Scrape loading has been used to assess the GJIC status of many cell types in various biological circumstances (Table 2). Sai et al. reported an in vivo GJIC assay, the incision loading/dye transfer method (IL/DT) using LY and RhD (44). RhD, which does not pass through the channels, is used to identify the incision sites or damaged cells. With this double dye transfer, authors examined whether drinking green tea might prevent GJIC inhibition by a tumorigenic compound, pentachlorophenol, in the liver of mice. Romualdi et al. described an improvement to this dye transfer method in which precision-cut slices (250 µm cross-section) from mouse liver were used to quantify GJIC after iontophoretic injection of the fluorescent dye Alexa Fluor 488 directly into hepatocytes (35). Dye injection and spreading were monitored in real time and quantified by fluorescence microscopy. The integrity and viability of the cells were controlled by monitoring the membrane potential.
The IL/DT method illustrates the potential benefit of detecting and predicting effective doses for tumorigenesis. This method has many advantages for studying tumor-promoting activity in vivo, because even though the Cx expression level recovered at an early stage after treatment with the tested compound, a low level of GJIC persisted for a longer time, indicating that an analysis of the expression level of Cx, such as for protein, mRNA, or its localization, does not always reflect gap junction function in vivo. With respect to devising a much simpler and quicker procedure compared with the microinjection method, IL/DT may be useful for rapidly screening tumorigenic compounds for setting doses for studies of carcinogenesis (44).
The analysis of GJIC by microinjection or scrape loading can also be used with sections of other tissues, such as skin (45), testis (46), and central nervous system tissue (47). An exhaustive protocol was described by Meda's group in primary tissues (13).ElectroporationPrinciple
Raptis et al. proposed a new way to introduce nonpermeable fluorescent dye into adherent cells on a partly conductive slide, called electroporation. (48). Cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. An electric pulse that opens transient pores on the plasma membrane is applied in the presence of the fluorescent dye LY, causing the dye's penetration into the cells that are growing on the conductive part of the slide. Cells growing on the nonconductive slide are not permeabilized as it does not receive any current (Figure 2C). Migration of the dye through gap junctions to nonelectroporated cells is then microscopically observed under fluorescence illumination (49). To quantitate gap junctional coupling, the number of cells into which dye transferred through gap junctions per electroporated border cells was calculated by dividing the total number of labeled cells on the nonconductive slide by the number of cells growing at the border with the conductive coating (Figure 2C) (50).Advantages
Lucifer yellow can enter simultaneously into large numbers of cells with minimal disturbance to cellular metabolism (45). This technique can be applied to a large variety of adherent cell types (50). According to De Vuyst et al., electroporation loading and dye transfer (ELDT) is a robust technique to investigate gap junctional coupling that combines minimal cell damage with accurate probing of the degree of cell-to-cell communication. It is a satisfactory method for loading a narrow longitudinal strip of cells for subsequent studies on the temporal and spatial spread of fluorescent dyes via gap junctions (51).Limitations
This technique is not recommended to examine cell types that do not adhere well to their solid support. If cells are grown to high confluence, they may detach due to the turbulence and suction forces created as the top electrode is removed after electroporation and GJIC study becomes impracticable. GJIC evaluation is also difficult when a small part of the cell layer is separated during removal of the electrode because the dye will diffuse under the cell sheet (52). An accurate determination of optimal voltage is important to not induce damage of the electroporated cells at the border with the nonconductive area (50).Significant studies and applications
Recently, Raptis et al. have described a slide configuration that eliminates the upper electrode, which can induce cell detachment when it is removed. Cells are grown on two co-planar electrodes, an approach that is valuable for the examination of cells that do not adhere closely to their solid support (52). To investigate the extent of dye spread between C6 glioma cells wild-type and transfected with Cx43 and Cx32 coupled by gap junctions, De Vuyst et al. proposed bipolar-pulsed high frequency electroporation by a small two-wire electrode positioned close to the cells (51).