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.

<table><tbody><tr><td>DMEM</td><td>Cellgro http://www.cellgro.com/</td><td>50-013-PB</td><td></td></tr><tr><td>DMEM without Calcium</td><td>Hyclone (Thermo scientific: http://www.thermoscientific.com)</td><td>SH30319-01</td><td></td></tr><tr><td>Donor Calf Serum&amp;nbsp;</td><td>PAA: http://www.paa.com/</td><td>Cat.# B15-008</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>PAA: http://www.paa.com/</td><td>Cat.# A15-751</td><td></td></tr><tr><td>Electroporation apparatus</td><td>Cell Projects Ltd UK: http://www.cellprojects.com/</td><td>ACE-100</td><td></td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-04-CC</td><td>4-wells</td></tr><tr><td>Chambers</td><td>Cell Projects Ltd UK</td><td>ACE-08-CC</td><td>8-wells</td></tr><tr><td>Lucifer Yellow</td><td>Cell Projects Ltd UK</td><td>ACE-25-LY</td><td>High purity</td></tr><tr><td>CelTak</td><td>BD Biosciences</td><td>354240</td><td>Cell and tissue adhesive</td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen</td><td>BD Biosciences</td><td>354236</td><td></td></tr><tr><td>Poly-Lysine</td><td>Sigma Aldrich</td><td>P8920</td><td></td></tr></tbody></table>

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

9.9K 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

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Controllabile Espressione Ion Canale attraverso inducibile trasfezione transiente

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Ricerca

Genetica

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Adesione saggio di frequenza per In Situ Analisi cinetica delle interazioni molecolari incrociati giunzionale alla cellula-cellula di interfaccia

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioingegneria

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Sviluppo di un<em&gt; In Vitro</em&gt; Test per valutare funzione contrattile del mesenchimali Le cellule che sono stati sottoposti a transizione epitelio-mesenchimali

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Biologia dello sviluppo

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

Un test Galvanotaxis Analisi della Cinetica precursori neurali migrazione cellulare in un campo applicato esternamente diretto Corrente elettrica

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

Neuroscienze

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 ...

Biologia

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Transfection strategia ottimizzata per espressione e registrazione elettrofisiologica di ricombinante tensione canali ionici in cellule HEK-293T

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Microiniezione cella singola per l&#39;analisi cellulare comunicazione

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

<meta charset="utf8"></head><body>Metodo TEEI per l'analisi dell'elettroporazione e la previsione della consegna in celle A431

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

<meta charset="utf8"></head><body>Autore in primo piano: Ottimizzazione dell'elettroporazione del substrato poroso attraverso micro e nanocanali per un monitoraggio avanzato e la caratterizzazione

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Elettroporazione basata su substrato poroso con monitoraggio dell'impedenza elettrica transepiteliale

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

Un test funzionale per Gap giunzionale esame; Elettroporazione di cellule aderenti su indio-Tin Oxide

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Capitoli in questo video

Controllabile Espressione Ion Canale attraverso inducibile trasfezione transiente

Adesione saggio di frequenza per In Situ Analisi cinetica delle interazioni molecolari incrociati giunzionale alla cellula-cellula di interfaccia

Sviluppo di un

Un test Galvanotaxis Analisi della Cinetica precursori neurali migrazione cellulare in un campo applicato esternamente diretto Corrente elettrica

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

Transfection strategia ottimizzata per espressione e registrazione elettrofisiologica di ricombinante tensione canali ionici in cellule HEK-293T

Microiniezione cella singola per l'analisi cellulare comunicazione

Elettroporazione basata su substrato poroso con monitoraggio dell'impedenza elettrica transepiteliale

Elettroporazione basata su substrato poroso con monitoraggio dell'impedenza elettrica transepiteliale

Elettroporazione basata su substrato poroso con monitoraggio dell'impedenza elettrica transepiteliale