2.4. Flow cytometric analysis

To detect apoptotic events, cells were cultured in the absence or presence of DNR or VCR in combination with or without verapamil, cyclosporine A (CyA), or nilotinib for 24 hours, and stained with a fluorescein isothiocyanate‐conjugated Annexin‐V (BioLegend, San Diego, CA) and actinomycin‐D (Sigma‐Aldrich, St Louis, MO). Cell surface expression of P‐gp was analyzed using a phycoerythrin‐conjugated anti‐P‐gp antibody. For the functional assay of P‐gp‐mediated efflux of calcein‐AM (CAM), HALO1 cells were incubated with 0.25 mmol/L of CAM for 10 minutes at 37°C in the absence or presence of velapamil, CyA, or nilotinib. The stained cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA).