Gap junction-mediated diffusion of calcein is inhibited by carbenoxolone. Self-assembled KGN spheroids were incubated with the gap junction inhibitor carbenoxolone (CBX) or its vehicle control for 5 h prior to the addition of 2.5 µM of calceinRO. Confocal z-stacks of spheroids were acquired at 30, 45, 70, and 80 min after the addition of calceinRO. To quantify CBX inhibition of gap junction intercellular communication, the equatorial image at 80 min were taken for further analysis by automated batch processing. Representative equatorial images of calceinRO fluorescence (561 nm) were rendered for spheroids. Scale = 50 µm.