Levels of intracellular ROS in RAW 264.7 cells were determined using a DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, Raw 264.7 macrophages (5.0 × 105 cells in 1 ml per well) were seeded on 12-well plates and stimulated with silica particles (100 μg/ml) 24 h later. After 3 h stimulation, cells were incubated with 20 μM DCFDA for 30 min. Cells were then trypsinized and washed once with ice-cold PBS. An FACS Canto II flow cytometer were used to determine intracellular ROS levels by DCF fluorescence. In each experiment, the samples were biologically triplicated. The results are shown as mean ± SEM of 3 independent experiments. In the experiments assessing cellular and endosomal ROS, we employed silica particles without FITC labeling in order to measure fluorescent signals. We confirmed that FITC labeling of particles did not induce significant differences in cellular response by induction of chemokine expressions (Supplementary Fig. 16).

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