Dye transfer assay, fluorescence microscopy, & image analysis

Gap junction functionality was assessed by loading Lucifer Yellow in RFPEC, using a scratch technique [50], and observing the extent of Lucifer Yellow transfer from loaded cells to neighboring cells (S2B Fig). Lucifer Yellow has a molecular weight of 457.3 and can only enter cells via broken membranes or via gap junctions. The scratch loading technique involved pre-incubation of RFPEC (for 1 min) with calcium and magnesium free Hanks’ Balanced Salt Solution (HBSS; Life Technology) containing 5 mg/ml Lucifer Yellow dye (Life Technology). We then used a 5μm diameter tip scribe (Ted Pella, Inc, USA) to carefully create a straight scratch in the RFPEC monolayer and to allow the dye to enter the scratched cells. After the dye was loaded, it spread via open gap junctions to adjacent intact cells, with the extent of Lucifer Yellow spread reaching a plateau by 10 minutes after loading. At that point, excess dye was washed out and RFPEC were imaged at 10X magnification (dry objective) using a Zeiss Observer Z1 fluorescence microscope. From recorded images of dye transfer after scratch loading, ImageJ randomly selected dye spread locations that would be quantified along the scratch. At those random locations, lines perpendicular to the scratch axis were drawn along the perpendicular lines, the intact fluorescent cells (scratched cells excluded) were counted. The resultant values were taken to represent the extent of Lucifer Yellow spread across the cell monolayer.