Intra-endosomal ROS production was detected using OxyBURST H2HFF Green BSA (Thermo Fisher Scientific) as described previously [51, 52]. Briefly, Raw264.7 cells were incubated in the presence of 100 μg/ml OxyBURST H2HFF Green BSA for 1 h at 37 °C and then stimulated by the addition of phorbol myristate acetate (PMA) (300 nM) or each silica particle (100 μg/ml, no FITC label). For the assessment of NOX2 inhibition, gp91ds-tat (5 μM) was added simultaneously with PMA or silica particles. Cells were briefly washed with PBS and then fixed in 4% paraformaldehyde for 15 min and evaluated by confocal laser scanning microscopy (TiEA1R). For quantification of the endosomal ROS signal, the green puncta observed by confocal laser scanning microscopy in each cell were counted, and the average number of puncta per cell was calculated by assessing 20 cells per experiment. Experiments were performed three times, and data were expressed as mean ± SEM.

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