Resident peritoneal macrophages from CD68-GFP mice express high levels of GFP. (A) Flow cytometry analysis of resident peritoneal macrophages isolated from CD68-GFP mice revealed 3 distinct populations with different levels of GFP expression. These 3 populations were positive for CD11b, F4/80, and CD68 (myeloid/macrophage markers). In contrast, resident peritoneal macrophages isolated from CX₃CR1^GFP mice displayed no GFP expression, although they were positive for CD11b, F4/80, and CD68. (Representative flow cytometry plots are shown; analysis was carried out in 5 to 8 mice.) (B) Three GFP resident macrophages populations (GFP^{hi, int, lo}) from a pool of 10 CD68-GFP mice were sorted on the basis of GFP expression. (C) Total RNA was isolated, and quantitative RT-PCR was carried out to measure GFP mRNA expression in control cells and cells featuring LPS and interferon-γ. (D-F) Cytokine and chemokine levels were measured in LPS/interferon-γ–stimulated supernatants. (D) IL-6 (E) tumor necrosis factor-α (TNF-α), and (F) CCL2 levels showed no difference across all 3 populations. Data are mean ± SEM of 3 independent experiments, with 3 technical replicates per experiment. Statistical significance was assessed by 1-way analysis of variance and Dunnett’s multiple comparison posttest. ND, not detectable.