THP-1 differentiated macrophages were cholesterol-loaded with 50 μg/mL acetylated LDL or 50 μg/mL oxLDL (Intracel, Frederick, MD) for 24 h and subjected to conditions described above for another 24 h in the presence/absence of modified LDL. For Oil Red O staining, cells were fixed in 4% paraformaldehyde and then washed and stained with 0.2% Oil Red O (Sigma) for 30 min. After the PBS wash, cell nuclei were stained with hematoxylin (Sigma) for 5 min. After a final wash with PBS, coverslips were mounted on slides using Permount solution (Sigma).
Foam cells, recognized as macrophages stained with Oil Red O, were visualized via light microscopy (Axiovert 25; Carl Zeiss, Gottingen, Germany) with 40x magnification and photographed using a DC 290 Zoom digital camera (Eastman Kodak, Rochester, NY). The number of foam cells formed in each condition was calculated in triplicate manually and presented as percentage of total cells.
Foam cells, recognized as macrophages stained with Oil Red O, were visualized via light microscopy (Axiovert 25; Carl Zeiss, Gottingen, Germany) with 40x magnification and photographed using a DC 290 Zoom digital camera (Eastman Kodak, Rochester, NY). The number of foam cells formed in each condition was calculated in triplicate manually and presented as percentage of total cells.