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 Placage des cellules dans la électroporation Chambers <ol><li> Dans une hotte à flux laminaire, trypsiniser les cellules en utilisant une technique stérile, comme d&#39;habitude. NOTE: Il est très important d&#39;éliminer par centrifugation toutes les traces de la trypsine, car ils peuvent entraver la propagation des cellules sur la vitre, d&#39;où la formation d&#39;adhérentes et les jonctions communicantes. </li><li> Introduire à la pipette 1 ml de la suspension de cellules dans des chambres d&#39;électroporation stérile fourni avec l&#39;électroporation in situ (Figure 1), et les placer dans un 37 o C, incubateur à CO 2. NOTE: L&#39;adhésion cellulaire peut être améliorée par placage sur de la fibronectine, le collagène, la poly-lysine ou de la cellule et du tissu adhésif (voir le tableau des matériaux). </li><li> Lorsque les cellules ont formé une couche confluente ils sont prêts pour l&#39;examen de GJIC. </li></ol> 2. procédure électroporation L&#39;électroporation pour examen GJIC peut être effectuée en dehors d&#39;une hotte à flux laminaire,car il ne prend que quelques minutes. Si des temps d&#39;incubation plus longs sont nécessaires pour une expérience spécifique, il peut être réalisé entièrement dans une hotte à flux laminaire. Dans tous les cas, les chambres dans lesquelles les cellules sont cultivées doivent être stériles. <ol><li> Préparer une / ml solution jaune Lucifer 5 mg: Dissoudre 10 mg de poudre LY (fourni avec le électroporation) dans 2 ml de milieu de croissance exempt de calcium. Pour les expériences qui nécessite de plus longues durées d&#39;incubation, filtre stériliser la solution et conserver à 4 o C. </li><li> Aspirer le milieu de croissance et laver les cellules avec du milieu sans calcium, en faisant attention à ne pas rayer la monocouche ou sécher les cellules. Si les cellules sont séchées, ils ont généralement des noyaux sombres et ramasser LY sans électroporation. L&#39;effet est plus prononcé dans le milieu de la diapositive qui est plus exposé aux courants d&#39;air (figure 4F). </li><li> Avec une pipette Eppendorf, pipeter la solution de colorant sur les cellules (400 ul pour l&#39;ensemble de chambre), au niveau du bord de la chambre, being attention à ne pas toucher la couche de cellules. </li><li> Placer la chambre dans le support fourni avec le électroporateur et appliquer une impulsion de la force appropriée (voir Discussion). </li><li> Aspirer délicatement une partie de la solution LY. Il peut être réutilisé une deuxième fois pour des expériences moins importants. </li><li> Ajouter DMEM sans calcium contenant 10% de sérum dialyse. </li><li> Incuber les cellules pendant 3-5 minutes dans l&#39;incubateur pour permettre le transfert de colorant à travers des jonctions communicantes. NOTE: L&#39;inclusion de sérum dialyse à ce stade permet la fermeture des pores. </li><li> Laver le colorant non constituée en société DMEM sans calcium pour l&#39;observation de cellules vivantes. En variante, les cellules peuvent être fixées à ce stade, par l&#39;addition de formaldehyde à 4% à la source, puis lavées avec du PBS (solution saline tamponnée au phosphate). Dans tous les cas, le lavage doit supprimer tous fond. NOTE: Dans des expériences préliminaires afin de déterminer les conditions optimales, si la tension est trop faible, il est possible de laisser les cellules récupèrent dans le incubator pendant quelques minutes et l&#39;électroporation de la même lame d&#39;une deuxième fois, à enregistrer dans le coût de diapositives. </li></ol> 3. examen microscopique <ol><li> Observer les cellules sous illumination par fluorescence en utilisant un microscope inversé équipé d&#39;un filtre approprié pour la teinture utilisé. Pour jaune Lucifer, l&#39;excitation est 423nm, émission 555 nm, filtre VTC pour un microscope Nikon IX70. </li><li> Éliminer les effets du ménisque en plaçant une lamelle couvre-objet en verre sur le dessus de la matière plastique et, après l&#39;avoir rempli de liquide avec la partie supérieure, afin d&#39;examiner la morphologie cellulaire en contraste de phase. Pour de meilleures images, le puits peut être détachée de la lame, et la lamelle couvre-objet placé sur le châssis. Pour les cellules fixées, le puits peut être rempli avec du glycérol pour l&#39;observation au microscope, tandis que pour des cellules vivantes, il doit être rempli avec un milieu de croissance. NOTE: Le lavage et la photographie est plus facile si les cellules sont fixées avec du formaldéhyde après le transfert du colorant (étape 2.7 ci-dessus). Si les cellules ne sont pas d&#39;t fixée, la fluorescence est éliminé des cellules à l&#39;intérieur d&#39;environ 60 minutes en fonction du type de cellule, la tension et la quantité de LY introduit alors dans les cellules fixes fluorescence est conservée pendant plusieurs heures. Cependant, la fluorescence se fane ou se dissipe après O / N incubation, même dans les cellules fixes. Pour cette raison, il faut prendre des images, peu de temps après l&#39;électroporation (figure 2). </li></ol> 4 La quantification de la communication intercellulaire <ol><li> Photographier les cellules avec un objectif 20x sous fluorescence et contraste de phase (figure 3). </li><li> Identifier et marquer d&#39;une étoile les cellules par électroporation à la frontière avec la zone non-électroporation (Figure 2, flèche, bord d&#39;électroporation). </li><li> Identifier et marquer avec un point cellules fluorescentes sur le côté non-électroporation, où le colorant a transféré par les jonctions communicantes (figures 3A et 3B). </li><li> Diviser le nombre total decellules fluorescentes sur la zone non-électroporation par le nombre de cellules électroporées le long du bord (figures 3A et 3B). REMARQUE: Le transfert d&#39;au moins 200 cellules par électroporation frontières contiguës est calculé pour chaque expérience. Le nombre obtenu est la valeur 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.

