Gap-junctional coupling measured by flow cytometry

A method is described which reliably quantifies the degree of intercellular communication via gap junctions by combining a dye-loading technique with fluorescence-activated how cytometry. Our experiments expand former measure ments of other groups by analyzing the time- and density-dependent onset of coupling with a fixed ratio of donor to recipient cells. The high sensitiv ity of this technique provides a better resolution than the microelectrode technique and allows the detection of small changes in gap-junctional coupl ing by examining a large number of cells in a single experiment. Suspended cells were loaded with the membrane-permeable dye calcein AM which is intra cellularly hydrolyzed by nonspecific esterases, and the resulting polyanion ic calcein is thus trapped inside these donor cells. Gap junctions, however , are permeable for this fluorescent dye, as can be observed when suspended donor cells are added to recipient cells (i.e., monolayer cultures) in whi ch case cell-cell contact is established within less than 60 min. In additi on, one of these two cell populations can also be stained with a membrane-r esident dye (e.g., DiI), which facilitates the identification of different cell populations (donors, recipients, and noncoupled cells) not only by epi fluorescence microscopy but also by how cytometry. Our analyses reveal that junctional coupling depends not only on the connexin type (homo- or hetero typic junction) but also on the origin (species) of the contacting cells (h omo- or heterospecific contact). We confirm earlier reports in which homoty pic-homospecific coupling was demonstrated with different techniques in con nexin-transfected HeLa and RIN cells as well as in BICR/M1R(k) and 3T3/SV40 cells. In contrast to other publications, we show that a significant heter otypic-homospecific coupling between Cx40- and Cx43-HeLa transfectants can be resolved, whereas no coupling was detected for heterotypic-heterospecifi c contacts between Cx40-HeLa transfectants and the Cx43-expressing cell lin es BICR/M1R(k), 3T3/SV40, and RIN. (C) 2000 Academic Press.