*2.6. Bi-Directional Transport Study*

For the evaluation of the in vitro inhibitory potential of human BCRP by quercetin, the basolateral-to-apical (B-to-A) and apical-to-basolateral (A-to-B) permeability coefficients (Papp) of prazosin (the stereotypical substrate of BCRP) were determined in BCRP-overexpressing MDCKII cells in the presence of various concentrations of quercetin. Briefly, MDCKII cells were seeded on Transwell® filters (12 mm diameter, 0.4 µm pore size; Corning, NY, USA) at a density of 0.5 × 10<sup>6</sup> cells·mL−<sup>1</sup> and then cultured for 5 days before being used in the transport assays. The confluence and integrity of the tight junctions were confirmed via microscopic observations as well as the measurement of transepithelial resistance [29]. The cells were washed twice and pre-incubated with transport buffer (9.7 g/L Hanks' balanced salt solution, 2.38 g/L HEPES, and 0.35 g/L sodium bicarbonate, pH adjusted to 7.4) for 30 min at 37 ◦C. Transport was initiated by adding transport buffer containing 10 µM prazosin in the presence or absence of quercetin (in a final concentration range of 0.1–300 µM) to the donor compartment (500 µL for the apical chamber or 1.5 mL for the basolateral chamber), followed by incubation at 37 ◦C for 120 min. At the end of the incubation, aliquots (300 µL for the apical chamber and 500 µL for the basolateral chamber) of the incubation mixture were collected from the donor and receiver chambers and subjected to LC-MS/MS assays.
