Pgp glo assay system kit

The ATPase activity of P-gp induced by blank micelles was measured via P-gp-Glo™ Assay System kit (Promega Corporation).36 (link) The assay relied on the ATP-related light-generating reaction of firefly luciferase, which could be detected by luminometer (spectra Max M5; Molecular Devices LLC). ATP consumption of sample was calculated from a standard curve. Specifically, various concentrations of verapamil (control), sodium orthovanadate (Na₃VO₄, acted as a selective inhibitor of P-gp), or different compounds were mixed with recombinant human P-gp membranes in the white opaque 96-well plate (Corning Incorporated, Corning, NY, USA). After preincubation at 37°C for 5 minutes, MgATP was added, and the light-generating reaction started. After sustained reaction at 37°C for 40 minutes, the samples were moved to room temperature, and then ATP detection reagent was added to each hole. The plate was maintained at room temperature for another 20 minutes to develop luminescent signal, and then detected by luminometer. The experiments were performed in triplicate.