Scrape-loading and dye transfer

Gap junction-mediated intercellular communication has been recognized in cells from different tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new, direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication-competent cells the dye transmission occurred within minutes after loading. The involvement of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate, rhodamine dextran (MW 10,000). Once introduced intracellularly the rhodamine dextran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communication were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition, it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action.