Resazurin reduction assay

Resazurin reduction assay was conducted to test the cytotoxicity of the compounds. This assay is based on the reduction of resazurin to resorufin by viable cells [64]. Non-viable cells do not show a blue staining because they lost their metabolic capacity which causing reduction of resazurin. Briefly, aliquots of 0.5×10⁴ adherent cells which were allowed to attach overnight and 1×10⁴ suspension cells per well were seeded in 96-well-plates with or without the addition of varying concentrations of the test substance to get a total volume of 200 μL/well. After 72 h incubation and addition of resazurin (Sigma-Aldrich) for 4 h, staining was measured by an Infinite M2000 ProTM plate reader (Tecan, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was independently performed for at least three times, with six parallel replicates each. The protocol has been recently reported [65]. Fifty percent inhibition concentrations (IC₅₀) represent the drug concentrations required to inhibit 50% of cell proliferation, which were fitted with nonlinear regression using GraphPad^® Prism7.