Fig. 6: TLR9-signaling induced cytokines increase phagocytosis of pHrodo-labeled Aβ42 in human iPSC-derived microglia.
Conditioned media were collected from PBMCs after treatment with 1 μM TLR9 agonist ODN2216 (Donor#_TLR9; containing mixture of TLR9-signaling induced cytokines) or vehicle (Donor#_CT) and were diluted 10-fold to pre-treat human iPSC-derived microglial cultures for ~14 h. After the pre-treatment, microglial cultures were incubated with pHrodo-labeled Aβ42 (1 μg/mL) for 4 h before live-cell scanning with the presence of CellMask™ deep red stain. Phagocytosis of pHrodo-Aβ42 were quantified by dividing total fluorescence intensities of pHrodo by total cell numbers (A). Alternatively, recombinant cytokines (IFNα-2a, IFN-β, IFN-λ1, IFN-γ, IL-1RA, IL-10, SCF) at concentration of 50 ng/ mL were used to pre-treat microglial cultures preceding phagocytosis assay, phagocytosis of pHrodo-Aβ42 were quantified accordingly (B). Biological replicates from n = 3 or 4 experiments (each experiment with 6 biological replicates) are plotted in small-sized orange squares, mean values of biological replicates per experiment were plotted in black dots. Mean ± SD, One-way ANOVA with Fisher’s LDS pairwise comparisons (A) or Dunnett’s multiple comparisons (B) of mean values from independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns (not significant).
