We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1. Plating las células en las cámaras de electroporación <ol><li> En una campana de flujo laminar, trypsinize las células utilizando una técnica estéril como de costumbre. NOTA: Es muy importante eliminar por centrifugación todos los rastros de la tripsina, ya que pueden impedir la propagación de las células en el cristal, por lo tanto, la formación de adherentes y uniones gap. </li><li> Pipetear 1 ml de la suspensión de células en las cámaras de electroporación estériles proporcionadas con el en electroporador situ (Figura 1), y colóquelo en un 37 o C, CO 2 incubadora. NOTA: La adhesión celular se puede mejorar en placas sobre fibronectina, colágeno, poli-lisina o de células y el tejido adhesivo (ver Tabla de Materiales). </li><li> Cuando las células han formado una capa confluente de que estén listos para el examen GJIC. </li></ol> 2. Procedimiento electroporación La electroporación para el examen GJIC puede llevarse a cabo fuera de una campana de flujo laminar,ya que toma sólo unos pocos minutos. Si se requieren tiempos de incubación más largos para un experimento específico, entonces puede llevarse a cabo enteramente en una campana de flujo laminar. En todos los casos, las cámaras donde se cultivan las células deben ser estériles. <ol><li> Prepare una solución de color amarillo ml 5 mg / Lucifer: Disolver 10 mg de polvo LY (suministrado con el electroporador) en 2 ml de medio de cultivo libre de calcio. Para los experimentos que requieren tiempos de incubación más largos, filtro-esterilizar la solución y se almacena a 4 º C. </li><li> Aspirar el medio de cultivo y se lavan las células con medio libre de calcio, teniendo cuidado de no rayar la monocapa o secar las células. Si se secan las células por lo general tienen núcleos oscuros y recoger LY sin electroporación. El efecto es más pronunciado en el centro de la diapositiva que está más expuesta a corrientes de aire (Figura 4F). </li><li> Con una pipeta Eppendorf, pipeta la solución de colorante a las células (400 l para toda la cámara), en el borde de la cámara, ber cuidado de no tocar la capa de células. </li><li> Coloque la cámara en el soporte suministrado con el electroporador y aplicar un pulso de la fuerza apropiada (ver Discusión). </li><li> Aspirar cuidadosamente algo de la solución LY. Puede ser reutilizado una segunda vez para experimentos menos importantes. </li><li> Añadir DMEM libre de calcio que contiene 10% de suero dializado. </li><li> Se incuban las células durante 3-5 minutos en la incubadora para permitir la transferencia de colorantes a través de uniones. NOTA: La inclusión de suero dializado en este punto ayuda de cierre de los poros. </li><li> Lavar el colorante no incorporado con DMEM libre de calcio para la observación de células vivas. Alternativamente, las células pueden fijarse en esta etapa, a través de la adición de 4% de formaldehído al pozo, después se lavaron con PBS (solución salina tamponada con fosfato). En todos los casos el lavado debe eliminar todo el fondo. NOTA: En experimentos preliminares para determinar las condiciones óptimas, si el voltaje es demasiado bajo, entonces es posible hacer que las células se recuperan en el incubator durante unos minutos y electroporar la misma diapositiva una segunda vez, para ahorrar en el coste de diapositivas. </li></ol> 3. Examen microscópico <ol><li> Observar las células bajo iluminación de fluorescencia, usando un microscopio invertido equipado con el filtro apropiado para el tinte que se utiliza. Para Lucifer amarillo, excitación es 423nm, 555nm emisión, filtro WBV para un microscopio Nikon IX70. </li><li> Eliminar los efectos de menisco mediante la colocación de un cubre de vidrio en la parte superior del plástico y, después de llenar a la cima con el líquido, para examinar la morfología celular bajo contraste de fase. Para obtener mejores imágenes, el pozo puede ser separado de la corredera, y el cubreobjetos colocado en el bastidor. Para las células fijas, el pozo puede ser llenado con glicerol para la observación microscópica, mientras que para las células vivas que debe ser llenado con medio de crecimiento. NOTA: Lavado y fotografiar es más fácil si las células se fijan con formaldehído tras la transferencia del colorante (paso 2.7). Si las células no sont fijo, la fluorescencia se elimina de las células dentro de aproximadamente 60 min dependiendo de la introdujo en células fijadas, mientras que la fluorescencia se mantiene durante varias horas tipo de célula, la tensión y la cantidad de LY. Sin embargo, la fluorescencia se desvanece o se disipa después de O / N de incubación, incluso en células fijadas. Por esta razón, las fotografías deben tomarse pronto después de la electroporación (Figura 2). </li></ol> 4. La cuantificación de la comunicación intercelular <ol><li> Fotografiar las células con un objetivo de 20x bajo de fluorescencia y de contraste de fase (Figura 3). </li><li> Identificar y marcar con una estrella las células sometidas a electroporación en la frontera con la zona de no-electroporación (Figura 2, flecha, borde electroporación). </li><li> Identificar y marcar con un punto células fluorescentes en el lado no-electroporación, donde el colorante ha transferido a través de uniones (Figuras 3A y 3B). </li><li> Se divide el número total decélulas fluorescentes en la zona de no-electroporación por el número de células sometidas a electroporación a lo largo del borde (Figuras 3A y 3B). NOTA: La transferencia de al menos 200 células del borde contiguas a electroporación se calcula para cada experimento. El número obtenido es el valor GJIC. </li></ol>

