Measurement of reactive oxygen species (ROS)

ROS production in macrophages was evaluated using a ROS assay kit (BestBio, Shanghai, China; cat. no. BB-4705-2), in accordance with the manufacturers’ instructions. Briefly, DCFH-DA was diluted (1:1500) with RPMI-1640 medium without fetal bovine serum. Coverslips with adherent cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum. The medium was changed to diluted DCFH-DA (2 mL/well) after treatment with different concentrations of CO₂ and O₂; cells were incubated in this medium for 20 minutes at 37°C with 5% CO₂. Coverslips were then washed using RPMI-1640 medium without fetal bovine serum. Finally, coverslips were mounted with a fluorescent mounting medium and detected using a fluorescence microscope (Olympus DP73 Microscope, Olympus, Tokyo, Japan). This experiment was repeated four times.