPex - pila YFPem con 3 usando: Proceso-Matemáticas-Multiplicar Este paso es opcional, ya que sólo se diseñó para aumentar la claridad visual de las imágenes asegurando que las razones no están en el rango entre 0 y 1. </li><li> En el YFPex - pila YFPem, dibuja una región de interés (ROI) en un área que carece de células, pero que está cerca de una célula de medición. Células diferentes en la misma imagen puede requerir diferentes regiones, ya que tanto el fondo y la intensidad de la señal por lo general es más fuerte en el centro de la imagen. </li><li> Agregue el retorno de la inversión ROI de manager: Analizar las herramientas de ROI-manager. </li><li> Medir la intensidad promedio de la rentabilidad, tanto en el YFPex - YFPem y la CFPex - pila YFPem: Analizar, Medir </em&gt;. </li><li> Repetir a los puntos de tiempo múltiples y verificar que las intensidades de fondo cerca de la célula de interés son similares en toda la película. </li><li> Establecer medidas para incluir un mínimo de intensidad: Analizar las mediciones-Set-Min y el valor máximo de gris. </li><li> Dibuja una región de interés que cubren la mayor parte de una célula y medir la intensidad mínima, tanto en el YFPex - YFPem y CFPex el - pila YFPem. Tomar la diferencia entre la intensidad medida mínima y la intensidad de fondo de 4,6 y se divide por dos. Esto proporciona una estimación de partida para un valor de corte. </li><li> Además Relación abierta. Seleccione la YFPex - YFPem y CFPex el - pila YFPem. Para FRET relación, seleccione CFPex - YFPem como una pila, para visualizar relación invertida FRET sondas FRET, donde disminuye la eficiencia de la fosforilación, seleccione YFPex - YFPem como una pila. </li><li> Inserte las intensidades de fondo medido y los valores de recorte. </li><li> Establecer la escala de la pila ratio resultante y aplicar una u look adecuadop tabla (LUT) para visualizar los cambios de relación. </li></ol> 5. Resultados representante Plk1 actividad es visible por primera vez en el núcleo en la fase G2 y picos durante la mitosis. La Figura 3 muestra un experimento con la configuración mínima fototoxicidad como se describe en la sección 2. Tenga en cuenta que este es un resultado inicial de representación y que las condiciones de exposición o el tiempo entre las imágenes puede ser modificado para aumentar la relación señal a ruido o la resolución temporal. Figura 3D muestra que la mayoría de las células que expresan la sonda proliferan con un tiempo de ciclo celular de entre 20 y 25 horas, lo que indica que las condiciones de imagen y los niveles de expresión de la sonda no afectan a los tiempos del ciclo celular. Aunque hay mucho ruido, la tendencia de aumento de la actividad Plk1 en G2 y en horas pico en la mitosis 14 es claramente visible (Figura 3a). Procesamiento de los datos en bruto, aquí por medio de filtrado presenta como una quimógrafo (Fig. 3B), o la cuantificación de la media ra invertido<html> tio (Fig. 3C) puede mejorar la claridad. <img alt="Figura 1" src="/files/ftp_upload/3410/3410fig1.jpg" /> Figura 1. Esquema de una sonda FRET para monitorear las actividades de quinasa y fosfatasa. Dos fluoróforos, por lo general PPC (azul) y YFP (verde), están conectados por un dominio de unión a fosfato (naranja) y una secuencia phosphorylatable (amarillo). Fosforilación (rojo) media de unión al dominio de unión a fosfato, lo que induce un cambio conformacional en la sonda. Los resultados de cambio en la conformación de una diferencia en la distancia y orientación entre los dos fluoróforos, que afecta a la eficiencia de FRET entre PPC y YFP. FRET puede ser visualizado por PPC emocionante y control de las emisiones YFP (líneas punteadas). <img alt="Figura 2" src="/files/ftp_upload/3410/3410fig2.jpg" /> Figura 2. Esquema de los del procedimiento experimental. p_upload/3410/3410fig3.jpg &quot;/&gt; Figura 3. Plk1 actividad es visible por primera vez en el núcleo en la fase G2 y picos durante la mitosis. Las células que expresan una U2OS FRET seguimiento basado en la actividad de la sonda Plk1 9,14 fueron filmados durante 60 horas utilizando un DeltaVision Spectris sistema de imágenes equipado con un objetivo de 20x NA aire 0,7 y una lámpara de mercurio. Condiciones imágenes fueron seleccionadas para causar fototoxicidad mínimo, como se indica en la sección 2, con binning 4x y filtros de densidad neutra que bloquea el 99% de la luz entrante. Una representación, en falso color de la invertida FRET-relación, a raíz de un celular a través de cuatro divisiones. B, quimógrafo de la celda se muestra en la A después de aplicar un filtro de media. C, la cuantificación de la proporción invertida FRET de la celda se muestra en A. D, entrada mitótico acumulado de 50 FRET-sonda de las células que expresan, incluyendo las divisiones de las células hijas.

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No tenemos nada que revelar.

