Name: a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide
Uploaded: 2014-10-18T15:00:00.000+00:00
Duration: 11 min 2 s
Description: Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

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. plaqueamento das células do Eletroporação Chambers <ol><li> Numa capela de fluxo laminar, as células trypsinize usando uma técnica estéril, como de costume. NOTA: É muito importante para eliminar por centrifugação todos os vestígios de tripsina, porque eles podem prejudicar propagação das células no vidro, daí a formação de adherens e junções comunicantes. </li><li> Pipetar 1 mL da suspensão de células nas câmaras de electroporação estéril fornecido com o electroporador em situ (Figura 1), e em lugar de um de 37 o C, incubadora de CO 2. NOTA: A adesão celular pode ser melhorada através de plaqueamento em fibronectina, colagénio, poli-lisina ou de células e tecido adesivo (ver Tabela Materiais). </li><li> Quando as células formaram uma camada confluente eles estão prontos para análise CIJH. </li></ol> 2. Eletroporação Procedimento Eletroporação para exame GJIC pode ser realizada fora de uma capela de fluxo laminar,uma vez que leva apenas alguns minutos. Se forem necessários tempos de incubação mais longos para uma experiência específica, em seguida, pode ser realizado inteiramente num exaustor de fluxo laminar. Em todos os casos, as câmaras onde são cultivadas as células têm de ser estéreis. <ol><li> Prepara-se uma / ml solução amarelo Lucifer 5 mg: Dissolver 10 mg de pó de LY (fornecida com o electroporador) em 2 ml de meio de crescimento isento de cálcio. Para os experimentos que requerem tempos de incubação mais longos, filtro de esterilizar a solução e armazenar a 4 ° C. </li><li> Aspirar o meio de crescimento e lavar as células com meio livre de cálcio, tendo o cuidado para não arranhar a monocamada ou secar as células. Se as células são secas têm geralmente núcleos mais escuras e pegar LY sem electroporação. O efeito é mais pronunciado no meio do slide que está mais exposto a correntes de ar (Figura 4F). </li><li> Com uma pipeta de Eppendorf, pipetar a solução corante para as células (400 ul para o conjunto da câmara), na extremidade da câmara, being cuidado para não tocar a camada de células. </li><li> Coloque a câmara no suporte fornecido com o electroporador e aplicar um impulso de força adequada (ver discussão). </li><li> Aspirar cuidadosamente um pouco da solução LY. Ele pode ser reutilizada uma segunda vez para experiências menos importantes. </li><li> Adicionar DMEM livre de cálcio com 10% de soro dialisado. </li><li> Incubar as células durante 3-5 minutos na incubadora para permitir a transferência de corante através de junções de hiato. NOTA: A inclusão de soro dialisado, neste ponto ajuda encerramento dos poros. </li><li> Lavar o corante não incorporado com DMEM isento de cálcio para a observação de células vivas. Alternativamente, as células podem ser fixadas nesta fase, por meio da adição de 4% de formaldeído para o bem, em seguida, lavadas com PBS (solução salina tamponada com fosfato). Em todos os casos, a lavagem deve remover todo o fundo. NOTA: Em experimentos preliminares para determinar as condições ideais, se a tensão é muito baixa, então é possível para permitir que as células se recuperar no incubator durante alguns minutos e electroporate mesma lâmina uma segunda vez, para economizar no custo de lâminas. </li></ol> 3. exame microscópico <ol><li> Observar as células sob iluminação de fluorescência, utilizando um microscópio invertido equipado com o filtro apropriado para o corante a ser utilizado. Para Lúcifer amarelo, excitação é 423nm, emissão de 555nm, filtro VCI para um microscópio Nikon IX70. </li><li> Eliminar efeitos de menisco, colocando uma lamela de vidro na parte superior do poço de plástico, depois do enchimento ele para o topo com o líquido, para examinar a morfologia das células sob contraste de fase. Para melhores imagens, o poço pode ser isolada a partir da lâmina, e a lamela colocada sobre a moldura. Para as células fixas, o poço pode ser preenchido com glicerol para observação microscópica, enquanto para as células vivas, deve ser preenchido com o meio de crescimento. NOTA: Lavagem e fotografando é mais fácil se as células são fixadas com formaldeído após a transferência do corante (passo 2.7 acima). Se as células não sãot fixo, a fluorescência é eliminada das células dentro de aproximadamente 60 min, dependendo do tipo de célula, da tensão e da quantidade de LY introduzido enquanto em células fixas fluorescência é mantida durante várias horas. No entanto, a fluorescência ou dissipa após se desvanece O / N a incubação, até mesmo em células fixadas. Por esta razão, deve ser feita fotografias logo após a electroporação (Figura 2). </li></ol> 4. quantificação de comunicação intercelular <ol><li> Fotografar as células com uma objectiva de 20x sob contraste de fase e fluorescência (Figura 3). </li><li> Identificar e marcar com uma estrela as células eletroporados na fronteira com a área não-electroporados (Figura 2, seta, borda eletroporação). </li><li> Identificar e marcar com um ponto de células fluorescentes no lado não-electroporado, em que o corante foi transferida através de junções de hiato (Figuras 3A e 3B). </li><li> Dividir o número total decélulas fluorescentes na área de não-electroporado pelo número de células electroporadas ao longo da borda (Figuras 3A e 3B). NOTA: A transferência de, pelo menos, 200 células de fronteira electroporadas contíguos é calculado para cada experiência. O valor obtido é o valor CIJH. </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.

