Figure 1
Basic characterization of C/EBPβ -/- mixed glial cultures. Secondary mixed glial cultures from C/EBPβ+/+ (white bars) and C/EBPβ-/- (black bars) show similar total cell numbers and microglial density in control conditions and after 16 h of LPS or LPS+IFNγ. A. C/EBPβ+/+ and C/EBPβ-/- total cell density was estimated by Hoechst-33258-positive nucleus counting. No significant differences were observed between genotypes. Wild-type cultures show a statistically significant increase of cell density after 16 h of LPS and LPS+IFNγ treatment compared to control; C/EPBβ-null cultures show no difference after treatments. Two-way ANOVA, followed by Bonferroni's test was applied. *p < 0.05; compared to C/EBPβ+/+ control. (n = 4). B. Microglia as a percentage of total cells was estimated by CD11b-positive cell counting in C/EBPβ+/+ and C/EBPβ-/- cultures after 16 h treatments with LPS, LPS+IFNγ or vehicle. Significant differences among treatments groups or genotypes are not observed. Two-way ANOVA, followed by Bonferroni's test was applied. (n = 4). C. Secondary mixed glial cultures were immunostained for CD11b (green). Nuclei are stained with Hoechst-33258 (blue). Microglial cell numbers were similar for C/EBPβ+/+ and C/EBPβ-/- cultures. LPS and LPS+IFNγ induced morphological changes in microglial cells in both genotypes. Bar = 50 μm. D. A representative western blot shows levels of GFAP in C/EBPβ+/+ and C/EBPβ-/- mixed glial protein extracts 16 h after vehicle (control), LPS and LPS+IFNγ treatments. β-Actin was used for normalization. E. Densitometric analysis was used to quantify GFAP protein levels versus β-actin in 4 independent western blots in arbitrary units (a.u.). Changes in GFAP protein levels are not observed. Two-way ANOVA, followed by Bonferroni's test was applied. (n = 4). F. Secondary mixed glial cultures were immunostained for GFAP (red) showing a confluent astrocytic layer. Overlapping of astroglial cell bodies makes counting very difficult and imprecise. No differences in astroglial morphology or density among genotypes are observed. Nuclei are stained with Hoechst-33258 (blue). Bar = 50 μm. E. Lack of NOS2 expression in activated astrocytes. C/EBPβ+/+ and C/EBPβ-/- secondary mixed glial cultures were immunostained for GFAP (green) and NOS2 (red), and stained for Hoechst 33258 (blue), after 16 h of LPS+IFNγ treatment. A marked reduction in number of NOS2-positive cells is seen in C/EBPβ-null cultures. The representative merge images show clearly that NOS2-positive cells do not colocalize with GFAP-positive cells. Bar = 50 μm.