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

Electrode and slide assembly for the study of intercellular, junctional communication: standard slide. (A) Top panel: side view. A coating of ITO (1a) (shown greatly enlarged for clarity) on the upper surface of the glass slide (1) makes a conductive path from the positive contact bar (2), through the cells (4) and the electroporation solution to the bottom surface of the negative electrode (3).+and − denote connections to the positive and negative poles of the pulse source, respectively. (5), insulating Teflon frame, (6), electrode holder. Note that the negative electrode is inclined to compensate for the resistance of the coating (Raptis et al., 2006). Lower panel: top view. The outline of the positive (7) and negative (8) electrodes and their relative position on the slide in relation to the frame (5) and the window where the cells are grown are indicated. The lightly shaded area (a) represents the conductive coating. (b), area where the conductive coating has been removed. Arrowhead points to the transition line between conductive and non-conductive areas. (B) Examination of gap junctional communication using the above apparatus. Human lung carcinoma A549 cells were plated in the window (a,b) as shown in (A) and electroporated in the presence of 5mg/ml Lucifer yellow. After washing the unincorporated dye, cells from the same field were photographed under fluorescence (right panel) or phase contrast (left panel) illumination. Arrows point to the transition line between conductive and non-conductive areas. Note the absence of fluorescence in cells growing on the non-conductive part of the slide (Tomai et al., 1999).

Electroporation in the absence of an upper electrode. (A) Top view. Cells were grown on an ITO-coated slide from which the coating was removed in a strip (c) as shown. The two conductive sides, (b, e), serving as electrodes, continue outside the cell growth area (a, f), and are connected to the positive (2) and negative (3) poles of the pulse generator. (B) Side view. The slide with the cells growing on the ITO-coated and the bare glass regions is schematically shown. Current from the pulse generator connected to contact point (3) passes into the thin conducting ITO layer (1a), under the wall of the chamber (4) to area (e) to the opposite electrode (b) as shown. (8), electric field lines. An electrical potential is created across the cells growing on (e) and those on the glass area (c) (Fig. 2A), immediately next to the edge as current passes into the electroporation buffer, which acts as an electrolyte, around and through the electroporated cells. Bottom panel, enlargement of the edge of the conductive coating. Dotted lines with arrows indicate the electric field lines along the surface of the slide, around the cells and through the surrounding fluid. The gray-scale represents the relative intensity of Lucifer yellow fluorescence; darker gray cells have been electroporated with higher fields than their lighter neighbors. (C) A549 cells were plated in a chamber as in (A) above and electroporated in the presence of Lucifer yellow (30V, 0.2μF, 6 times, with the polarity reversed on alternate pulses). After washing the unincorporated dye, cells from the same field were photographed under fluorescence (b) or phase contrast (a) illumination. Note the gradient of fluorescence in cells growing on the non-conductive part of the slide, despite the fact that these cells do not have gap junctions, as shown in Fig. 1B (Tomai et al., 1999). Arrows point to the transition line between conductive and non-conductive areas.

Electroporation on two co-planar ITO electrodes, with a barrier separating the two areas. (A) Top view. Cells were grown on an ITO-coated slide from which the coating was removed in a strip as shown. The two conductive sides (a, f), serving as electrodes, were connected to the positive and negative poles of the pulse generator (2) and (3). A non-conductive barrier (5) divides the strip of bare glass in half and separates the chamber into two sections. (B) Side view. The slide with the cells growing on the ITO coated and the bare glass regions is shown. When electroporation buffer is added to the chamber to a level above the height of the barrier (5) then an electrical path between the electrodes (e and b) is formed. Note that the ITO layer (1a) is shown with dramatically exaggerated thickness for clarity, although its actual thickness is much less than the thickness of the cells. (C) A549 cells were plated on the slide in (A) above and electroporated in the presence of Lucifer yellow as in Fig. 2 above. After washing the unincorporated dye, cells from the same field were photographed under fluorescence (b) or phase contrast (a) illumination. Note the absence of fluorescence in cells growing on the non-conductive part of the slide. Arrows point to the transition line between conductive and non-conductive areas. (D) 3T3 L1 preadipocytes were plated in the window shown in (A) above and electroporated in the presence of Lucifer yellow. After washing the unincorporated dye, cells from the same field were photographed under fluorescence (b) or phase-contrast (a) illumination. Note the gradient of fluorescence, indicating extensive dye transfer through gap junctions (). To quantitate gap junctional communication, electroporated cells growing at the border with the non-conductive zone (stars), and fluorescing cells growing on the non-conductive side of the slide, into which the dye had transferred through gap junctions (dots) were identified. The number of cells into which the dye has transferred per electroporated border cell can be calculated by dividing the total number of fluorescing cells on the non-conductive side by the number of cells growing at the border with the conductive coating. Arrows on the conductive side point to the transition line between conductive and non-conductive areas. Magnification: 200×. (E) 3T3L1 preadipocytes were plated in the window shown in (A) above and induced to differentiate by the addition of IBMX, insulin and dexamethasone at confluence (see Section 3). Ten days later, terminally differentiated adipocytes were electroporated in the presence of Lucifer yellow. Note the absence of dye transfer through gap junctions. Arrows point to the transition line between conductive and non-conductive areas. Arrowheads point to a terminally differentiated adipocyte growing on the transition line between conductive and non-conductive areas. Magnification: 200×.