Additional file 3 of Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures

Additional file 3: Supplementary Fig. 3 αS aggregates in αS pff and tg brain homogenate. (A, B) HTRF-FRET immunoassay analysis of aggregated αS in αS pff (light grey), tg brain homogenate (dark grey), and wt homogenate (white) at different sample dilutions. Data is reported as ratio of 665 nm / 620 nm × 10,000. Note that αS pff showed Hook effect at dilutions < 1: 51200 (Hook effect: high aggregate concentrations will capture all antibodies leading to a plateau and to a decrease of the signal) (A). Dotted line (red) illustrates exemplary comparison of dilutions that resulted in similar aggregation signal (B). Aggregation levels that gave a signal ratio of ~ 20,000 for ratio of 665 nm / 620 nm × 10,000. αS pff (light grey) needed to be diluted approximately 204,800-fold, and tg brain homogenate (dark grey) 25-fold. Although the signal cannot be attributed to an absolute amount of aggregated αS given that the conformations of aggregated αS in pff and tg brain extract are likely to be different, results appear consistent with tg brain-derived αS seeds to be more seeding potent compared to αS pff seeds. Mean ± SEM; n = 3 triplicate measurements per dilution.