Iodixanol Gradient Fractionation and SEC

OptiPrep (Axis‐Shield, Oslo, Norway) density gradients were prepared as previously described (Tulkens et al., 2020a (link); Van Deun et al., 2014 ). Briefly, a discontinuous iodixanol gradient was prepared by layering 4 ml of 40%, 4 ml of 20%, 4 ml of 10% and 3.5 ml of 5% iodixanol in a 16.8 ml open top polyallomer tube (Beckman Coulter, Fullerton, California, USA). One millilitre of crude extract was placed on top of the gradient, followed by 18 h ultracentrifugation at 100 000 g and 4°C using a SW 32.1 Ti rotor (Beckman Coulter, Fullerton, California, USA). Fractions of 1 ml were collected and ODG fractions corresponding to a density of 1.04‐1.07 g/ml were pooled, as well as ODG fractions corresponding to a density of 1.09‐1.10 g/ml. SEC was performed on these pooled ODG fractions (identical to the first SEC) to remove iodixanol (Tulkens et al., 2020a (link); Vergauwen et al., 2017 (link)). SEC fractions 4–7 were pooled, concentrated to 100 μl, stored at ‐80°C until further use and referred to as the LPP extract (1.04‐1.07 g/ml) and EV extract (1.09‐1.10 g/ml).