To identify the mechanisms by which IFN-gamma reprograms early macrophage differentiation and subsequent response to TLR ligands.

Peripheral blood mononuclear cells were isolated from the blood of two independent healthy human donors by centrifugation on a Ficoll density gradient, and monocytes were positively selected using anti-CD14 magnetic beads. Monocytes were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 10 ng/mL M-CSF in the presence or absence of 100 U/mL IFN-gamma for 24 h, and then stimulated with 10 ng/mL Pam3Cys for 6 h. Where indicated, RNA was extracted from fresh monocytes immediately upon isolation. Total RNA extraction was performed using TRIzol reagent (Invitrogen) and RNeasy mini kit (Qiagen). Labeled cRNA was prepared with the Affymetrix GeneChip IVT Labeling Kit, and samples were hybridized to Affymetrix HG U133 Plus 2.0 microarrays. Data was processed using Affymetrix protocols and further analyzed using GeneSpring software.