Quantitative determination of gap junction intercellular communication using flow cytometric measurement of fluorescent dye transfer

Gap junction intercellular communication (GJIC) is involved in several aspe cts of normal cell behaviour, and disturbances in this type of communicatio n have been associated with many pathological conditions. Reliable and accu rate methods for the determination of GJIC are therefore important in studi es of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R. (1993) Journal of Cell Biology, 122, 157-167)reported some years ago the use of flow cytometer to determine transfer between cells of a mob ile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliab le determination of GJIC, it has been modestly used, possibly due to techni cal difficulties. In the present work we have illustrated several ways to u se flow cytometric data to express cell communication through gap junctions . The recipient cells were pre-stained with the permanent lipophilic dye PK H26, and the donor cell population were loaded with the gap junction permea ble dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give ad ditional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the m ore potent effects of DMSO on GJIC measured by the present method than on a lready existing GJIC measured by microinjection or quantitative scrape load ing. We also show that the problem related to the mobile dye calcein not be ing fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the firs t hours after formalin cell fixation.