Foam Cell Formation
Resident mouse peritoneal macrophages were obtained from male ApoE-/- mice fed a WD for 16 weeks. Sterile ice-cold phosphate-buffered saline (PBS) was injected into the cavity of each mouse by peritoneal lavage. This fluid was carefully collected and centrifuged at 1,000 rpm for 6 min. After centrifugation, the supernatant was then withdrawn, and the cell pellet was resuspended in RPMI 1640 medium (containing 100 IU ml-1 of penicillin, 100 μg ml-1 of streptomycin and 100 μg ml-1 of l-glutamine) and plated in 6-well tissue culture plates (Costar) at 1 × 106 cells per well. Cells were incubated in a humidified CO2 (5%) incubator at 37 °C for 2–3 h to allow adherence, and non-adherent cells were rinsed away with pre-warmed RPMI 1640 and 2 ml of complete RPMI 1640 medium (supplemented with 10% fetal bovine serum) was added. Medium with all additions were replaced daily and macrophages were used within 5 days from harvesting. Then, peritoneal macrophages were stimulated with 50 μg mL-1 of oxidized LDL (ox‐LDL) in the presence or absence of As2O3 (2.5 μM or 5.0 μM ) for 24 hours. Cells were then fixed in 4% paraformaldehyde for 15 min, washed with PBS, and incubated with a 0.5% working solution of Oil Red O for 15 min. Images were captured using Nikon microscope equipped with a digital camera (Tokyo, Japan).