IFN-γ production and STAT4 activation of unselected versus sorted PE-MΦs or BMMΦs of C57BL/6 mice. CD68⁺ PE-MΦs (A-C) or CD11b⁺F4/80⁺ or CD68⁺ BMMΦs (D-F) were purified from total PEC or BMMΦ populations by MoFlo sorting (purity ≥ 99%). (A,D) Unseparated and sorted PECs, unseparated and sorted BMMΦs, and PE-MΦs (obtained after adherence of PECs and removal of nonadherent cells) were stimulated under adherent conditions with IL-12 plus IL-18 or IFN-γ (20 ng/mL) plus LPS (200 ng/mL) for 72 hours before culture supernatants were analyzed for IFN-γ and TNF. Means (± SEM) of 3 experiments are shown. ^*Significantly different from unseparated PECs (P ≤ .01, unpaired Student t test). (B,E) The presence of IFN-γ⁺ cells in purified CD68⁺ PE-MΦs or CD11b⁺F4/80⁺ BMMΦs in response to IL-12/IL-18 was analyzed by ICS and FACS. Representative for 3 experiments. (C,F) Activation of STAT4 as analyzed by EMSA. Unsorted PE-MΦs or purified CD68⁺ PE-MΦs and unsorted or purified CD11b⁺F4/80⁺ BMMΦs were cultured without or with IL-12/IL-18 for 2 hours. As controls, the nuclear extracts were combined with the [³²P]-labeled DNA probe in the presence of a 100-fold excess of the unlabeled probe, the nuclear extract was omitted from the gel shift-reaction, or nuclear extracts were prepared from unsorted STAT4^-/- PE-MΦs. Representative of 3 experiments.