Differentiation and Activation of Macrophages

RAW264.7 mouse macrophages and THP-1 human monocytes were purchased from American Tissue Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM or RPMI containing 10% fetal bovine serum (FBS) and antibiotics, respectively, as previously reported. THP-1 culture medium also contained 0.05 mM β-mercaptoethanol. THP-1 differentiation into macrophages was achieved by treating the cells in their culture medium with 40 nM phorbol 12-myristate 13-acetate (PMA) for 48 h, detached, PMA removed, and cells plated in 12 well plates for experiments. Differentiation into macrophages was confirmed by CD14 mRNA expression. For experiments, when cells were at approximately 80% confluence, FBS concentration was reduced to 0.1% at the time treatments were initiated and for the 48 h duration of the studies. LPS and PMA were purchased from Sigma-Aldrich (St. Louis, MO, USA), TGF-β from R&D Systems (Minneapolis, MN), MPLAs, CLI-095, and Poly(I:C) from InvivoGen (San Diego, CA, USA), and CU-T12-9, HPI-1, HPI-4, and TO901317 from Cayman Chemical (Ann Arbor, MI, USA). All oxysterols and sterols were purchased from Sigma. Oxysterols were prepared in-house according to our published protocols and those outlined in the present manuscript [34 (link),36 (link)].