Oxidative stress was evaluated by the level of intracellular ROS measured using the dye dichlorodihydrofluorescin diacetate (DCFH-DA) [39]. Briefly, 2 × 105 PBMC per well were seeded in a 96-well cell culture plate and treated with or without nanoparticles. After 24 h, cells were extensively washed with PBS and incubated with 10 µM DCFH-DA in PBS, for 30 min, at 37 °C. As positive control, 1 mM H2O2 was added to another duplicate of cultures. Fluorescence of the oxidized form of DCFH-DA (DCF) was measured using a FacsCanto cytofluorimeter (excitation wavelength: 485 nm; emission wavelength: 530 nm). For each sample, 10,000 events within the viable cells gate were acquired. Each condition was tested in quadruplicate.

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