2.3. Macrophage lipid engulfment

Residential peritoneal macrophages were isolated from WT and Cnn2^−/− mice as above and seeded onto pre-cleaned coverslips in a 48-well culture plate in RPMI 1640 medium containing 10% FBS. Cells were allowed to adhere to the coverslips at 37°C overnight. Non-adherent cells were removed by gentle washing with pre-warmed RPMI 1640 medium. The adherent macrophages were processed for experiments as described (Zhang et al., 2008). To load the macrophages with lipid, the culture medium was switched to RPMI 1640 containing 10% FBS and 25 μg/mL acetylated LDL (BT-906, Alfa Aesar). The cells on coverslips were fixed at 4 hrs, 8 hrs and 24 hrs of lipid loading and the formation of lipid laden foam cells was examined by staining the intracellular lipid droplets with 60% Oil Red O (O0625, Sigma-Aldrich) in isopropanol at room temperature for 5 minutes. Cell nucleus was counter-stained with Mayer’s hematoxylin (26043-06, Sigma-Aldrich) for 5 min. The stained coverslips were mounted on glass slides and photographed using a Zeiss Axiovert 100 microscope. The formation of foam cells was quantified in at least 10 representative view fields in different areas of each coverslip using ImageJ 64 software (NIH, Bethesda, MD). The assay was performed in a genotype-blinded manner.