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. Plating the Cells in the Electroporation Chambers
<ol>
	<li>In a laminar-flow hood, trypsinize the cells using sterile technique as usual. 
	NOTE: It is very important to eliminate by centrifugation all traces of trypsin, because they may hinder spreading of the cells on the glass, hence the formation of adherens and gap junctions.</li>
	<li>Pipette 1 ml of the cell suspension in the sterile electroporation chambers provided with the in situ electroporator (Figure 1), and place in a 37 oC, CO2 incubator. 
	NOTE: Cell adhesion can be improved by plating on fibronectin, collagen, poly-lysine or cell and tissue adhesive (see Materials Table).</li>
	<li>When the cells have formed a confluent layer they are ready for GJIC examination.</li>
</ol>
2. Electroporation Procedure
Electroporation for GJIC examination can be conducted outside a laminar-flow hood, since it takes only a few minutes. If longer incubation times are required for a specific experiment, then it can be conducted entirely in a laminar-flow hood. In all cases, the chambers where the cells are grown must be sterile.
<ol>
	<li>Prepare a 5 mg/ml Lucifer yellow solution: Dissolve 10 mg LY powder (provided with the electroporator) in 2 ml Calcium-free growth medium. For experiments that requires longer incubation times, filter-sterilize the solution and store at 4oC.</li>
	<li>Aspirate the growth medium and wash the cells with Calcium-free medium, being careful not to scratch the monolayer or dry the cells. If the cells are dried they usually have darker nuclei and pick up LY without electroporation. The effect is more pronounced in the middle of the slide which is more exposed to air drafts (Figure 4F).</li>
	<li>With an Eppendorf pipettor, pipette the dye solution to the cells (400 &#956;l for the whole chamber), at the edge of the chamber, being careful not to touch the cell layer.</li>
	<li>Place the chamber in the holder supplied with the electroporator and apply a pulse of the appropriate strength (see Discussion).</li>
	<li>Carefully aspirate some of the LY solution. It can be reused a second time for less important experiments.</li>
	<li>Add Calcium-free DMEM containing 10% dialysed serum.</li>
	<li>Incubate the cells for 3-5 min in the incubator to allow dye transfer through gap junctions. 
	NOTE: The inclusion of dialysed serum at this point helps pore closure.</li>
	<li>Wash the unincorporated dye with Calcium-free DMEM for live cell observation. Alternatively, cells can be fixed at this stage, through the addition of 4% formaldehyde to the well, then washed with PBS (phosphate-buffered saline). In all cases the washing must remove all background. 
	NOTE: In preliminary experiments to determine the optimal conditions, if the voltage is too low, then it is possible to let the cells recover in the incubator for a few minutes and electroporate the same slide a second time, to save in the cost of slides.</li>
</ol>
3. Microscopic Examination
<ol>
	<li>Observe the cells under fluorescence illumination, using an inverted microscope equipped with the appropriate filter for the dye being used. For Lucifer yellow, excitation is 423nm, emission 555nm, WBV filter for a Nikon IX70 microscope.</li>
	<li>Eliminate meniscus effects by placing a glass cover-slip on top of the plastic well, after filling it to the top with liquid, to examine cell morphology under phase contrast. For better pictures, the well can be detached from the slide, and the coverslip placed on the frame. For fixed cells, the well can be filled with glycerol for microscopic observation, while for live cells it must be filled with growth medium. 
	NOTE: Washing and photographing is easier if the cells are fixed with formaldehyde following the transfer of the dye (step 2.7 above). If the cells are not fixed, fluorescence is eliminated from the cells within approximately 60 min depending upon the cell type, voltage and amount of LY introduced while in fixed cells fluorescence is retained for several hours. However, fluorescence fades or dissipates after O/N incubation, even in fixed cells. For this reason, photographs must be taken soon after electroporation (Figure 2).</li>
</ol>
4. Quantitation of Intercellular Communication
<ol>
	<li>Photograph the cells with a 20x objective under fluorescence and phase contrast (Figure 3).</li>
	<li>Identify and mark with a star the electroporated cells at the border with the non-electroporated area (Figure 2, arrow, electroporation edge).</li>
	<li>Identify and mark with a dot fluorescing cells on the non-electroporated side, where the dye has transferred through gap junctions (Figures 3A and 3B).</li>
	<li>Divide the total number of fluorescing cells on the non-electroporated area by the number of electroporated cells along the edge (Figures 3A and 3B). 
	NOTE: The transfer from at least 200 contiguous electroporated border cells is calculated for each experiment. The number obtained is the GJIC value.</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.