Les étapes critiques dans le protocole Matériau électroporation La pureté de la matière à une électroporation est très important. Si les cellules sont très plates, des concentrations plus élevées alors du colorant de suivi doivent être utilisés (jusqu&#39;à 20 mg / ml pour LY) et dans de tels cas, la pureté est encore plus important que dans les cellules avec une forme plus sphérique 1. Outre LY une grande variété d&#39;autres colorants ou d...

L&#39;application d&#39;un courant électrique à une cellule provoque la formation de pores de la membrane de la cellule, par un processus appelé électroporation. Les pores permettent le passage d&#39;une variété de molécules à travers la membrane nonpermeant. Le champ électrique peut être commandée avec précision, de sorte que les pores formés sont très petites et se referment rapidement, avec un minimum de perturbation de la physiologie cellulaire. Fait intéressant, les cellules adhérentes sont cultivées sur une lamelle de verre revêtue d&#39;oxyde d&#39;indium-étain conductrice et transparente (ITO) et une électroporation in situ, qui se trouve sur la surface, où elles poussent, sans se détacher à une électroporation en suspension. Les cellules peuvent se développer très bien sur cette surface, et comme ils sont attachés et étendus, l&#39;observation microscopique détaillée est possible. En utilisant cette technique, de petites molécules peuvent être introduites nonpermeant directement et essentiellement en 100% des cellules, ce qui rend cette technique particulièrement appropriée pour des études sur l&#39;activation de la components une voie de stimulation suivantes ligand d&#39;un récepteur (revue dans 1). L&#39;électroporation a été utilisé principalement pour l&#39;introduction d&#39;ADN (également appelé électrotransfection). Cependant, l&#39;électroporation in situ peut être utile pour l&#39;introduction d&#39;une grande variété de molécules, telles que des peptides, des oligonucléotides 2-4, tels que l&#39;ARN antisens, des oligonucleotides double brin de l&#39;ADN leurre pour inhiber la liaison du facteur de transcription, ou siRNA 3,5, 6, 7-10 nucléotides radioactifs, des protéines 11 12 ou pro-médicaments 13. Après l&#39;électroporation, les cellules peuvent être lysées par des analyses biochimiques ou fixées et colorées avec des anticorps. Le revêtement d&#39;ITO conductrice est très mince, 800-1,000Å, de sorte que la croissance de la cellule n&#39;est pas perturbée par la différence de hauteur des deux surfaces, comme les cellules se développent à travers le bord de la couche conductrice. Ceci offre l&#39;avantage que le non-electropfilmée cellules peuvent être cultivées à côté de ceux électroporation, pour servir de témoins. La même approche peut être utilisée pour l&#39;examen des jonctions lacunaires, la communication intercellulaire (GJIC), comme décrit dans la vidéo. Les jonctions lacunaires sont des canaux reliant les intérieurs des cellules adjacentes 14. Gap jonction, la communication intercellulaire joue un rôle important dans la formation des tumeurs et des métastases, alors que les oncogènes Src telles que supprimer GJIC 15,16. Pour examiner GJIC un colorant fluorescent tel que le jaune Lucifer (LY) est souvent introduit dans des cellules en culture à travers la micro-injection ou gratter de chargement 17 et de la diffusion du colorant dans les cellules voisines est observée au microscope sous éclairage fluorescent. Ces techniques mais provoquent invariablement des dommages cellulaires. Nous décrivons maintenant une technique dans laquelle les cellules sont cultivées sur une lamelle de verre, qui est partiellement revêtue d&#39;ITO 18. Une impulsion électrique est appliquée en présence de LY (ou d&#39;autres colorants) cal&#39;aide de sa pénétration dans les cellules en croissance sur la partie conductrice de la glissière, tandis que la migration de colorant aux cellules adjacentes, non électroporation a été observée au microscope par l&#39;illumination de fluorescence. Pour éviter que les cellules sensibles perturbateurs peuvent avoir tendance à se détacher de la monocouche, un ensemble a été