Monitoreo FRET todo el ciclo celular requiere de consideraciones que son menos importantes cuando se evalúa a corto plazo respuestas a los estímulos externos. En primer lugar, progresión del ciclo celular es fácilmente perturbada por las señales de estrés, lo que requiere que la fototoxicidad se mantiene al mínimo. En segundo lugar, todos los periodistas pueden afectar los procesos celulares en la titulación de quinasas, fosfatasas o dominios de la interacción. La forma en que probablemente más sencilla de eva...

<table border="1"><tbody><tr><td> Reactivo </td><td> Número de catálogo </td><td> Empresa </td></tr><tr><td> Leibovitz L-15, sin rojo fenol </td><td> 21083-027 </td><td> GIBCO, por Tecnologías de la Vida </td></tr><tr><td> DMEM + Glutamax-I </td><td> 31966 </td><td> GIBCO, por Tecnologías de la Vida </td></tr><tr><td> De suero fetal bovino (FBS) </td><td> SV30160.03 </td><td> HyClone </td></tr><tr><td> 0,05% de tripsina EDTA </td><td> SH30236.01 </td><td> HyClone </td></tr><tr><td> Penicilina-estreptomicina </td><td> SV30010 </td><td> HyClone </td></tr><tr><td> DPBS </td><td> 14287 </td><td> GIBCO, por Tecnologías de la Vida </td></tr><tr><td> Puromicina </td><td> P8833 </td><td> Sigma-Aldrich </td></tr></tbody></table>

monitoring kinase and phosphatase activities through the cell cycle by ratiometric fret

Förster transferencia de energía por resonancia (FRET) basado en una prensa permite la evaluación de la quinasa endógena y las actividades de la fosfatasa en las células vivas. Estas sondas consisten normalmente en las variantes de la PPC y YFP, intervenido por una secuencia phosphorylatable y un dominio de unión a fosfato. Tras la fosforilación, la conformación de la sonda cambios, que se traduce en un cambio de la distancia y orientación entre la PPC y YFP, dando lugar a un cambio en la eficiencia de FRET (Figura 1). Varias sondas han sido publicados en la última década, el seguimiento del balance de la actividad de múltiples quinasas y fosfatasas, incluyendo reporteros de la PKA 2, PKB 3, 4 PKC, PKD 5, 6 ERK, JNK 7, Cdk 18, Aurora B 9 y Plk1 9 . Dada la estructura modular, sondas adicionales es probable que surjan en el futuro cerca de 10. Progresión del ciclo celular se ve afectada por el estrés signaling vías 11. Cabe destacar que el ciclo celular está regulado de manera diferente durante imperturbable crecimiento en comparación con cuando las células se están recuperando del estrés 12. Time-lapse de imágenes de células a través del ciclo celular por lo que requiere especial precaución. Esto se convierte en un problema particularmente cuando se emplean imágenes radiométrica, ya que dos imágenes con una elevada relación señal ruido están obligados a interpretar correctamente los resultados. Radiométrica de imágenes de FRET los cambios dependiente del ciclo celular en las actividades de quinasa y fosfatasa ha sido predominantemente restringida a sub-secciones del ciclo celular 8,9,13,14. En este caso, hablamos de un método para supervisar FRET basado en sondas utilizando imágenes radiométrica todo el ciclo de la célula humana. El método se basa en un equipo que está disponible para muchos investigadores en ciencias de la vida y no requiere conocimientos técnicos de la microscopía y procesamiento de imágenes.

Watch this Scientific Journal Video about Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET at JoVE.com

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

FRET basada en los periodistas están cada vez más utilizado para monitorear las actividades de quinasa y fosfatasa en las células vivas. Aquí se describe un método sobre el uso de FRET basada en la prensa para evaluar el ciclo celular dependiente de los cambios en la fosforilación de destino.

Seguimiento de las actividades de quinasa y fosfatasa través del ciclo celular por Proporcional FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

monitoring-kinase-phosphatase-activities-through-cell-cycle

Research

JoVE Journal

Biology

14.2K vistas.  Karolinska Institutet. FRET basada en los periodistas están cada vez más utilizado para monitorear las actividades de quinasa y fosfatasa en las células vivas. Aquí se describe un método sobre el uso de FRET basada en la prensa para evaluar el ciclo celular dependiente de los cambios en la fosforilación de destino.

Video: Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3.
Here, we present two techniques for drawing blood from rats, each with its specific applications.
Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5, determining plasma chemistry, or blood counts 6.
Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture.

La extracción de sangre de las ratas a través de la vena safena y por punción cardiaca

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: ...

Investigación

Biología

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 ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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 ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

El estudio de ADN Looping por FRET sola molécula

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Sincronización de<em&gt; Caulobacter crescentus</em&gt; Para la Investigación del Ciclo Celular Bacteriana

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

Los enfoques experimentales para estudiar mitocondrial localización y función de un ciclo celular quinasa nuclear, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

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, ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

Conformaciones GPCR Proteína G-selectivos mide usando sensores FRET en un fluorómetro Ensayo de suspensión de células vivas

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Combina la sincronización celular mitótica y microscopia Confocal de alta resolución para estudiar el papel de las proteínas multifuncional ciclo celular durante la Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...