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supernatant was then withdrawn, and the cell pellet was resuspended in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).