FLT3 ITD mediates generation of high levels of ROS. ROS production in 32D cells (A) or human myeloid cell lines (B) expressing either WT FLT3 or FLT3 ITD (as indicated) was measured with 2 × 10⁶ cells per point using 5-(and-6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (carboxy-H₂DFFDA) in a fluorescence plate reader (mean ± SD, determination in triplicates, representative experiment of 3 with consistent results). (C) Elevated ROS production in AML patient blasts harboring FLT3 ITD. AML patient blasts were isolated from freshly taken blood samples. ROS levels were measured in a fluorescence plate reader using carboxy-H₂DFFDA. Values for 10 or 7 patients, harboring FLT3 WT or FLT3 ITD, respectively, are shown (significance tested by t test). Further data of the individual patients are provided in supplemental Figure 4.