4.4. Rhodamine 123 Exclusion Assay

The assay was performed as previously described [30] with some modifications. Briefly, for each sample of DU-145 or 22Rv1 cells and DCT^R clones, 2 aliquots of 4 × 10⁴ cells/mL were incubated at 37 °C with 1 μL of Rhodamine 123 (Rh123) (1 mM, SIGMA) and with or without 1 μL of Verapamil (50 mM, SIGMA). After 30 min, cells were harvested and centrifuged and each aliquot was split into two aliquots and analyzed with FACScalibur cytofluorimeter (BD Biosciences, San Jose, CA, USA) immediately (aliquot 1) and after 1 h (aliquot 2). Rh123 green was recorded on FL1 channel and the gate was set to exclude fluorescence of both aliquot 1 samples and verapamil minus aliquot 2 sample. The Rh123 extrusion was detected in the verapamil plus aliquot 2 sample and expressed as percentage of cells with a fluorescence different from the one excluded from the gate.