Figure 9.

Time-lapse live imaging shows PHF and 6B2 in pre-treated DC, but in separate compartments. DC were pre-incubated with 50 μg/mL tagged PHF-AF488 for 16 h, washed, incubated with Hoechst stain, washed again, and then incubated with Cypher5E-tagged 6B2 tau antibody for up to 150 min. Cells were analyzed using live time-lapse imaging at 5 min intervals. (A) Shows still frames from the live imaging of the 55–150 min time points, with all analyzed channels. The 6B2-Cypher5E signal increased over time, while the PHF signal did not change. In addition, an intensity co-localization analyses was performed between the 6B2 and PHF signals, which generates a co-localization heat map and intensity correlation coefficient (R²). The colocalization heat map (PDM) showed no change in intensity over time. Internalization of 6B2 occurred primarily in the neurites, while the PHF resided in the soma. (B) Shows quantification of the 6B2-Cypher5E signal, where the signal increased over time, and began to plateau near 100 min. (C) Shows quantification of the PHF-AF488 signal, which showed no change within the 150 min period. (D,E) The 150 min Merge and Merge + Brightfield images were magnified to show more detailed morphology and localization of 6B2 tau antibody and PHF in the cells. Most of the antibody signal was localized in the neurites of the cells, and did not colocalize with PHF, as indicated by the white arrows. (F) Shows the intensity correlation coefficients of PHF and 6B2 (R² = 0.058 to −0.059), which did not correlate over time (r² = 0.5334).