Microtiter assay useful for screening of cell-differentiation agents

Promyelocytic leukemia HL-60 cells induced to differentiate along the granulocytic and monocytic pathways respond to stimulation with phorbol myristate acetate by producing superoxide radicals. The amount of superoxide radical generation can be monitored by spectrophotometric measurement of cytochrome c reduction. We have developed a microtiter assay that assesses differentiation of HL-60 cells on the basis of cytochrome c reduction. HL-60 cells were incubated with known standards or unknown samples, including crude fermentation broths, for 6 days; then cytochrome c reduction was quantified as a function of increasing absorbance at 550 nm on a microtiter plate reader. HL-60 cells induced to differentiate showed up to a 10-fold increase in absorbance over that of control cells. Differentiation was confirmed by morphological assessment and by flow cytometric analysis of the DNA cell-cycle distribution and the cell-surface transferrin receptor. Analysis of 198 crude fermentation broth samples confirmed the feasibility of using this assay for large-scale drug screening.