Contaminating microglia rapidly produce TNF-α and IL-1β after IL-6 stimulation. (A–C) Triple immunostaining using GFAP (blue), CD68 (green), and pSTAT3 (red) antibodies. Before IL-6 stimulation, pSTAT3 accumulation was rarely observed (A), but pSTAT3 accumulation was observed in microglia-contaminated (B) and microglia-eliminated astrocyte cultures (C) 1 h after IL-6 stimulation. pSTAT3 accumulation was observed in GFAP- and CD68-positive cells. Inset shows high magnification image of nuclear accumulation of STAT3 in microglia. Scale bar, 100 μm. (D) Time course of the gene expression of pro-inflammatory cytokines in microglia-contaminated or microglia-eliminated astrocytes after IL-6 stimulation (n = 5). (E) Time course of the gene expression of pro-inflammatory cytokines in BV2 cells after IL-6 stimulation (n = 3). (F) Gene expression of pro-inflammatory cytokines in FACS-purified CD11b-positive and CD11b-negative populations from conventional microglia-contaminated primary astrocyte cultures 1 h after IL-6 stimulation (n = 3). *P <0.05, Kruskal-Wallis test with Dunn’s multiple-comparison test (D, E), Mann–Whitney U test (F). Data are presented as the mean ± SEM.