As etapas críticas no protocolo Material de electroporados A pureza do material a ser electroporada é muito importante. Se as células são muito plana, então as concentrações mais elevadas do corante de rastreio devem ser usados ​​(até 20 mg / ml para LY) e em tais casos, a pureza é mais importante do que em células com uma forma mais esférica 1. Além LY uma grande variedade de outros corantes ou moléculas nonpermeant têm sido empregues, tais...

A aplicação de corrente eléctrica para uma célula provoca a formação de poros na membrana da célula, por um processo denominado electroporação. Os poros permitem a passagem de uma variedade de moléculas nonpermeant através da membrana. O campo eléctrico pode ser controlada com precisão, de modo a que os poros que se formam são muito pequenas e religar-se rapidamente, com uma perturbação mínima para a fisiologia celular. Interessantemente, as células aderentes podem ser cultivadas numa lâmina de vidro revestido com óxido de índio-estanho condutora e transparente (ITO) e electroporado in situ, isto é sobre a superfície de onde elas crescem, sem ser retirado para ser electroporada em suspensão. As células podem crescer muito bem nesta superfície, e como eles estão ligados e ampliado, a observação microscópica detalhada é possível. Utilizando esta técnica, as pequenas moléculas nonpermeant pode ser introduzido imediatamente e essencialmente em 100% das células, o que faz com que esta técnica especialmente adequado para estudos sobre a activação do components de um caminho a seguir a estimulação do ligando de um receptor (revisto em 1). A electroporação foi usado principalmente para a introdução de ADN (também chamada electrotransfection). No entanto, a electroporação in situ pode ser valiosa para a introdução de uma grande variedade de moléculas, tais como péptidos 2-4, oligonucleótidos, tais como ARN anti-sentido, oligonucleótidos de cadeia dupla de DNA de engodo para inibir a ligação do factor de transcrição, ou siRNA 3,5, 6, nucleotídeos radioativos 7-10, proteínas 11 12 ou pró-drogas 13. Após a electroporação, as células podem ser lisadas para análise bioquímica ou fixadas e coradas com anticorpos. O revestimento condutor de ITO é muito fina, 800-1,000Å, de modo que o crescimento celular não é perturbado pela diferença em altura das duas superfícies, como as células crescem em toda a borda do revestimento condutor. Isto oferece a vantagem de que não electropAs células podem ser cultivadas orated lado a lado com os electroporadas, para servir como controlos. A mesma abordagem pode ser usada para o exame de junções de hiato, a comunicação intercelular (CIJH), como descrito no vídeo. Junções comunicantes são canais que ligam o interior das células adjacentes 14. Das junções de hiato, a comunicação intercelular desempenha um papel importante na formação de tumores e metástases, enquanto que os oncogenes tais como Src suprimir CIJH 15,16. Para examinar a CIJH um corante fluorescente, tal como amarelo Lucifer (LY) é muitas vezes introduzido em células cultivadas através de microinjecção ou raspar-carregamento 17 e a difusão do corante para as células vizinhas é observada microscopicamente sob luz de fluorescência. Estas técnicas contudo invariavelmente causar dano celular. Descreve-se agora uma técnica em que as células são cultivadas sobre uma lâmina de vidro, que é parcialmente revestida com ITO 18. Um impulso eléctrico foi aplicada na presença de LY (ou outros corantes) cautilizando a sua penetração nas células que crescem na parte condutora da lâmina, enquanto que a migração do corante para as células adjacentes, não electroporadas foi observada microscopicamente através de iluminação de fluorescência. Para evitar as células sensíveis perturbadores que possam tender a separar da monocamada, foi concebido um conjunto que não requerem um eléctrodo a ser colocado no topo das