Cellrox oxidative stress reagents

Commercial PM_2.5 (Diesel particulate matter; NIST^® SRM^® 1650b, Sigma-Aldrich, St. Louis, MO, USA) and tetrahydrochloride (NSC-23766; RAC1 inhibitor; N5412, Sigma-Aldrich) were purchased. The reagent 8-OHdG was donated by Myung-Hee Chung (Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Republic of Korea). CellRox^® oxidative stress reagents (Sigma-Aldrich) were used in the intracellular oxidative stress detection experiment. We also purchased anti-NOX1 (PA5-39281, Invitrogen, Waltham, MA, USA), anti-NOX2 (ab129068, Abcam, Waltham, MA, USA), anti-NOX3 (ab81864, Abcam), anti-NOX4 (ab109225, Abcam), anti-RAC1 (05-389, Millipore, MA, USA), anti-p22^phox (#27967S, Cell Signaling Technology, Danvers, MA, USA), anti-NLRP3 (#15101, Cell Signaling Technology), anti-pro-caspase-1 (#3866, Cell Signaling Technology), anti-cleaved-caspase-1 (#4199T, Cell Signaling Technology), anti-precursor-IL-1β (#12703, Cell Signaling Technology), anti-mature-IL-1β (#83186T, Cell Signaling Technology), and anti-ASC (A96472, Antibodies.com, St Louis, MO, USA) antibodies. β-Actin (SC-47778, Santa Cruz, TX, USA) was used as an internal control for the Western blot analysis. The secondary antibodies HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG were obtained from Cell Signaling Technology. Human IL-1β, IL-6, and IL-18 ELISA kits were purchased from R&D (Santa Clara, CA, USA).

CellRox^® oxidative stress reagents (Sigma-Aldrich) were used according to the manufacturer’s instructions to measure the intracellular ROS levels. BEAS-2B cells were seeded at a density of 2 × 10⁴ cells/well in an eight-well chamber slide and incubated for 24 h, followed by treatment with PM_2.5 or 8-OHdG for another 24 h. After washing twice with PBS, 5 μM CellROX^® green reagent was added to the cells, which were incubated for 30 min at 37 °C away from light. DAPI was used to stain the nuclei. Next, the cells were incubated in 3.7% paraformaldehyde for 15 min at 37 °C to perform cell fixation. Observations were made using an inverted microscope (Flowview FV1000, Olympus, Tokyo, Japan), and intracellular fluorescence values (excitation = 485 nm; emission = 520 nm) were measured using Image J software (version 1.53t, 24 August 2022 (upgrade)).