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 placcatura le cellule del elettroporazione Chambers <ol><li> In una cappa a flusso laminare, trypsinize le celle utilizzando una tecnica sterile, come al solito. NOTA: E &#39;molto importante eliminare mediante centrifugazione tutte le tracce di tripsina, perché possono ostacolare la diffusione delle cellule sul vetro, quindi la formazione di adherens e giunzioni. </li><li> Pipettare 1 ml di sospensione cellulare nelle camere di elettroporazione sterili forniti con il elettroporatore in situ (Figura 1), e posto a 37 ° C, CO 2 incubatore. NOTA: l&#39;adesione delle cellule può essere migliorata placcatura su fibronectina, collagene, poli-lisina o di cellule e tessuti adesivo (vedi Materiali Tabella). </li><li> Quando le cellule hanno formato uno strato confluente sono pronti per l&#39;esame GJIC. </li></ol> 2 elettroporazione Procedura Elettroporazione per l&#39;esame GJIC può essere condotta al di fuori di una cappa a flusso laminare,dal momento che richiede solo pochi minuti. Se sono necessari tempi di incubazione più lunghi per un esperimento specifico, allora può essere condotta interamente in una cappa a flusso laminare. In tutti i casi, le sezioni dove si coltivano le cellule devono essere sterili. <ol><li> Preparare una / ml soluzione gialla Lucifero 5 mg: sciogliere 10 mg di polvere LY (fornito con il elettroporatore) in 2 ml di terreno di coltura privo di calcio. Per esperimenti che richiedono tempi di incubazione più lunghi, filtro-sterilizzare la soluzione e conservare a 4 ° C. </li><li> Aspirare il terreno di crescita e lavare le cellule con terreno privo di calcio, facendo attenzione a non graffiare il monostrato o asciugare le cellule. Se le cellule sono asciugati solito hanno nuclei più scuri e raccogliere LY, senza elettroporazione. L&#39;effetto è più pronunciato nel mezzo della slitta che è più esposto a correnti d&#39;aria (Figura 4F). </li><li> Con una pipetta Eppendorf, pipetta la soluzione colorante per le cellule (400 microlitri per tutta la camera), sul bordo della camera, being attenzione a non toccare lo strato di cellule. </li><li> Posizionare la camera nel supporto fornito con il elettroporatore e applicare un impulso della forza appropriata (vedi discussione). </li><li> Aspirare accuratamente alcuni della soluzione LY. Può essere riutilizzato una seconda volta per esperimenti meno importanti. </li><li> Aggiungi DMEM privo di calcio contenente siero dializzato del 10%. </li><li> Incubare le cellule per 3-5 minuti in incubatrice per consentire il trasferimento colorante attraverso giunzioni gap. NOTA: L&#39;inclusione di siero dializzato, a questo punto aiuta chiusura dei pori. </li><li> Lavare il colorante costituite in società con DMEM senza calcio per l&#39;osservazione di cellule vive. In alternativa, le cellule possono essere fissate in questa fase, attraverso l&#39;aggiunta di 4% di formaldeide al pozzo, poi lavate con PBS (soluzione salina tamponata con fosfato). In tutti i casi il lavaggio deve rimuovere tutta sfondo. NOTA: In esperimenti preliminari per determinare le condizioni ottimali, se la tensione è troppo bassa, allora è possibile lasciare che le cellule recuperare nel incubator per alcuni minuti e l&#39;elettroporazione stesso vetrino una seconda volta, per risparmiare nel costo di diapositive. </li></ol> 3 Esame microscopico <ol><li> Osservare le cellule sotto illuminazione a fluorescenza, utilizzando un microscopio invertito equipaggiato con il filtro appropriato per il colorante utilizzato. Per Lucifero giallo, eccitazione è 423nm, 555nm emissione, filtro WBV per un microscopio Nikon IX70. </li><li> Eliminare effetti menisco mettendo un vetro di copertura antiscivolo sulla parte superiore della plastica e, dopo il riempimento al top con il liquido, per esaminare la morfologia delle cellule in contrasto di fase. Per le foto migliori, il pozzo può essere staccato dalla diapositiva, e il coprioggetto posto sul telaio. Per cellule fissate, il pozzo può essere riempito con glicerolo per l&#39;osservazione microscopica, mentre per le cellule vive deve essere riempito con terreno di crescita. Attenzione: Il lavaggio e la fotografia è più facile se le cellule vengono fissate con formaldeide a seguito del trasferimento del colorante (precedente punto 2.7). Se le cellule sonot fissato, la fluorescenza viene eliminata dalle cellule entro circa 60 minuti a seconda del tipo di cellula, la tensione e la quantità di LY introdotto mentre nelle cellule fissate fluorescenza viene mantenuta per diverse ore. Tuttavia, fluorescenza svanisce o si dissolve dopo O / N incubazione, anche in cellule fissate. Per questo motivo, illustrazioni, devono essere prese subito dopo l&#39;elettroporazione (Figura 2). </li></ol> 4. quantificazione di comunicazione intercellulare <ol><li> Fotografa le cellule con un obiettivo 20x in fluorescenza e contrasto di fase (Figura 3). </li><li> Identificare e contrassegnare con una stella le cellule elettroporate al confine con la zona non elettroporate (figura 2, freccia, bordo elettroporazione). </li><li> Identificare e segnare con un punto le cellule fluorescenti sul lato non-elettroporate, in cui il colorante è trasferito attraverso giunzioni gap (Figure 3A e 3B). </li><li> Dividere il numero totale dicellule fluorescenti sulla zona non elettroporate per il numero di cellule elettroporate lungo il bordo (Figure 3A e 3B). NOTA: Il trasferimento di almeno 200 cellule elettroporate di frontiera contigue viene calcolato per ogni esperimento. Il numero ottenuto è il valore 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.

