OptiPrep density gradient.

For the discontinuous iodixanol gradient, a slightly modified form of a previously published protocol was used (71, 72). Briefly, 8.3 ml of the 60% (wt/vol) stock OptiPrep solution (Sigma) was mixed with 1 ml of 10× PBS and 0.7 ml of Milli-Q water to a final concentration of 50%. This solution was diluted to final OptiPrep concentrations of 40, 20, 10, and 5% in 1× PBS. The gradient was formed by carefully layering 3 ml of the 40, 20, and 10% solutions and 2.5 ml of the 5% solution in 14- by 89-mm polyallomer tubes (Beckman Coulter). For OptiPrep density gradient isolation of exosomes, the 100,000 × g pellet from 630 ml of culture supernatant (18 T175 flasks) was resuspended in 500 µl of PBS and overlaid on the gradient. The gradient was subjected to high-speed centrifugation at 100,000 × g for 19 h at 4°C with an SW41Ti rotor (Beckman Coulter). Twelve fractions of 1 ml each were collected from the top of the gradient, diluted with PBS in 5 ml, and centrifuged at 100,000 × g for 1 h at 4°C (SW55Ti rotor; Beckman Coulter). The pelleted fractions were resuspended in 100 µl of PBS and then used for further experiments. As a control, 500 µl (100 µM, monomer equivalent) of in vitro-formed recombinant Sup35 NM fibrils or 500 µl of insoluble pellet isolated from s2E cell lysate from two T175 flasks were loaded onto the OptiPrep gradient. The insoluble pellet was separated from soluble proteins via centrifugation at 20,000 × g for 20 min. To determine the density of each fraction, a negative-control gradient with 500 µl of PBS instead of samples was performed in parallel. Fractions were diluted 1:4 with water, and the absorbance at 340 nm was measured in an F-bottom 96-well PS plate (Brand) with a microplate reader (BMG Labtech). The density of the iodixanol solutions was calculated according to Table 3 of application sheet C52 (Axis-Shield PoC).