Evaluating P-glycoprotein Function in BBB Model

The P-gp functionality was evaluated using rhodamine 123 (Sigma-Aldrich), a substrate of the P-gp transporter [19 (link),29 (link)]. Both channels were pretreated with 5 µM cyclosporine A (CsA, P-gp inhibitor, Sigma-Aldrich) for 1 h. rhodamine 123 was added to the vascular channel and incubated in the presence or absence of cyclosporine A at a flow rate of 120 µL/h. [¹³C₁₂] Sucrose (100 µg/mL) was simultaneously added to monitor the barrier integrity. Effluents were collected from the apical and basal channels and quantified using fluorescence intensity. The fluorescence was measured at 485/530 nm to quantify the rhodamine 123 concentration via a SynergyMX2 ELISA plate reader (BioTek Instruments, Winooski, VT, USA). The BBB permeability of rhodamine 123 and sucrose was measured in the presence or absence of the inhibitor.