Murine macrophage-like RAW 264.7 cells were obtained from the American Type Culture Collection. Primary peritoneal macrophages were isolated from WT Balb/C, WT C57BL/6, or mutant C57BL/6 mice defective in either TLR4 or both TLR4 and RAGE (7 to 8 weeks, 20 to 25 g, male or female) at 3 days after intraperitoneal injection of 2 ml of thioglycolate broth (4%) as previously described (23 (link), 24 (link), 49 (link)). Human blood was purchased from the New York Blood Center (Long Island City, NY, USA), and human PBMCs were isolated by density gradient centrifugation through Ficoll (Ficoll-Paque PLUS) as previously described (23 (link), 24 (link), 49 (link)). Murine macrophages and human PBMCs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum or 10% human serum. When they reached 70 to 80% confluence, adherent cells were gently washed with, and immediately cultured in, Opti-MEM I before stimulating with crude LPS (E. coli 0111:B4; #L4130, Sigma-Aldrich), recombinant human SAA (catalog no. 300-13, PeproTech), HMGB1, or pCTS-L. The intracellular and extracellular concentrations of pCTS-L or various other cytokines/chemokines were determined by Western blotting analysis, cytokine antibody arrays, or enzyme-linked immunosorbent assay (ELISA) as previously described (23 (link), 24 (link), 49 (link)).
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