2.4. MDR Reversal Analysis with MTT Assay

Cells were seeded in Nunc 96-well plates (2000 or 8000 cells/well for the MDCK-II and HT29 cells, respectively). Following cell attachment (12–24 h), drugs were added to a total volume of 200 µL. Control conditions consisted of full growth medium. All treatments were performed in triplicate. Following 72 h of drug exposure, MTT reagent was added for either 1 h (MDCK-II) or 3 h (HT29). For cell lysis and solubilization of the formazan crystals, DMSO was added to the MDCK-II cells, whereas acidified (0.02 M HCl) sodium dodecyl sulphate was added to the HT29 cells. Optical densities were measured with a microplate spectrophotometer at either 544 nm and 710 nm (background) for the MDCK-II cells or 570 nm and 670 nm (background) for the HT29 cells. Background optical density values were subtracted, and the average optical densities were calculated. Cell viability was calculated as percentage of untreated control cells. The mean IC₅₀ values were determined using GraphPad Prism (version 6.0, San Diego, CA, USA). The drug sensitivity analysis was carried out at least three independent times and representative data is shown.