Scrape Loading/Dye Transfer Assay

The scrape loading/dye transfer (SL/DL) assay was used to assess gap junctional intercellular communication (GJIC). Lucifer yellow (LY, MW 457), a membrane impermeable fluorescent dye that can go across junctional channels, is the most popular dye in research use. Rhodamine-dextran (Rhd, MW 10000), which is too large to transverse the junctional channels, is often used as an additional control to identify the cells initially loaded after the scrape [35]. After treatment with high glucose and apelin, cells seeded in 35 mm dishes were rinsed gently three times with PBS (pre-warmed to 37°C). Sufficient 1 mg/mL LY-dye and 1 mg/mL rhodamine-dextran mixed solution (37°C) was added to the dishes, and then the cells were scraped with a surgical steel blade. After incubation at 37°C for 4 min, the cells were washed three times with warmed PBS and then fixed by adding 4% paraformaldehyde before visualization with a fluorescent microscope (Zeiss, Oberkochen, Germany). National Institute of Health (NIH) imaging system was used to calculate the fluorescence area. The fraction of the control (GJIC-FOC) was used to evaluate the GJIC capacity [35].

GJIC FOC_Treatment = (Area_Treatment^LY - Area_Treatment^Rhd) / (Area_Control^LY - Area_Control^Rhd)