Fig. 5

BG-EVs elevate Glial Fibrillary Acidic Protein (GFAP) levels: 10,000 primary mouse astrocytes per well were seeded in a glass-bottom 96 well plate 24-h prior to experiment. Cells were treated with vehicle PBS or with 100 µg/mL pooled BG-EVs (n = 4, 25 µg/pool from each group) for 24 h, in triplicate wells and 9 field of views. Cells were then fixed, immuno-stained for GFAP, and imaged using an automated scope (Lionheart FX, Biotek). A, C Representative 10 × images for A WT and C CX3CR1 + GFP + astrocytes. Scale bar = 50 µm. B, D Mean fluorescence intensity (MFI) of GFAP calculated using single-cell analysis for B WT and D CX3CR1 + GFP + astrocytes. Ordinary one-way ANOVA multiple comparison test (Dunnett’s test) was used to assess statistical differences in B and D. *p < 0.05; ***p < 0.001; ****p < 0.0001. Error bars represent S.E.M. of 600–1000 cells per treatment. E Graph depicting genotype-dependent differential response to the treatments between WT and CX3CR1 + GFP + astrocytes. Unpaired T test with Welch’s correction was used to assess statistical differences. **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars represent S.E.M. of 600–1000 cells per treatment