Supplementary Figure 9: Microglia maturation is independent from the TLRs2,3,4,7,9.

(a) Left: View of caecum from a SPF-TLR^wt (control, left) and SPF-TLR2,3,4,7,9-deficient (SPF-TLR^ko) mouse (right). Ruler scale is shown. Representative pictures are shown. Right: Relative caecum weight of control SPF-TLR^wt and SPF-TLR^ko mice. Symbols represent individual mice, with six animals per group. Means ± s.e.m. are shown. No statistically significant differences were found by an unpaired t test.

(b) Iba-1 immunohistochemistry in the cortex of SPF-TLR^wt and SPF-TLR^ko animals (left) and quantification thereof (right). Every symbol represents one mouse. Six animals per group were analyzed and three to four sections per mouse were examined. Data are representative of two independent experiments. Means ± s.e.m. are indicated. No significant differences could be determined by an unpaired t test.

(c) Ddit4 mRNA measured by qRT-PCR in microglia from SPF-TLR^wt (black bar) or SPF-TLR^ko (white bar) mice. Means ± s.e.m. with six samples in each group are displayed. No significant differences could be determined by an unpaired t test. Data are representative of two independent experiments.

(d) Three-dimensional reconstruction (Scale bars: 15µm) and Imaris-based automatic quantification of cell morphometry (e) of Iba-1⁺ microglia. Individual symbols represent one mouse per group with at least three measured cells per mouse. Six mice per group were analyzed. Means ± s.e.m. are indicated. No significant differences were detected by an unpaired t test.

(f) Flow cytometry of isolated microglia from control six SPF-TLR^wt and six SPF-TLR^ko individuals. Percentages and mean fluorescence intensities (MFI) of the indicated surface molecules on microglia are shown. Each symbol represents one mouse. Means ± s.e.m. are indicated. Significant differences were evaluated by an unpaired t test and labelled with asterisks (*P < 0.05). P value: CD31 (MFI): 0.0419. Data are representative of two independent experiments.