ciBECs were cultured onto cell-culture inserts as described above. The media were replaced with HBSS supplemented with 10 mM HEPES and 0.1% BSA containing either 100 μM verapamil or vehicle (0.5% DMSO), followed by incubation at 37°C for 30 min. Cells were dye-loaded by removing buffer from the upper chamber and replacing it with fresh buffer containing 200 ng/mL rhodamine-123 at 37°C for 30 min. The cells were washed three times in HBSS supplemented with 10 mM HEPES and 0.1% BSA. Fresh assay buffer was added onto the cell-culture inserts and incubated at 37°C for 1 hr to allow dye efflux. At the end of the incubation the inserts were transferred to a fresh plate, and the cells were washed three times in PBS and lysed for 20 min in cell lysis buffer. The fluorescence was measured with an Envision fluorescence plate reader (PerkinElmer). The cellular uptake of rhodamine-123 was calculated using standard curves generated from stock solutions of the dye.
