Fig. 2

Sema 3A activates the microglia proinflammatory M1 phenotype 48 h after transfection. A Representative immunofluorescence pictures of the percentage of microglia cells transfected with Sema 3A-GFP (Sema 3A) or GFP empty vector (GFP). As control, we used non-transfected microglia cells (Ctrl). Scale bar: 55 µm. 10 × objective. B ELISA assay on media from Sema 3A, GFP and Ctrl microglia 48 h after transfection. Sema 3A levels were expressed as ng/ml and normalized on the number of alive cells (mean ± SEM from 10 different fields) for each experimental point. Data are the mean ± SEM of three independent experiments performed in triplicate. ****P < 0.0001 vs Sema 3A media. C Extent of cell survival obtained by counting the number of DAPI positive nuclei before and after Sema 3A transfection as well as in GFP and Ctrl microglia. Data are the mean ± SEM of three independent experiments in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. ****P < 0.0001 vs Sema 3A. D Quantitative analysis of TMEM 119, Iba1, CD86, CD68, and E iNOS and TNFα positive cells. MFI was performed on the entire slide using Zeiss Celldiscoverer7. Values are normalized on the number of DAPI positive cells for each slide and expressed as % of Ctrl. Data are the mean ± SEM of three independent experiments performed in quadruplicate. One-way ANOVA followed by Tukey’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Representative images of iNOS (red) and TNFα (red) and Sema 3A or GFP (green) staining are reported in F. Scale bar: 10 µm. 63 × objective