Passaggi critici nel protocollo Materiale elettroporate La purezza del materiale da elettroporate è molto importante. Se le cellule sono molto piatto, devono essere utilizzate concentrazioni più elevate di poi il colorante inseguimento (fino a 20 mg / ml per LY) e in tali casi, la purezza è ancora più importante che nelle cellule con una forma più sferica 1. Oltre LY una grande varietà di altri coloranti o molecole nonpermeant sono stati impiegati, come...

L&#39;applicazione di corrente elettrica a una cella provoca la formazione di pori nella membrana cellulare, da un processo chiamato elettroporazione. I pori permettono il passaggio di una varietà di molecole nonpermeant attraverso la membrana. Il campo elettrico può essere controllata con precisione, in modo che i pori formati sono molto piccole e richiudersi rapidamente, con il minimo disturbo per la fisiologia cellulare. È interessante notare, cellule aderenti possono essere coltivate su un vetrino rivestito con ossido di indio-stagno conduttivo e trasparente (ITO) e elettroporate in situ, che si trova sulla superficie dove crescono, senza essere distaccato per essere elettroporate in sospensione. Le cellule possono crescere molto bene su questa superficie, e come sono collegati ed estese, osservazione microscopica dettagliata è possibile. Usando questa tecnica, piccole molecole nonpermeant possono essere introdotti istantaneamente e in sostanza il 100% delle cellule, che rende questa tecnica particolarmente adatto per studi sull&#39;attivazione di components di un percorso seguito stimolazione ligando di un recettore (recensito in 1). L&#39;elettroporazione è stata utilizzata principalmente per l&#39;introduzione di DNA (chiamato anche electrotransfection). Tuttavia, elettroporazione in situ può essere utile per l&#39;introduzione di una grande varietà di molecole, come peptidi 2-4, oligonucleotidi antisenso, come RNA, oligonucleotidi a doppio filamento di DNA esca per inibire fattore di trascrizione vincolante, o siRNA 3,5, 6, 7-10 nucleotidi radioattivi, proteine ​​11 12 o pro-farmaci 13. A seguito di elettroporazione, le cellule possono essere lisate per analisi biochimiche o fissate e colorate con anticorpi. Il rivestimento conduttivo ITO è molto sottile, 800-1,000Å, in modo che la crescita cellulare non è disturbato dalla differenza di altezza delle due superfici, come le cellule crescono lungo il bordo del rivestimento conduttivo. Questo offre il vantaggio che non electropciente cellule possono essere coltivate fianco a fianco con quelli elettroporate, per servire come controlli. Lo stesso approccio può essere utilizzato per l&#39;esame di spazi di giunzione, la comunicazione intercellulare (GJIC), come descritto nel video. Giunzioni sono canali che collegano gli interni di celle adiacenti 14. Gap giunzionale, comunicazione intercellulare svolge un ruolo importante nella formazione del tumore e metastasi, mentre oncogeni quali Src sopprimono GJIC 15,16. Per esaminare GJIC un colorante fluorescente, come Lucifero giallo (LY) è spesso introdotto in cellule in coltura tramite microiniezione o raschiare carico 17 e la diffusione del colorante nelle cellule vicine all &#39;osservazione microscopica sotto illuminazione a fluorescenza. Queste tecniche però invariabilmente causano danni alle cellule. Descriviamo ora una tecnica in cui le cellule sono coltivate su un vetrino che è parzialmente ricoperto di ITO 18. Un impulso elettrico è stato applicato in presenza di LY (o altri coloranti) cautilizzando la sua penetrazione nelle cellule in crescita da parte conduttiva della diapositiva, mentre la migrazione colorante per le adiacenti, cellule non elettroporate è stata osservata al microscopio con illuminazione a fluorescenza. Per evitare di cellule sensibili inquietanti che possono tendere a staccarsi dalla monostrato, un&#39;assemblea è stato progettato che non richiedono un elettrodo per essere posizionato sulla parte superiore delle cellule di applicare la corrente elettrica 19. Questo approccio offre la possibilità di quantificare GJIC in un gran numero di cellule, senza alcun disturbo rilevabile al metabolismo cellulare, come indicato dall&#39;assenza di un effetto sulla durata della fase G1 seguente siero stimolazione 12, aumento dei livelli di FOS protooncogene proteine ​​(Raptis, inedito) o due chinasi associate a stress cellulare, il maiale