*4.5. Detection of Intracellular ROS*

Either DMSO- or KJ-28d-treated cells (5 <sup>×</sup> <sup>10</sup><sup>5</sup> ) were further treated with 10 µM CM-H2DCFH-DA (Thermo Fisher Scientific, Rockford, IL, USA) for 30 min and then washed with phosphate-buffered saline (PBS). After trypsinization, cells were collected, washed, and resuspended in PBS. Inhibition of ROS was evaluated by treating cells with 5 mM NAC 2 h prior to KJ-28d treatment. Intracellular ROS levels were detected using a CyFlow cube 6 flow cytometer (Sysmex Partec, Gorlitz, Germany) at excitation/emission wavelengths of 488/525 nm.
