Quantification of Reactive Oxygen Species in Hematopoietic Malignancies

Various types of ROS were determined in untreated and drug-treated hematopoietic malignant cells by flow cytometry using live-cell permeant specific fluorogenic probes. Dihydroethidium (DHE, Marker Gene Technologies, M1241) was used as probe for detection of the cytosolic superoxide anion (cO2•-), MitoSox (Molecular Probes, M36008) was used as probe for detection of the mitochondrial superoxide anion (mO2•-) and 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Molecular Probes, C-400) was used as probe for detection of H₂O₂. DHE was oxidized to red fluorescent ethidium by cytosolic superoxide and MitoSox was selectively targeted to mitochondria, where it was oxidized by superoxide and exhibited red fluorescence. Carboxy-H2DCFDA was cleaved by esterase to yield DCFH, a polar nonfluorescent product, but in the presence of hydrogen peroxide, the latter is oxidized to a green fluorescent product, dichlorofluorescent (DCF). For cell staining, cells were centrifuged and the pellets were resuspended in PBS with a final concentration of 5 μM for each probe. The mixture was incubated in the dark at 37 °C for 15 min. Then, the cell suspension was analyzed using flow cytometry within 20 min.