células para aplicar a corrente eléctrica 19. Esta abordagem oferece a possibilidade de quantificar a CIJH em ​​um grande número de células, sem qualquer perturbação detectável para o metabolismo celular, como indicado pela ausência de um efeito sobre a duração da fase G1 seguindo estimulação sérica 12, em aumentar os níveis de fos proteína proto-oncogene (Raptis, não publicado) ou duas quinases associadas ao estresse celular, o porco p38 ou JNK / SAPK quinase 1. Esta abordagem possibilitou a análise da relação entre os níveis de oncogene expressão, transformação e GJIC 18, bem como o efeito da Src e Stat3 sobre CIJH em ​​uma variedade de tipos de células, incluindo as células cultivadas de fresco a partir de espécimes de tumor de pulmão de 20-23. Além disso, em electroporação in situ com a configuração descrita, a qual não tem um eléctrodo de topo tem sido empregue com sucesso para a demonstração de fecho das junções de hiato na diferenciação dos adipócitos, embora a adesão celular ao substrato é reduzido, nessa 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.

A Figura 2 mostra as células epiteliais de fígado de rato 22 T51B, electroporadas com LY e fotografadas sob fluorescência (painel A e B) ou de contraste de fase (C), na sequência de fixação de iluminação e de lavar roupa. No painel A, a borda da área de electroporação é marcado a vermelho. O gradiente de fluorescência para a direita da linha vermelha indica a transferência através de junções de hiato. Na Figura 3A e 3B, a quantificação da c...

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.

Um ensaio funcional para juncional Exame; Eletroporação de aderentes em células no Índio-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 visualizações.  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.

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

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.

Ensaio de aderência de freqüência para In Situ Análise Cinética de Cross-juncional interações moleculares na interface célula-célula

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

Bioengenharia

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.

Desenvolvimento de um<em&gt; In Vitro</em&gt; Ensaio para avaliar contrátil Função de células mesenquimais que a transição epitelial-mesenquimal Sofreu

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

Biologia do Desenvolvimento

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.

Um Ensaio Galvanotaxis para Análise de Precursores Neurais Cinética migração celular em um campo aplicado externamente Corrente Contínua Elétrica

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 ...

Neurociência

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.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

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).

Estratégia de Transfection otimizado para gravação de Expressão e eletrofisiológicos de Recombinante dos canais de íon em HEK-293T Células

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.

Microinjeção de uma única célula para análise da comunicação celular

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>Método TEEI para análise de eletroporação e previsão de entrega em células 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>Autor em destaque: Otimizando a eletroporação de substrato poroso por meio de micro e nanocanais para monitoramento aprimorado e caracterização de estágio intermed

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.

Eletroporação Baseada em Substrato Poroso com Monitoramento de Impedância Elétrica Transepitelial

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

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Um ensaio funcional para juncional Exame; Eletroporação de aderentes em células no Índio-Tin Oxide

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