Fig. 2. Fluorescence of internalized Aβ^pH is retained in fixed cells. (A) Confocal images of fixed HMC3, N9, and BV2 cells showing the uptake of Aβ^pH (green). Cells are stained for acidic intracellular organelles (LysoTracker Red, confirming co-localization of the Aβ^pH within the acidic intracellular organelles) and nuclei (DAPI, blue). No antibody is required to detect Aβ^pH. (B) Primary mouse microglia grown in defined, reduced-serum media phagocytose Aβ^pHex vivo. Cells are fixed and stained for nuclei and show Aβ^pH colocalized in the acidic organelles with LysoTracker Red. (C) The phagocytic uptake of Aβ^pH by primary microglia is measured and quantified via flow cytometry analysis. Dot plot shows live (ZV⁻) and Aβ^pH+ cells. No green fluorescence is measured in unstained cells (UC) or dead cells stained with the ZV live/dead stain only whereas green fluorescence is measured in cells treated with 0.5, 1.0, and 2.0 μM Aβ^pH for 1 hour. Data shown in terms of % max, by scaling each curve to mode = 100% (y-axis). (D) Primary immunopanned rat astrocytes also phagocytose Aβ^pH in serum-free conditions. Cells are fixed and stained for astrocyte specific GFAP antibody (red) and nuclei. (E) Uptake of Aβ^pH over time by primary immunopanned astrocytes as observed in live cells in real time. (F) Quantification of uptake of 0.5, 1.0, and 2.0 μM Aβ^pH by primary astrocytes. Data are mean ± SEM, n = 8 separate wells per group/timepoint.