Fig. 2

CSF inhibits α-syn aggregation in unseeded and seeded conditions in a patient-dependent manner. A Six different human HC CSF samples (40 µL) spiked with 20 fg synthetic preformed α-syn fibrils (seeds) and analysed by diagnostic SAAs conditions: 0.3 mg/mL (19.6 µM) of recombinant α-syn in 100 mM PIPES pH 6.5 and 500 mM NaCl, 200 µL final volume. B Protein aggregation assay performed using 0.7 mg/mL of recombinant α-syn in PBS with (black) and without (red) 40 μL of NC CSF pool (final volume of 200 μL). C Seed amplification assay in PBS of different amounts of synthetic seeds (0.5, 5, 50, 500 and 5,000 pg) with and without NC pooled CSF (only 3 seed masses shown). D Graphical description of the fitting function used. A2 fits the fluorescence value of the second plateau, A1 fits the fluorescence value of the first plateau and A0 fits the baseline fluorescence. The time parameters t1 and t2 fit the first and the second inflection points, respectively, while d1 and d2 represent the slopes of the sigmoids. E Protein aggregation assay performed using 0.7 mg/mL of recombinant α-syn in PBS (final volume of 200 μl). Six glass beads with a diameter of 1 mm were added in each well. The shaking/incubation protocol consisted in 1 min shaking at 500 rpm and 14 min rest at 37 °C. The experiment was performed in quintuplicate; three replicates were used to produce the above reported average aggregation profile, the other two replicates were collected from the plate at t = 35 h and t = 165 h, and analysed by TEM to produce the representative images shown in the bottom of panel E). All ThT fluorescence traces are represented as average intensity over 3 replicates with error bars representing SEM