4.8. Rhodamine 123 Efflux Assay

P-gp efflux was evaluated by the rhodamine 123 assay, according to previous published methods [16], using verapamil as a standard P-gp inhibitor. Briefly, confluence cells (1 × 10⁶ cells/well) were subjected to a pre-treatment with the test substances or the positive control verapamil (5, 50 and 100 μM) for 2 h, then rhodamine 123 was added. After harvesting and washing, the rhodamine 123 fluorescence was measured at the FL1 emission spectrum (485 nm excitation wavelength; 528 nm emission wavelength) by a BD Accuri™ C6 flow cytometer.