*5.12. Calcein-AM Assay*

P-glycoprotein activity was measured using the calcein-AM assay. CHO-APP and SK-N-SH cells were seeded into 96-well clear-bottom black-walled plates at 4 × 10<sup>4</sup> cells/well in full culture medium and incubated overnight. The next day when cells reached ~80–90% confluency, media was discarded, and the cells were washed with phenol red-free HBSS or MEM. To assess the inhibitory effect of verapamil and nicardipine on P-gp activity, the inhibitors and DMSO control were prepared at 2× concentrations in HBSS buffer and

added to the cells at 100 µL/well. Calcein-AM substrate was also prepared at 2× concentration, and 100 µL was added to each well using a multi-channel pipette to achieve the final working concentrations. Fluorescence measurements were obtained every minute for 20 min, starting immediately after addition of calcein-AM, using the Perkin Elmer Victor X plate reader. The excitation and emission wavelengths were set at 485 and 535 nm, respectively. Temperature was maintained at 37 ◦C. Measurements were recorded in relative fluorescence units (RFU) and computed using Graphpad Prism software.
