Rhodamine123 efflux assay

Rhodamine123 (rh123) efflux assays with transiently transfected HEK293 cells were performed after a 24 h incubation at 28°C or 37°C. Cells were harvested by trypsinization after washing with phosphate‐buffered saline (PBS), and centrifuged at 500 g followed by resuspension of the pellet with DMEM (pH 7.8) containing rh123 (Sigma Chemical Co., St. Louis) at a final concentration of 0.2 μg/mL (0.53 μmol/L). Loading with rh123 was carried out in a water bath under continuous shaking at 37°C for 30 min. Afterwards cell were chilled on ice and subsequently washed with ice‐cold DMEM (pH 7.4) to remove extracellular rh123. Cell pellet was resuspended in DMEM at 37°C (pH 7.4) to start efflux of rh123. The cellular fluorescence was continuously monitored in a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, Vienna, Austria) and the first order rate constants (k‐values) were calculated as described previously (Donmez Cakil et al. 2014).