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The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

Los pasos críticos en el protocolo Material de electroporados La pureza del material que se va a electroporación es muy importante. Si las células son muy planas, se deben utilizar concentraciones más altas de entonces el colorante de seguimiento (hasta 20 mg / ml para LY) y en tales casos, la pureza es incluso más importante que en las células con una forma más esférica 1. Además LY han empleado una gran variedad de otros colorantes o moléculas non...

La aplicación de corriente eléctrica a una célula provoca la formación de poros en la membrana celular, mediante un proceso denominado electroporación. Los poros permiten el paso de una variedad de moléculas nonpermeant través de la membrana. El campo eléctrico puede ser controlada con precisión, de modo que los poros formados son muy pequeñas y volver a cerrar rápidamente, con la mínima perturbación a la fisiología celular. Curiosamente, las células adherentes pueden ser cultivadas en un portaobjetos de vidrio recubierto con óxido de indio-estaño conductora y transparente (ITO) y electroporación in situ, que está en la superficie donde crecen, sin ser individual para ser a electroporación en suspensión. Las células pueden crecer muy bien en esta superficie, y como están unidos y extendidos, la observación microscópica detallada es posible. Usando esta técnica, moléculas pequeñas nonpermeant se pueden introducir de forma instantánea y en esencialmente el 100% de las células, lo que hace esta técnica especialmente adecuada para estudios sobre la activación de un borradoronents de una vía después de la estimulación del ligando de un receptor (revisado en 1). La electroporación se ha utilizado principalmente para la introducción de ADN (también llamado electrotransfección). Sin embargo, la electroporación in situ puede ser valiosa para la introducción de una gran variedad de moléculas, tales como péptidos, oligonucleótidos 2-4, tales como ARN antisentido, oligonucleótidos de doble cadena de ADN de señuelo para inhibir la unión del factor de transcripción, o siRNA 3,5, 6, 7-10 nucleótidos radiactivos, proteínas 11 12 o profármacos 13. Después de la electroporación, las células pueden ser lisadas para los análisis bioquímicos o fijaron y se tiñeron con anticuerpos. El recubrimiento de ITO conductor es muy delgada, 800-1,000Å, de modo que el crecimiento celular no se ve perturbado por la diferencia de altura de las dos superficies, ya que las células crecen a través del borde del revestimiento conductor. Esto ofrece la ventaja de que no electropevaporaron células pueden ser cultivadas lado a lado con los electroporación, para servir como controles. El mismo enfoque se puede utilizar para el examen de la unión brecha, la comunicación intercelular (GJIC), como se describe en el vídeo. Gap cruces son canales que conectan los interiores de células adyacentes 14. Unión Gap, la comunicación intercelular juega un papel importante en la formación de tumores y metástasis, mientras que los oncogenes tales como Src suprimen GJIC 15,16. Para examinar GJIC un colorante fluorescente como el amarillo Lucifer (LY) a menudo se introduce en células cultivadas a través de microinyección o raspar de carga 17 y la difusión del colorante en las células vecinas se observa microscópicamente bajo iluminación de fluorescencia. Estas técnicas, sin embargo, invariablemente causan daño celular. Describimos ahora una técnica donde las células se cultivan en un portaobjetos de vidrio que está parcialmente recubierto con ITO 18. Un pulso eléctrico se aplica en la presencia de LY (u otros colorantes) cautilizando su penetración en las células que crecen en la parte conductora de la corredera, mientras que la migración de colorante a las células adyacentes no se observó microscópicamente electroporación través de la iluminación de fluorescencia. Para evitar células sensibles perturbadores que pueden tender a separarse de la monocapa, un conjunto fue diseñado que no requieren un electrodo que se coloca en la parte superior de las células para aplicar la corriente eléctrica 19. Este enfoque ofrece la posibilidad de cuantificar la GJIC en un gran número de células, sin ninguna perturbación detectable con el metabolismo celular, como se indica por la ausencia de un efecto sobre la duración de la fase G1 después de la estimulación de suero 12, el aumento de los niveles de los fos proteína protooncogén (Raptis, inédito) o dos quinasas asociadas con el estrés celular, el cerdo p38 o JNK / SAPK quinasa 1. Este enfoque hace posible el examen de la relación entre los niveles de expresión de oncogenes, transformación y GJIC 18, así como el efecto de Src y Stat3 sobre GJIC en una variedad de tipos de células, incluyendo células recién cultivadas a partir de muestras de tumor de pulmón 20-23. Además, en la electroporación in situ con la configuración descrita que carece de un electrodo superior ha sido empleado con éxito para la demostración de cierre de brecha de la salida a la diferenciación adipocítico, aunque la unión celular al sustrato se reduce en esa etapa 19,24.

