Kevin L. Firth

Examination of gap junctional, intercellular communication by in situ electroporation on two co‐planar indium‐tin oxide electrodes

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 LYs 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.

Applications of electroporation of adherent cells in situ, on a partly conductive slide

Nontraumatic, simple, and reproducible procedures for the introduction of nonpermeant molecules into adherent mammalian cells byin situ electroporation are described. Ctells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse is applied in the presence of the molecules to be introduced, and their effect on the cellular phenotype can be observed. The cells growing on the nonconductive side of the slide do not receive any pulse and serve as controls. Careful adjustment of electric field strength can achieve the introduction of the molecules into essentially 100% of the cells, and this treatment causes no detectable disruption to cellular metabolism. This is applied in the presence of the fluorescent dye, Lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide. 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

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.

Electrode Assemblies Used for Electroporation of Cultured 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.

Dissecting Pathways; in Situ Electroporation for the Study of Signal Transduction and Gap Junctional Communication

Publisher Summary This article describes a technique where cells are grown on a glass surface coated with electrically conductive, optically transparent indium-tin oxide (ITO) at the time of pulse delivery. This coating promotes excellent cell adhesion and growth, allows direct visualization of the electroporated cells, and offers the possibility of ready examination due to their extended morphology. The instant introduction of the molecules into essentially 100% of the cells makes this technique especially suitable for kinetic studies of effector activation. Unlike other techniques of cell permeabilization, under the appropriate conditions, in situ electroporation does not affect cell morphology, the length of the G1 phase of serum-stimulated cells. The technique of in situ electroporation can be used equally effectively for large-scale biochemical experiments. Growth factors such as the epidermal growth factor (EGF) stimulate cell proliferation by binding to, and activating, membrane receptors with cytoplasmic tyrosine kinase domains. Assessment of Erk activity by Western blotting following electroporation of the Grb2-SH2 blocking peptide can reveal the involvement of this domain in growth factor-mediated Erk activation.

In situ electroporation of radioactive compounds into adherent cells

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.

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. 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 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.

Growth on Indium-Tin Oxide-Coated Glass Enhances 32P-Phosphate Uptake and Protein Labelling of Adherent Cells

ABSTRACT A method to improve the efficiency of labelling of adherent cells with radioactive 32P is described. Cells are grown on a glass surface which is coated with indium-tin oxide, a commercially available, transparent material which permits excellent cell adhesion and growth. The results show that a 2 to 3-fold increase in 32P uptake by the cells can be achieved by growing cells on this material, compared to conventional tissue culture plastic.

Application of Tablet Computers in a Capstone Design Course

Tablet PCs look much like regular laptop computers, except their digitized screens can be swiveled around, folded over, and written on with a stylus. Instructors have recognized that this simple write-on feature gives them the opportunity to change the way in which they lecture in a classroom. This paper examines the application of tablet PCs outside of the lecture hall. Specifically, it describes the application of tablet computers to a final year capstone design course. Particular applications include the replacement of the traditional individual design notebook with a shared electronic notebook and enhancement of sketching as a communication tool. A description of the capstone course is given to provide a context for how the tablets were used. The impact of the tablet PCs is discussed and the results of a user survey are presented.Copyright

In Situ Electroporation of Radioactive Nucleotides: Assessment of Ras Activity or 32P Labeling of Cellular Proteins

Publisher Summary This chapter focuses on in situ electroporation of radioactive nucleotides to examine ras activity or 32p labeling of cellular proteins. Unlike other techniques of cell membrane permeabilization, such as streptolysin-O (SLO) treatment, in situ electroporation does not detectably affect cellular metabolism, presumably because the pores reseal rapidly so that the cellular interior is restored to its original state. Cells are grown on conductive and transparent glass slides, which are placed in a petri dish to maintain sterility. The cell growth area is defined by a “window” formed with an electrically insulating frame made of Teflon. The frame creates a gap between the conductive coating and the negative electrode so that current can only flow through the electroporation fluid and cells growing in the window. A large number of signal transducers are present in small amounts in the cell so that a large number of cells may be required to obtain a strong signal.