Cells were seeded on 24-well fluorescence imaging plates and after 24 h of cultivation in standard conditions, treated with medium containing IONPs for 24 h. To determine mitochondrial ROS levels, the cells were incubated with 5 µM of CellROX Orange reagent (Invitrogen, 35050061) for 30 min. Subsequently, the medium was replaced by FluoroBrite DMEM (Invitrogen, A1896702), supplemented with 10% FBS and 1% GlutaMax (Invitrogen, 35050061) and examined with Leica DMI6000B fluorescence microscope equipped with a temperature/CO2 chamber. Fluorimetric analysis was performed in ImageJ software34,35.
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