4.5. Estimation of P-gp Transport Activity by Calcein/AM Retention Assay

P-gp transport activity was measured using a calcein retention assay [42,43]. The cells were spun down (500× g) and washed two times in phosphate-buffered saline (PBS, Sigma-Aldrich) containing 0.2% BSA to remove the drugs. The cells were then resuspended in 0.5 cm³ of the same solution. Calcein/AM (at final concentration: 0.1 μM) was added directly to the solution with the cells. Calcein retention assays were performed in the absence or presence of TBT-Br, TPT-NCS (both in concentration 0.25 µM), and verapamil (10 μM) added directly to the solution with the cells. The samples were subsequently incubated for 20 min at 37 °C in a CO₂ incubator. After incubation, propidium iodide (Sigma-Aldrich) was added at a final concentration of 0.9 μM, and the cells were incubated for an additional 10 min. The cells were then washed twice with ice-cold PBS. Fluorescence was measured using an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Only viable, non-propidium iodide-stained cells (more than 92% in each case) were counted.