conçu qui ne nécessite pas une électrode à être placé au-dessus des cellules pour appliquer le courant électrique 19. Cette méthode offre la possibilité de quantifier GJIC dans un grand nombre de cellules, sans aucune perturbation détectable pour le métabolisme cellulaire, comme indiqué par l&#39;absence d&#39;un effet sur ​​la durée de la phase G1 suite à une stimulation de sérum 12, l&#39;augmentation des niveaux de la fos protéine oncogène (Raptis, non publié) ou deux kinases associées au stress cellulaire, le porc de p38 ou JNK / SAPK kinase 1. Cette approche rendue possible l&#39;examen du lien entre les niveaux d&#39;expression oncogène, la transformation et GJIC 18, ainsi que l&#39;effet de la Src et Stat3 sur GJIC dans une variété de types de cellules, y compris des cellules fraîchement cultivées à partir d&#39;échantillons de tumeur du poumon de 20 à 23. En outre, dans l&#39;électroporation in situ avec la configuration décrite qui manque une électrode supérieure a été utilisé avec succès pour la démonstration de l&#39;écart jonction lors de la différenciation adipocytaire fermeture, bien que la fixation cellulaire au substrat est réduite à ce stade 19,24.

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			<td style="width:428px;">Company</td>
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			<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>
			<td></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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			<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>
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		<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 figure 2 montre foie de rat cellules épithéliales de T51B électroporation avec 22, LY et photographiées sous fluorescence (panneau A et B) ou à contraste de phase (C), ci-après la fixation d&#39;éclairage et de lavage. Dans le panneau A, le bord de la zone électroporation est marqué en rouge. Le gradient de fluorescence à la droite de la ligne rouge indique transfert à travers les jonctions communicantes. Dans la figure 3A et 3B, la quantificati...

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.

Une analyse fonctionnelle pour les jonctions lacunaires examen; Électroporation des cellules adhérentes sur 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-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.8K vues.  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.

Vidéo: 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.

Cellules Patterning sur des électrodes d&#39;indium optiquement transparents Tin Oxide

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

Recherche

Biologie

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.

Étiquetage CSEh et hMSCs d&#39;oxyde de fer nanoparticules pour les non-invasives dans le suivi in ​​vivo avec IRM

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.

En électroporation in vivo et ex utero pour l&#39;expression génique dans les cellules ganglionnaires de la rétine de la souris

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 fractionnement subcellulaire situ des adhérentes et non-adhérents des cellules de mammifères

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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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.

Test clonogénique: cellules adhérentes

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.

L&#39;identification des cibles de microARN humaine avec le système LightSwitch Luciferase Assay utilisant 3&#39;UTR-reporter Construit et un microARN dans les cellules adhérentes Mimic

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.

Examen de<em&gt; Drosophila</em&gt; Larvaires cellules terminales trachéales par microscopie optique

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.

Mesure et analyse de la production extracellulaire acide pour Déterminer glycolytique Taux

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.

Néphrotoxine microinjection dans Zebrafish au Modèle lésion rénale aiguë

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 ...