Analysis of total PECs and unseparated BMMΦs of C57BL/6 mice for the expression of IFN-γ by ICS and FACS after stimulation with IL-12/IL-18. Brefeldin A was added during the final 8 hours of the 36-hour stimulation period. (A) Total PECs: a dominant IFN-γ⁺ cell population is located within region R3 (Figure 1A). In addition a few IFN-γ⁺ cells were detectable localized within region R1. (B) Total PECs: dot plot analysis of the surface phenotype of the IFN-γ⁺ cells within region R3. The percentage of IFN-γ⁺ cells is given in the plot panels. (C) Total BMMΦs: dot plot analysis of the surface phenotype of IFN-γ⁺ cells within BMMΦs. The cells that specifically stained with anti-IFN-γ compared to normal rat IgG (left 2 panels) are negative for CD11b and F4/80 (2 panels in the middle) and positive for the T-cell markers CD3 and CD8 (4 panels on the right). Panels A and B represent one of 5 independent experiments; panel C, one of 3 independent experiments.