Figure 4

IFNβ enhances microglia association with myelin debris and phagocytotic activity. a Co-cultures of sorted primary adult microglia with demyelinated OSCs. Microglia from C57BL/6 N mice treated with rIFNβ and labeled with CMTMR (red) and untreated DiD-labeled (blue) microglia were mixed 1:1 and co-transferred onto LPC-demyelinated OSCs (n = 16) from PLP-EGFP mice. For better visualization transplanted cells were marked using Adobe Photoshop software. Diagrams show percentages of rIFNβ-treated (red) and control (blue) microglia associated with myelin debris. b Expression of phagocytosis associated genes in WT, IFNβ^−/− and IFNAR1^−/− primary adult microglia after stimulation with rIFNβ (6 h). n = 2-4. c WT, IFNβ^−/− and IFNAR1^−/− primary adult microglia were left untreated or stimulated with rIFNβ (24 h). DII-labeled myelin was added for the last hour of stimulation. Uptake of myelin was quantified by flow cytometry. d Quantification of phagocytosis activity measured in (c) (n = 4–7). e Experimental setup for analysis of myelin phagocytosis in OSCs (f,g). OSCs were LPC-demyelinated, treated with rIFNβ or PBS (24 h), and analysed for myelin debris content. DIV, Days In Vitro. f OSCs from PLP-EGFP mice treated as in (e) were imaged. g PLP-EGFP-intensity was analyzed by ImageJ software. (n = 16–22). h OSCs from WT, IFNβ^−/− and IFNAR1^−/− mice, cultured for 3 days before LPC-demyelination were treated with rIFNβ (24 h) or left untreated and stained for MBP. i Quantification of myelin debris in OSCs from WT, IFNβ^−/− and IFNAR1^−/− mice after rIFNβ treatment. Scoring as described in Additional file 6: Figure S6. (n = 3-4). j Confocal Z-stack image from a phagocytic microglia in an WT OSC after poly (I:C) treatment and demyelination. Arrows indicate internalized myelin. Error bars represent SEM (a,b,g,i) and SD (d). Scale bars represent 10 μm (j), 100 μm (a,h), 1 mm (f).