p38 o JNK / SAPK chinasi 1. Questo approccio ha reso possibile l&#39;esame del legame tra i livelli di espressione dell&#39;oncogene, trasformazione e GJIC 18, nonché l&#39;effetto di Src e Stat3 su GJIC in una varietà di tipi cellulari, comprese le cellule in coltura fresco da campioni di tumore del polmone 20-23. Inoltre, in elettroporazione situ con la configurazione descritta, che manca di un elettrodo superiore è stato impiegato con successo per la manifestazione di chiusura del gap junction sulla differenziazione adipocytic, anche se l&#39;attaccamento cellulare al substrato è ridotto in quella fase 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>
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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>
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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>
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			<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>
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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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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
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			<td>4-wells</td>
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			<td style="width:428px;">Cell Projects Ltd UK</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>
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			<td>High purity</td>
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			<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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			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
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			<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>
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		<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>
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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 mostra fegato di ratto cellule epiteliali T51B 22, elettroporate con LY e fotografati sotto fluorescenza (pannello A e B), o contrasto di fase (C) illuminazione, seguito fissaggio e lavaggio. Nel pannello A, il bordo dell&#39;area elettroporate è indicato in rosso. Il gradiente di fluorescenza a destra della linea rossa indica il trasferimento attraverso giunzioni gap. Nella Figura 3A e 3B, viene mostrata la quantificazione di comunicazione gap g...

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 test funzionale per Gap giunzionale esame; Elettroporazione di cellule aderenti su indio-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 Views.  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.

Cellule patterning su elettrodi Tin otticamente trasparente di ossido di indio

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

Ricerca

Biologia

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.

Etichettatura hESC e hMSCs con nanoparticelle di ossido di ferro per i non-invasiva in vivo inseguimento con la 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.

In elettroporazione in vivo ed ex utero per l&#39;espressione genica in cellule gangliari della retina del mouse

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.

In situ Frazionamento subcellulare di cellule di mammifero aderenti e non aderenti

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.

Mutagenesi e Analisi funzionale dei canali ionici Heterologously Espresso in cellule di mammifero

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.

Clonogenica saggio: cellule aderenti

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.

Obiettivi Identificazione di microRNA umani con il sistema di analisi Luciferasi LightSwitch utilizzando 3&#39;UTR-reporter Costruisce e un Mimic microRNA in cellule aderenti

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.

Esame della<em&gt; Drosophila</em&gt; Larvali tracheali Terminal cellule al microscopio ottico

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.

Misura e Analisi di extracellulare Acido prodotte per determinare glicolitica Tasso

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.

Nefrotossina microiniezione in Zebrafish per creare un modello danno renale acuto

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 ...