Fig. 1. Synthesis and characterization of Aβ^pH. (A) The Aβ^pH is synthesized by conjugating the amine-reactive pH-sensitive Protonex Green dye to the side chain amine groups of the lysine residues and the N-terminal of human Aβ_1–42 peptide. (B) The pH-sensitivity of the Aβ^pH probe characterized at different concentrations from 0.1 μM to 5.0 μM. Increased fluorescence is observed at acidic pH values of ∼5.0 to ∼2.0, covering the pH range of the intracellular acidic organelles. (C) Atomic force microscopy topographic images of Aβ^pH oligomers compared to synthetic Aβ oligomers. Left-2D topographic image of Aβ^pH and synthetic Aβ oligomers. Right-3D image (2 × 2 μm x–y). (D) Live cell imaging of the phagocytic uptake of 1 μM Aβ^pH by BV2 and N9 mouse microglia and by HMC3 human microglia over 24 hours. (E) Quantification of Aβ^pH phagocytic score by BV2, N9, and HMC3 microglial cells from the live cell images. (F) The phagocytic uptake of Aβ^pH by BV2 cells is measured and quantified via flow cytometry analysis. Dot plot shows live (PI⁻) and Aβ^pH+ cells. No green fluorescence is measured in unstained cells (UC) and in dead cells stained with the PI only whereas green fluorescence is measured in cells treated with 0.5 and 5.0 μM Aβ^pH for 1 hour (higher fluorescence is seen in cells exposed to the higher concentration of Aβ^pH). Data shown in terms of % max, by scaling each curve to mode = 100% (y-axis).