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. 2020 May 1;143(5):1476–1497. doi: 10.1093/brain/awaa090

Figure 1.

Figure 1

PFF treatment promotes the release of α-syn containing exosomes from microglia. Primary cultured microglia were treated with PFF (2 μg/ml) for 24 h, followed by treatment with or without LPS as described. (A) Primary cultured microglia were fixed and stained with an antibody to detect total α-syn. Scale bar = 20 μm. (B–E) The exosomes were extracted from the same volume of culture medium. Immunoblotting was used to detect exosomes as evidenced by the levels of the exosomal markers Alix and TSG 101 proteins (B–D). Exosomes were lysed with RIPA buffer and sonicated briefly. The same amount of exosomal protein from isolated exosomes were loaded and total α-syn levels were detected by western blotting (E). (F and G) Representative image of dot blot and scatterplots of densitometry analysis assessing misfolded α-syn in exosomes obtained from same volume of microglia culture media with different treatments. A conformation-specific antibody against α-syn oligomers was used. (H) LDH levels in culture media were measured after different treatment by ELISA. Ctrl = treatment with ATP before exosome collecting. All data represent mean ± SEM, n = 5–6 independent experiments, using one-way ANOVA followed by Newman-Keuls post hoc test, *P < 0.05 versus Ctrl, #P < 0.05 versus PFF. CM = conditioned medium.