The I-YFP GJIC assay was performed as previously described [17 (link)], with minor modifications. Briefly, a 1:4 mixture of LN215-YFP and LN215-SLC26A4 cells was plated on a 96-well plate at a density of 20,000 cells/well and incubated for 24 h. Culture media were aspirated and cells were washed twice with 200 μL of C-solution. Next, the cells were treated with vehicle or chemicals diluted in 100 μL C-solution and further incubated for the indicated period. The 96-well plate containing the cells was placed into a POLARstar microplate reader (BMG Labtech, Ortenberg, Germany). An equal volume of I-solution (10 mM HEPES [pH 7.4], 140 mM NaI, 10 mM glucose, 5 mM KCl, 1 mM MgCl2, and 1 mM CaCl2) was injected into each well at 1 s after each measurement was started at a rate of 135 μL/s using the machine-equipped automated injector. Fluorescence was measured for 20 s at 0.4 s intervals in kinetic mode using a 485 nm excitation/520 nm emission filter. The percentage (%) of YFP quenching and GJIC activity were calculated as follows:
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