Generation of ROS was determined by addition of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) (Invitrogen). Briefly, logarithmically growing H460 cells were incubated with 10 mM CMH2DCFDA for 1 hour according to manufacturer’s instructions. Afterward, cells were incubated with or without different concentrations of 2b during indicated time periods. After trypsinization and centrifugation, the cells were fixed in cold 80% methanol. Shortly before measurement, they were centrifuged and resuspended in PBS. The fluorescence of the product, developed by removal of the acetate groups from CM-H2DCFDA by intracellular esterases and oxidation, was measured by flow cytometry on BC Navios instrument (Beckman Coulter, Inc., Miami, FL, USA).
To further examine whether toxicity of 2b is coupled with formation of ROS, two ROS scavengers were used: NAC, a drug that has been known for years to directly reduce the level of ROS25 (link) or the new ROS scavenger tempol.26 (link) Their effect was determined by MTT assay or colony-forming assay as described in Cytotoxicity assay.
To further examine whether toxicity of 2b is coupled with formation of ROS, two ROS scavengers were used: NAC, a drug that has been known for years to directly reduce the level of ROS25 (link) or the new ROS scavenger tempol.26 (link) Their effect was determined by MTT assay or colony-forming assay as described in Cytotoxicity assay.