4.7. P-gp ATPase Activity Assay

The method has been described in our previous research [46]. For the evaluation of P-gp ATPase activity of caffeic acid, Pgp-Glo^TM Assay System from Promega (Madison, WI, USA) was used. In a 96-well untreated white plate, 25 μg of recombinant human P-gp membranes were incubated with Pgp-Glo^TM Assay Buffer (untreated control), 200 μM verapamil (positive control for drug induced P-gp ATPase activity), 100 μM sodium orthovanadate (selective inhibitor for P-gp ATPase activity), or a series of concentrations of caffeic acid. The reaction was initiated by adding 5 mM MgATP and incubated for 40 min at 37 °C, followed by stopping the reaction with 50 μL ATPase Detection Reagent for 20 min at room temperature. Luminescence was measured using a BioTek Synergy HT Multi-Mode Microplate Reader, and data were presented as Change in Luminescence (ΔRLU).