Real-time monitoring of reactive oxygen species production during differentiation of human monocytic cell lines (THP-1)

Human monocytic leukemia cell line THP-1 differentiates into macrophages by phorbol myristate acetate (PMA) treatment. We report real-time monitoring of reactive oxygen species (ROS) production during the differentiation process. The production of ROS by THP-1 with several hours time scale has been detected using electrochemical and chemiluminescence methods. The increase in oxidation current derived from ROS arising from THP-1 was observed between 1 and 10 h after the commencement of treatment with 20 nM PMA. The response was unusually slow compared to the quick oxidative response observed in the macrophages. Chemiluminescence methods were used to further investigate the ROS production rate by carrying out treatment with 20 nM to 100 μM PMA. The chemiluminescence responses were found to be faster for the cells treated with higher concentrations of PMA. The time scale of the oxidative response monitored using the chemiluminescence method correlated with that recorded using a microelectrode. The slow time scale of ROS production reflects the differentiation rate of THP-1 into macrophages. The effect of inhibitors on NADPH oxidase and NO synthase were also examined to find that the superoxide anion is the main radical produced from NADPH oxidase.