“Parachute” dye-coupling assay

GJIC function was evaluated by “Parachute” dye-coupling assay was performed as described in our previous study ²². Donor and receiver cells were grown to confluence. Donor cells were loaded with 5 µM calcein-acetoxymethyl ester, which is converted into the gap junction-permeable dye calcein and diffuse through GJ channel. The donor cells were then seeded onto the receiver cells at 1:150 donor/receiver ratio after trypsinization. The donor cells were allowed to contact with the monolayer of receiver cells for GJ formation for 4 h at 37 °C. The average amount of the receiver cells receiving dyes per donor cell was determined using a fluorescence microscope and normalized to the controls.