Fig. 7

Conditioned media from cholesterol-enriched SH-SY5Y cells exposed to Aβ alter the phagocytic capacity of microglia. Conditioned media were obtained from SH-SY5Y cells cholesterol-enriched (CHO:MCD complex, 1 h) and then treated with Aβ (10 μM, 24 h). In some cases, cells were pre-treated with GSH ethyl ester (GSHee, 4 mM) for 30 min before Aβ exposure. SIM-A9 cells were incubated with the conditioned media for 16 h and then HiLyte Fluor 488-labeled Aβ (1 μM) was added for 4 h. a and b Representative confocal micrographs from 3 independent experiments showing Aβ phagocytosis (green). In b, SIM-A9 cells were cholesterol-enriched with the CHO:MCD complex for 1 h and after 4-h recovery were primed with LPS (10 μg/ml) plus MDP (10 μg/ml) (L + M) for 16 h or Aβ (10 μM) for 24 h before exposure to conditioned media from SH-SY5Y cells. Cells were counterstained with CellMask (cytosol/plasma membrane, red) and DRAQ5 (nuclei, blue). Images from the green channel corresponding to fluorescent-labeled Aβ are shown in black and white. Scale bars: 15 μm. a and c Plots represent Aβ phagocytosed per cell, quantified as the corrected total cell fluorescence (CTCF) of the green channel (n = 9–12 non-overlapping images). d Trem2 and Clec7a mRNA expression levels analyzed by selfie qRT-PCR. Transcript copies were normalized to total genomic DNA and reported as relative levels referred to the expression in mock cells. (n = 4 independent experiments). One-way ANOVA followed by the Tukey–Kramer test was applied to calculate statistical significance (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)