TLR agonists and M-CSF induce CD14⁺HLA-DR⁺human monocytes to differentiate into macrophages. Monocytes from healthy volunteers were stimulated in vitro with optimized concentrations of M-CSF (500 ng/mL), PAM3 (1 µg/mL), IFN-γ (500 ng/mL), LPS (1 µg/mL), MPLA (1 µg/mL), R848 (3 µg/mL), PGN (1 µg/mL), or FSL-1 (10 ng/mL). Samples stimulated for 3 (n = 2) or 5 (n = 4) days yielded similar patterns of macrophage polarization. (A) Representative dot plots exemplifying changes in 25F9 and CD206 expression. (B) Fold change in the number of MAC_suppress (solid bars) or MAC_inflam (cross-hatched bars) present at the end of culture (mean ± standard deviation of 5-6 independently studied donors per data point). (C) Ratio of CD206⁺ to CD206⁻ 25F9⁺ macrophages in the samples described in panel B. *P < .05, **P < .01, ***P < .001 vs unstimulated cultures. Rx, treatment.