Exploring New Inflammatory Biomarkers and Pathways during LPS-Induced M1 Polarization

Figure 2

M1 polarized N9 microglia display amoeboid morphology, together with increased cell area, perimeter, and Feret’s diameter, while showing increased proliferation rate based on the high representation of CD11b and Ki-67 positive cells. Morphological analysis and detection of Ki-67 in the nucleus were performed by immunocytochemistry using anti-Iba1 and anti-Ki-67, respectively, as indicated in Materials and Methods. For morphology, representative results of one experiment are shown (a) and quantified as the percentage of cells with different morphologies, namely, round/oval, ramified, and amoeboid (b). Evaluation of cell area (c), perimeter (d), and Feret’s diameter (e) was performed using the computer program ImageJ. Cd11b expression was assessed by qRT-PCR (f). Representative results of Ki-67 immunostaining are shown (g) and expressed as the percentage of Ki-67 positive cells versus total number of cells (h). Results are mean ± s.e.m. from four independent experiments, except for Cd11b expression (f), where five experiments were performed. Comparison of more than two groups (b) was done by one-way ANOVA followed by multiple comparisons Bonferroni post hoc correction. Comparisons between lipopolysaccharide-treated (LPS-treated) N9 cells and nontreated cells (control) were made via two-tailed Student’s -test (f, h) or unpaired -test with Welch’s correction (c, d, e). , , and versus nontreated cells (control). Scale bar represents 20 µm (and 10 µm in the close-up shown inset in (g)).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)