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			<td height="42" style="height:42px;width:179px;">Name of Material/ Equipment</td>
			<td style="width:428px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
			<td style="width:119px;">50-013-PB</td>
			<td></td>
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			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
			<td></td>
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			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
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			<td height="21" style="height:21px;width:179px;">Electroporation apparatus</td>
			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
			<td style="width:119px;">ACE-100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-04-CC</td>
			<td>4-wells</td>
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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-08-CC</td>
			<td>8-wells</td>
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			<td height="21" style="height:21px;width:179px;">Lucifer Yellow</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-25-LY</td>
			<td>High purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CelTak</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Poly-Lysine</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
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a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

La Figura 2 muestra células de hígado de rata T51B epitelial 22, electroporación con LY y fotografiados bajo de fluorescencia (panel A y B), o de contraste de fase (C), la iluminación siguientes fijación y lavado. En el panel A, el borde de la zona de electroporación está marcado en rojo. El gradiente de la fluorescencia a la derecha de la línea roja indica la transferencia a través de uniones. En la figura 3A y 3B, la cuantificación de la brecha oc...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Un ensayo funcional para Gap Junctional examen; La electroporación de células adherentes de indio-óxido de estaño

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.8K vistas.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Video: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

Las células de patrón indio electrodos ópticamente transparente de óxido de estaño

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

Investigación

Biología

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Etiquetado y hESCs hMSCs con nanopartículas de óxido de hierro para no invasivo en el seguimiento in vivo mediante RM

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

La electroporación in vivo en el útero y el ex de la expresión génica en células de ratón ganglionares retinianas

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

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

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

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, ...

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. ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

Objetivos la identificación de los microARN humanos con el sistema de ensayo con luciferasa LightSwitch reportero 3&#39;UTR-Construye y microRNA un imitador de las células adherentes

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

El examen de<em&gt; Drosophila</em&gt; Larvas células traqueales de terminales por microscopía de luz

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...