Figure 2

Endocytic uptake of Aβ(1–40) and Aβ(1–42) in different cell lines. (a–c) Confocal fluorescence microscopy images of live cells. ((a) SH-SY5Y, (b) CHO-K1 and (c) NIH 3T3) incubated with HF488-labelled Aβ(1–40) (left panels) or Aβ(1–42) (right panels) (green) for 1 h with a peptide concentration of 1 µM. Cell nuclei (blue) were stained with Hoechst 33342 (5 µg/ml) and the cell membrane and endosomes (grey) were stained with FM 4–64 (5 µg/ml) 10 min prior to imaging. (d) Cellular uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF488-labelled dextran 10 kDa after 1 h incubation with respectively 1 µM Aβ(1–40), 1 µM Aβ(1–42) and 125 µg/ml dextran 10 kDa. (e,f) Scatter plots of the cell forward and side scatter (FSC/SCC) showing the fraction of the total number of counted cells gated within the live and dead gates after 2 h incubation at 4 °C (e) and after 2 h subsequent recovery at 37 °C (f). (g) Uptake of Aβ(1–40) and Aβ(1–42) in cells kept at 4 °C. The left bars show reduction in uptake relative to control (37 °C) upon 2 h incubation with 500 nM peptide and the right bars show uptake in cells that after 2 h incubation at 4 °C were allowed to recover, in presence of peptide for 2 h at 37 °C. (h) Uptake of Aβ(1–40) and Aβ(1–42) following ATP depletion with 10 mM 2-deoxy-D-glucose and 10 mM sodium azide in PBS. The cells were pre-treated with the ATP depletion solution for 2 h before addition of 1 μM Aβ(1–40) or Aβ(1–42) for an additional hour and cell uptake is reported relative to untreated control (left bars). The right bars represent uptake in cells that were allowed to recover in ATP-containing media for 2 h following depletion. The uptake is reported as mean cellular fluorescence intensity ± SD of the total number of gated live cells for three replicate samples (n = 3). All flow cytometry data were corrected for baseline contributions by subtracting the cellular autofluorescence.