Iodixanol Density Gradient Ultracentrifugation for EV Isolation

OptiPrep™ density gradient (ODG) ultracentrifugation was conducted, as previously reported by Van Deun et al. [8 (link)]. Briefly, appropriate amounts of a homogenization buffer (10 mM Tris-HCl (tromethamine-hydrochloric acid), 1 mM EDTA (Ethylenediaminetetraacetic acid) and 0.25 M sucrose (pH 7.4)) and an iodixanol working solution were mixed in order to prepare 5%, 10%, 20%, and 40% iodixanol solutions. The iodixanol working solution was made by adding a working solution buffer (60 mM Tris-HCl, 6 mM EDTA, 0.25 M sucrose (pH 7.4)) to a stock solution of OptiPrep™ (60% (w/v) aqueous iodixanol solution). Moreover, the gradient was prepared in a 16.8 mL open-top polyallomer tube (Beckman Coulter) by layering 4 mL of 40%, 4 mL of 20%, 4 mL of 10%, and 3.5 mL of 5% solutions on top of each other. One mL of embryo-conditioned medium was overlaid onto the top of the gradient. Subsequently, the gradient was centrifuged at 4 °C for 18 h at 100,000× g (SW 32.1 Ti rotor, Beckman Coulter). Then, all 16 gradient fractions were divided into six samples by pooling the fractions 1–4, 5–7, 8–9, 10–12, 13–16, respectively. The pooled fractions were added to 14 mL PBS. Subsequently, the separate suspensions were centrifuged at 4 °C for 3 h at 100,000× g. The resulting pellets were resuspended in 100 µL PBS and stored at −80 °C for further EV characterization.