Quantifying Cellular ROS Levels

For analysis of ROS, the samples were processed essentially, as previously described using Cell ROX Green kit (Life technologies, Grand Island, NY, USA) [46 (link)]. Briefly, AML PGF positive and negative samples were simultaneously thawed, incubated 3 h, and one million cells were taken for ROS measurement. Subsequently, Cell ROX solution and antibodies against surface markers were added, specifically CD45-V450 (BD biosciences) for lymphocyte cells, CD34-APC (Myltenyi Biotec, Bergisch Gladbach, Germany) for HSPCs, and 7-AAD (BD biosciences, San Jose, CA, USA) for dead cells. After 45 min. of incubation, the samples were analyzed by ImageStreamX-100 (Amnis). The data were analyzed via Inspire software. For positive control, cells were treated with Tert-butyl hydrogen peroxide (200 µM).