Figure 4

Long term primary hippocampal cell cultures naturally release Aβ1-42 in increasing amounts over time. Aβ1-42 is also seen to form an increasing amount of aggregates over time, and is present in live cells. A. Aβ1-42 immunoreactivity in the CM samples collected from individual cultures (n = 4 per time point) was measured by a direct ELISA. There was a marked increase in Aβ release beginning at DIV 21. Data are presented as mean values ± SEM. Cell viability in hippocampal cell cultures was determined using Live/Dead assay. B. Live cells stained with Congo red dye. Staining with Congo Red is a classical method of the detection of amyloids. Congo red exhibits very low non-specific binding. Congo Red positive staining increases as cells age, beginning at DIV 21 and increasing until DIV 40. C. Congo red staining can also be observed in what appears to be the inside of cells, as well as the network. D. Congo red (red) can also be observed in live cells at DIV 60. Live cells are marked with calcein (green), and an overlap of orange indicates an overlap of markers. Other Congo red marked cells may be dead at this time point. E. Counts of Congo red positive cells significantly (P < 0.05) increase over time.