Endotoxin contamination does not contribute to SAA-induced G-CSF secretion. Mouse BMDMs were stimulated with different reagents for 24 hours, and secretion of G-CSF was measured using ELISA. (A) Both C57BL/6 (WT) and Tlr4^lps-del BMDMs were used and incubated with LPS (1 μg/mL), SAA (1 μM), heat-treated LPS and SAA (100°C for 25 minutes), or polymyxin B (50 μg/mL, 1 hour)–treated SAA and LPS. (B) Mouse BMDMs were stimulated with the lipopeptides FSL-1 (1 μg/mL), Pam3CSK4 (1 μg/mL), or SAA (0.1, 1, and 5 μM, as indicated in parentheses), or with the same ligands treated with proteinase K (PK, 50 μg/mL) for 1 hour at 37°C and then heated at 100°C for 5 minutes for inactivating proteinase K. Short exposure of SAA to heat (100°C for 5 minutes), which caused only a small decrease on SAA activity, was used as a control. (C, D) Different concentrations of SAA (0.1 and 1 μM), Pam3CSK4 (0.01 and 0.1 μg/mL), PGN (1 and 10 μg/mL), and their mixtures were added to the cells. All data shown are mean plus or minus SEM from 3 experiments.