Optimized fluorescent probe combinations for evaluation of proliferation and necrosis in anthracycline-treated leukaemic cell lines

Proliferation and multidrug resistance status are key predictors of therape utic outcome in acute myeloid leukaemia (AML). Anthracyclines such as dauno rubicin (DNR) are typically used to treat AML and can induce drug resistanc e. The goal of the studies described here was to select a combination of fl uorescent probes that could be used in combination with flow cytometry to m onitor cell proliferation vs. cell death/necrosis as a function of anthracy cline uptake. Propidium iodide (PI), the most commonly used marker of membr ane integrity, cannot be used to evaluate necrosis in DNR-containing cells because of spectral overlap, A membrane integrity probe compatible with the use of a dye dilution method using PKH67 to study cell proliferation was a lso selected. The results show that DAPI and Cascade Blue (CB), like PI, we re able to detect necrotic cells when no DNR was present, although CB gave less resolution between viable and necrotic cells than PI or DAPI. In the p resence of DNR, DAPI cannot be used owing to the fluorescence quenching by DNR. However, it was found that a combination of DNR, CB, and PKH67 allows simultaneous identification of chemoresistant cells, based on reduced DNR a ccumulation, necrotic cells based on CB incorporation, and proliferating ce lls based on partitioning of PKH67 fluorescence between daughter cells. It was also found that unless a marker of necrosis is used in combination with the dye dilution assay, a moderate decrease of fluorescence as a result of necrosis may be incorrectly interpreted as proliferation.