Assessment of reactive oxygen species (ROS) production

CAL27 cells (1×10⁶) were seeded into a 60-mm dish and pre-cultured for 24 h. The cells were then treated with BTZ and/or RCS for 24 and 48 h. NAC was also used in combination with these drugs as a scavenger of ROS. The cells were stained using 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Inc.), a chemically reduced form of cell-permeable fluorescein that is used as an indicator of ROS, according to the manufacturer's instructions. Fluorescence intensities were assessed by flow cytometry using an Attune Acoustic Focusing Cytometer, and data analysis was performed using Attune^® Cytometer software version 2.1.0 (both from Thermo Fisher Scientific, Inc.).