Critical steps in the protocol
Electroporated material 
The purity of the material to be electroporated is very important. If the cells are very flat, then higher concentrations of the tracking dye must be used (up to 20 mg/ml for LY) and in such cases, purity is even more important than in cells with a more spherical shape1. Besides LY a large variety of other dyes or nonpermeant molecules have been employed, such as a series of Alexa dyes to use as probes for c...

The application of electrical current to a cell causes the formation of pores on the cell membrane, by a process termed electroporation. The pores allow the passage of a variety of nonpermeant molecules through the membrane. The electrical field can be controlled precisely, so that the pores formed are very small and reclose rapidly, with minimal disturbance to the cellular physiology. Interestingly, adherent cells can be grown on a glass slide coated with conductive and transparent indium-tin oxide (ITO) and electroporated in situ, that is on the surface where they grow, without being detached to be electroporated in suspension. Cells can grow very well on this surface, and as they are attached and extended, detailed microscopic observation is possible. Using this technique, small nonpermeant molecules can be introduced instantly and into essentially 100% of the cells, which makes this technique especially suitable for studies on the activation of components of a pathway following ligand stimulation of a receptor (reviewed in1).
Electroporation has been used mostly for the introduction of DNA (also called electrotransfection). However, electroporation in situ can be valuable for the introduction of a large variety of molecules, such as peptides2-4, oligonucleotides, such as antisense RNA, double-stranded DNA decoy oligonucleotides to inhibit transcription factor binding, or siRNA3,5,6, radioactive nucleotides7-10, proteins1112 or pro-drugs13. Following electroporation, cells can be lysed for biochemical analyses or fixed and stained with antibodies.
The conductive ITO coating is very thin, 800-1,000&#197;, so that cell growth is not disturbed by the difference in height of the two surfaces, as the cells grow across the edge of the conductive coating. This offers the advantage that non-electroporated cells can be grown side by side with electroporated ones, to serve as controls. The same approach can be used for the examination of gap junctional, intercellular communication (GJIC), as described in the Video.
Gap junctions are channels connecting the interiors of adjacent cells14. Gap junctional, intercellular communication plays an important role in tumor formation and metastasis, while oncogenes such as Src suppress GJIC15,16. To examine GJIC a fluorescent dye such as Lucifer yellow (LY) is often introduced into cultured cells through microinjection or scrape-loading17 and the diffusion of the dye into neighboring cells is microscopically observed under fluorescence illumination. These techniques however invariably cause cell damage. We now describe a technique where cells are grown on a glass slide which is partly coated with ITO18. An electric pulse was applied in the presence of LY (or other dyes) causing its penetration into the cells growing on the conductive part of the slide, while dye migration to the adjacent, non-electroporated cells was microscopically observed through fluorescence illumination. To avoid disturbing sensitive cells that may tend to detach from the monolayer, an assembly was designed that did not require an electrode to be placed on top of the cells to apply the electrical current19. This approach offers the ability to quantitate GJIC in a large number of cells, without any detectable disturbance to cellular metabolism, as indicated by the absence of an effect on the length of the G1 phase following serum stimulation12, increase in the levels of the fos protooncogene protein (Raptis, unpublished) or two kinases associated with cellular stress, the p38hog or JNK/SAPK kinase1. This approach made possible the examination of the link between levels of oncogene expression, transformation and GJIC18, as well as the effect of Src and Stat3 upon GJIC in a variety of cell types, including cells freshly cultured from lung tumor specimens20-23. In addition, in situ electroporation with the setup described which lacks a top electrode has been successfully employed for the demonstration of gap junction closure upon adipocytic differentiation, although cellular attachment to the substrate is reduced at that stage19,24.

<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.

Figure 2 shows rat liver epithelial T51B cells22, electroporated with LY and photographed under fluorescence (panel A and B), or phase-contrast (C) illumination, following fixation and washing. In panel A, the edge of the electroporated area is marked in red. The gradient of fluorescence to the right of the red line denotes transfer through gap junctions. In Figure 3A and 3B, the quantitation of gap junctional communication is shown: The cells at the edge of t...

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.

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

Universidad de Cartagena UDEC

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

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.

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

Bioengineering

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.

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

Developmental Biology

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.

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 ...

Neuroscience

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.

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 ...

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).

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.

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

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

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.

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

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.

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.

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

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

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

Summary

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Chapters in this video

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

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

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

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

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

Single-cell Microinjection for Cell Communication Analysis

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

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

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Recording Gap Junction Current from Xenopus Oocytes