Fig. 3
LysMCre-C/EBPβfl/fl microglial function in culture. a Primary microglial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice were treated with vehicle (C), LPS (100 ng/mL) ± IFNγ (0.1, 1, 10, 30 ng/mL) for 48 h. NO production was estimated by measuring nitrites in the conditioned medium by the Griess reaction. Data show mean + SEM of six independent experiments. Asterisks show the significance of the treatment effect (**p < 0.01; ***p < 0.001 compared with the respective control conditions) and number sign shows the significance of the genotype effect (##p < 0.01; ###p < 0.001 compared with the respective C/EBPβfl/fl condition). b Mixed glial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice were treated for 24 h with vehicle (C) or LPS (100 ng/mL) + IFNγ (1 ng/mL) and processed for immunocytochemistry for NOS2 (green), the microglial marker CD11b (red) and the nuclear counterstain DAPI (blue). LPS-induced NOS2 expression in C/EBPβfl/fl cultures is markedly attenuated in LysMCre-C/EBPβfl/fl cultures. NOS2 expression is always microglial as revealed by CD11b colocalization. Arrows show NOS2-positive cells. Scale bar, 100 μm. c Primary glial cultures from C/EBPβfl/fl and LysMCre-C/EBPβfl/fl mice were treated with vehicle (C), LPS (100 ng/mL) ± IFNγ (1 and 30 ng/mL). Five days after treatment, Iba1-positive microglial cells were counted as indicated in the “Methods” section. Data represent the percentage of microglial cells on day 0 shown as mean + SEM of three independent experiments. Note the marked AICD effect induced by LPS and particularly by LPS + IFNγ and the absence of differences between the two genotypes. d Primary microglial cell cultures from C/EBPβfl/fl and LysMCre C/EBPβfl/fl mice were treated with vehicle (C), LPS (100 ng/mL) or LPS + IFNγ (1 ng/mL) for 24 h prior to infection with fluorescent Salmonella for 30 min. Microglial cultures were fixed immediately (white bars) or 4 h later (black bars), and the percentage of infected microglial cells and the number of bacteria phagocytosed were analyzed. Data show mean + SEM of three independent experiments. The asterisk shows the significance of the differences between 30 min and 4 h (*p < 0.05; **p < 0.01 compared with the respective 30-min value) and the number sign shows the significance of the genotype effect (#p < 0.05; ##p < 0.01; ###p < 0.001 compared with the respective C/EBPβfl/fl condition)