Hiscreen capto core 700 column

EV-rich fraction of small-scale DGUC, i.e., 300 μL of F6 fraction with highest TSG101 signal was diluted to 5 mL with PBS, then ultracentrifuged for 3 h at 100,000 ×g, 4°C (SW55 rotor). Pellets were resuspended in 200 μL PBS (Figure 2).
Bind-elute SEC was performed with modifications to what has been previously described (Corso et al., 2017 (link)). EV-rich DGUC fractions (1.5 mL from the pool of F6 + F7 + F8 in case of large-scale DGUC) were loaded onto a HiScreen Capto Core 700 column (4.7 mL bed volume, GE Healthcare Life Sciences). Flow rates and elute conditions were chosen according to the manufacturer’s instructions. Content was eluted with 1× PBS (12 × 500 μL, EF1–12), then the column was washed with 0.1M NaOH in 30% 2-propanol, where CIPs were collected (CIP1-20, 20 × 500 μL; Figure 2). CIP1-20 fractions were immediately pH-adjusted to 7.4 with 0.1M HCl solution in order to assess the composition of bound proteins. Capto Core 700 column was regenerated with 1M NaOH in 30% 2-propanol solution.
Exo-Spin^TM gravity elution SEC was performed as previously described (Welton et al., 2015 (link)). Pool of DGUC F5-7 (0.7 mL) was loaded onto Exo-Spin^TM Midi Columns with 10 mL bed volume (Cell Guidance Systems; Cambridge, United Kingdom). Conditions were chosen according to the manufacturer’s instructions. Thirty EFs were collected (30 × 500 μL, ES1-30; Figure 2).

Plasma EVs were isolated and purified as previously described by Onódi et al.^{25 (link)}. In brief, extracellular vesicle isolation was performed by iodixanol (60 w/V% iodixanol in ultrapure water; Axis-Shield, Oslo, Norway) density gradient ultracentrifugation (24 h, 120.000 × g, 4 °C). The EV-rich DGUC fractions (Supplementary Figure S1) were loaded into a HiScreen Capto Core 700 column (GE Healthcare Life Sciences) and size exclusion chromatography-based purification was performed. Vezics system (vezics.com) was used to implement the isolation.