Macrophage Stimulation and Isolation

Macrophages were isolated as previously described (53 (link)). Briefly, mice were injected intraperitoneally with 4% thioglycollate media (Sigma, T9032) and 4 days later, peritoneal macrophages were isolated, washed, counted, and plated in DMEM supplemented with 10% FBS. Macrophages treated with LPS (100 ng/ml, Invivogen), cholesterol crystals (500 μg/ml), oxidized-LDL (50 μg/ml), silica crystals (200 μg/ml, Invivogen), alum (100 μg/ml, Invivogen), bafilomycin (100 nM, Sigma B1793), chloroquine (10 μM, Sigma C6628), PD98059 (30 μM, EMD Millipore 513000), SB 203580 (10 μM, EMD Millipore 559389), and/or JSH-23 (25 μM, EMD Millipore 481408) were harvested at various times for mRNA or protein using standard techniques or were fixed with 4% paraformaldehyde for immunofluorescence microscopy. Cholesterol crystals were generated by subjecting cholesterol powder (Sigma, C8667) to an ethanol precipitation technique as described (54 (link),55 (link)). Oxidized LDL was generated by Cu²⁺-oxidation of LDL (Sigma, L7914) as described (56 (link)).