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

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

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In situ electroporation of large numbers of cells using minimal volumes of material.

We previously developed a technique, termed in situ electroporation, where nonpermeant molecules are introduced through an electrical pulse into adherent cells, while they grow on electrically conductive, optically transparent, indium-tin oxide (ITO). Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Electroporation of radioactive material is faced with two important considerations: (1) potential for exposure of personnel to irradiation, and (2) the requirement for electroporation of a large number of cells. In this report, we describe a modification in the geometry of the slides and electrodes which permits the use of inexpensive ITO-coated glass of lower conductivity that can be discarded after use, to electroporate large numbers of cells using a minimum volume of radioactive nucleotide solution. The results demonstrate that, using this assembly, the determination of the Ras-bound GTP/GTP+GDP ratios through electroporation of [alpha32P]GTP can be conducted using approximately five times lower amounts of isotope than in previous designs. Moreover, this assembly permits efficient upscaling, which makes the determination of Ras-GTP binding in cells which are deficient in Ras activity possible. In addition, we demonstrate the labeling of two viral phosphoproteins--the Simian Virus 40 Large Tumor antigen, and Adenovirus E1A--through [gamma32P]ATP electroporation using this setup. In both cases, electroporation of the nucleotide can achieve a great increase in the efficiency and specificity of labeling compared to the addition of [32P]-orthophosphate to the culture medium, presumably because the immediate phosphate donor nucleotide itself is introduced, which can directly bind to the target proteins.

In situ electroporation of radioactive compounds into adherent cells.

Electroporation was initially developed for the introduction of DNA into cells which grow in suspension and was performed in a cuvette with two flat electrodes on opposite sides. Different configurations were subsequently developed for the electroporation of adherent cells in situ, while the cells were growing on nonconductive surfaces or a gold-coated, conductive support. We developed an assembly where the cells grow and are electroporated on optically transparent, electrically conductive indium-tin oxide (ITO). This material promotes excellent cell adhesion and growth, is inert and durable, and does not display spontaneous fluorescence, making the examination of the electroporated cells by fluorescence microscopy possible. The molecules to be electroporated are added to the cells and introduced through an electrical pulse delivered by an electrode placed on top of the cells. We describe several electrode and slide configurations which allow the electroporation of large numbers of cells for large-scale biochemical experiments or for the detection of changes in cell morphology and biochemical properties in situ, with control, nonelectroporated cells growing on the same type of ITO-coated surface, side by side with the electroporated ones. In a modified version, this technique can be adapted for the study of intercellular, junctional communication; the pulse is applied in the presence of a fluorescent dye, such as lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. An assembly is also described for the electroporation of sensitive cells without the use of an upper electrode.

Electrode assemblies used for electroporation of cultured cells.

Cultured adherent cells can be electroporated in situ, as they grow on a glass slide coated with electrically conductive, optically transparent indium-tin oxide (ITO). Although the introduction of DNA is a common use, the technique of electroporation in situ is valuable for studying many aspects of signal transduction. This is because, under the appropriate conditions, in situ electroporation can be remarkably nontraumatic, while a large variety of molecules, such as peptides, oligonucleotides, or drugs, are introduced instantly and into essentially 100% of the cells, making this technique especially suitable for kinetic studies of effector activation. Following the introduction of the material, the cells can be either extracted or biochemically analyzed, or their morphology and gene expression can be examined by immunocytochemistry. In this chapter, we describe the introduction of a peptide blocking the Src-homology 2 domain of the adaptor Grb2 to inhibit the activation of the downstream effector Erk1/2 by EGF. The setup includes nonelectroporated, control cells growing side by side with the electroporated ones on the same type of ITO-coated surface. In a modified version, this assembly can be used very effectively for studying intercellular, junctional communication: cells are grown on a glass slide half of which is ITO-coated. An electric pulse is applied in the presence of the fluorescent dye lucifer yellow, causing its penetration into the cells growing on the conductive part of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

Electroporation of adherent cells in situ for the study of signal transduction and gap junctional communication.

Gap junctions are plasma membrane channels between neighboring cells. We previously described a powerful technique where gap junctional, intercellular communication (GJIC) of adherent cells can be examined by in situ electroporation on a slide, part of which is coated with electrically conductive and transparent indium-tin oxide. An electric pulse is applied through an electrode placed on the cells in the presence of the tracking dye, Lucifer yellow (LY). The pulse causes LY's penetration into the cells growing on the conductive part of the slide, and the subsequent migration of the dye to the non-electroporated cells growing on the non-conductive area is microscopically observed under fluorescence illumination. Although this technique is adequate for a number of cell lines, the turbulence generated as the electrode is removed can cause cell detachment, which makes GJIC examination problematic. In this communication, we describe a slide configuration where junctional communication can be examined in the absence of an upper electrode: Cells are grown on two co-planar electrodes separated by a barrier which diverts the electric field, rendering it vertical to the cell layer. The elimination of an upper electrode is especially valuable for the electroporation of sensitive cells, such as terminally differentiated adipocytes. This technique can also be used for the introduction of other non-permeant molecules such as peptides or siRNA, followed by examination of the cellular phenotype or gene expression levels in situ.

Kevin Firth

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