3.3.1. In Vitro Cytotoxicity Assay

In vitro cytotoxicity was measured according to the Resazurin Cell Growth Inhibition Assay [53,55,59,60] Alamar Blue or Resazurin (Promega, Mannheim, Germany) reduction assay was used to assess the cytotoxicity of the studied samples. The assay tests cellular viability and mitochondrial function. Briefly, adherent cells were grown in tissue culture flasks, and then harvested by treating the flasks with 0.025% trypsin and 0.25 mM EDTA for 5 min. Once detached, cells were washed, counted, and an aliquot (5 × 10³ cells) were placed in each well of a 96-well cell culture plate in a total volume of 100 mL. Cells were allowed to attach overnight and then treated with samples. The final concentration of samples ranged from 0 to 100 mM. After 48 h, 20 mL Resazurin 0.01% w/v solution was added to each well and the plates were incubated at 37 °C for 1–2 h. Fluorescence was measured on an automated 96-well Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. 5-FU as well as erlotinib were used as standard treatment. Each assay was performed as triplicates. It should be noted that DMSO was used as a solvent control such that the vehicle concentration does not exceed 0.05% in all concentrations used. Cancer cells were treated with gradually increasing concentrations of our compounds from (0.01 μM to 100 μM). The viability was compared based on a comparison with untreated cells. IC₅₀ (on cancer cells) were the concentration of sample required to inhibit 50% of the cell proliferation and were calculated from a calibration curve by a linear regression using Microsoft Excel.