Improved Procedure for Examination of Gap Junctional Intercellular Communication by In Situ Electroporation on a Partly Conductive Slide

Gap junctions are plasma membrane channels that serve as conduits for the passage of small molecules and ions between the interiors of cells (6). Gap junctional intercellular communication (GJIC) has long been thought to play an important role in cell growth and differentiation, as well as in the metastatic potential of human cancer cells (6,23). The investigation of junctional permeability is usually conducted through the introduction of a fluorescent dye such as Lucifer yellow (LY), through microinjection, scrape-loading or preloading (13), followed by observation of its migration into neighboring cells or by measuring the recovery of fluorescence after photobleaching (22). These methods are expensive and time consuming; they may also introduce the potential complication of cellular damage. We have developed a novel technique for GJIC measurement that can overcome these problems (17). This technique, termed in situ electroporation on a partly conductive slide, takes advantage of the ability of short electric pulses to create transient “pores” on the cell membrane through which LY can enter simultaneously into large numbers of cells with minimal disturbance to cellular physiology (7,16–18). Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent indium-tin oxide (Applied Films, Boulder, CO, USA). The nonconductive area is created by removing the conductive coating with hydrochloric acid in the presence of metallic zinc powder, thus exposing the bare glass underneath (9,17,19). An electric pulse is applied in the presence of LY, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye through gap junctions to the non-electroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination (17) (Figure 1, A and B, and Figure 2). In this way, dye transfer through gap junctions can be precisely quantitated simultaneously and in a large number of cells, without any detectable disturbance to the cell (2,18). At the same time, contrary to microinjection or scrape-loading (8), there is no mechanical manipulation of the cells that might disturb cell-to-cell contact areas, interrupt gap junctions and cause artifactual uncoupling. This approach, which offers the ability to quantitate GJIC, made possible the examination of the link between levels of oncogene expression, transformation and GJIC, as well as observation of gap junction closure upon adipocyte differentiation (3–5,17). This technique was later adapted for GJIC examination of lines established from human lung carcinomas or cells freshly explanted from lung-tumor tissues (20,21). Senescence of human umbilical vein endothelial cells after a number of passages in culture induces a reduction in GJIC (24). During culture of human lung carcinoma cells (20,21), it became evident that, contrary to the fibroblasts present in the specimen, the tumor cells senesced and died within 10–15 days. Therefore, since senescence may downregulate GJIC, to obtain a true measurement of their junctional communication in vivo, GJIC must be examined within a few days after surgery, as soon as cells have attached and before they approach their limits of life in culture. This is especially important because the (normal) fibroblasts that invariably predominate after the tumor cells have died display extensive GJIC, which makes GJIC assessment of the tumor cells problematic (21). The slide configuration previously employed for GJIC examination consists of a window of 7 × 4 mm with a conductive area of 4 × 4 mm where the cells are grown, so that the transition line between conductive and nonconductive sides runs through the middle of the slide and is 4 mm long (17) (Figure 1B). This configuration is satisfactory for GJIC examination in most established cell lines. However, many cells are required because the cells need to be situated precisely at the transition line, which is a relatively small proportion of the total area, for examination of their communication to be possible. Therefore, for GJIC assessment when small amounts of tissue are available, the chances of the cells attaching at this line must be increased so that GJIC can be examined before they have divided in culture and senesced. This paper describes electrode and slide configurations that can be effectively employed for this purpose. The use of a very small window (2 × 2 mm, with a transition line of 2 mm in length and conductive area of 1 × 2 mm) (Figure 1C), requires very low voltage and capacitance settings that cannot be provided by the commercialBenchmarks