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. 2019 Feb 13;14(3):448–461. doi: 10.1007/s11481-019-09839-0

Fig. 3.

Fig. 3

Inhibition of BTK reduces microglial uptake of synaptic structures with minimal impact on migration and cytokine release. a Representative confocal microscopy images of primary microglia incubated with 1 μM of CC-292 for 24 h followed by incubation with pHrodo® zymosan particles for 1 h. Cells were stained with phalloidin and CellMask to allow for cell segmentation. Scale bar = 25 μm. b Phagocytosis of zymosan particles was significantly reduced following CC-292 treatment as measured by the phagocytic index. c Western blot of rat brain synaptosome preparations showing enrichment of SV2A (presynaptic marker) and PSD-95 (postsynaptic marker) with low MBP (myelin) signal in the synaptosome (Syn) fraction. d Representative confocal microscopy images of primary microglia incubated with 1 μM of CC-292 for 24 h followed by incubation with pHrodo®-conjugated purified synaptosomes for 1 h. Cells were stained with CellMask to allow for cell segmentation. Scale bar = 75 μm. e Synaptosome uptake was significantly reduced following CC-292 treatment as measured by co-localization with CellMask Blue. f Representative light microscopy images from scratch migration assay of primary microglia incubated with 1 μM of CC-292 for 24 h. Scale bar = 200 μm. g Ability of microglia to migrate into scratch was not significantly different between vehicle and CC-292 treatment. h Cytokine release from LPS-stimulated primary microglia incubated with 0.1 or 1 μM of CC-292 for 24 h. IL-6 levels were unchanged between vehicle and CC-292 treatment in LPS-stimulated cells. A small but significant decrease in TNF-α was observed with 1 μM of CC-292. Non-significant decreases in IL-1β were measured with CC-292 treatment. Data presented as mean ± s.e.m., 4 biological replicates for each condition, n = 300–350 analyzed cells per condition for phagocytosis assays, unpaired t-test (phagocytosis and migration assays), one-way ANOVA with Tukey’s post-hoc multiple comparison test (cytokine release), *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle control (for cytokine analysis, versus LPS vehicle control)