EVs were isolated from the U2OS cell lines mentioned above. The U2OS cells were pelleted, and the supernatant was passaged through 0.45 µm and 0.22 µm vacuum filtrations. The supernatant was then subject to cross-flow filtration on a filtration system equipped with a 750 kDa hollow-fiber cartridge (Cytiva UFP-750-C-H24LA). EVs were precipitated from the supernatant using 40 mg/mL polyethylene glycol, centrifuged at 1,200 g at 4°C for 1h, and resuspended in 500 µL PBS (McNamara et al., 2018a) .
EVs were then incubated with 50 µg/mL of CellMask Red or CellMask Green membrane intercalating dyes (Thermo Fisher C10046 and C37608), DNase (Promega, M6101), and RNase A (ThermoFisher 00787-333) in 1X PBS at 4°C for 1 h. Excess dye was removed via CaptoCore 700 column (GE Healthcare 17548151). EVs were affinity-selected using anti-CD81 magnetic beads equilibrated in PBS. After incubating EVs with magnetic beads and washing with PBS, CD81+ EVs were eluted with 100 mM Glycine at pH 2.0 for 30 min at room temperature. The EVs were then added to an equal volume of 100 mM Tris-HCl at pH 7.5 in 1X PBS.
EVs were then incubated with 50 µg/mL of CellMask Red or CellMask Green membrane intercalating dyes (Thermo Fisher C10046 and C37608), DNase (Promega, M6101), and RNase A (ThermoFisher 00787-333) in 1X PBS at 4°C for 1 h. Excess dye was removed via CaptoCore 700 column (GE Healthcare 17548151). EVs were affinity-selected using anti-CD81 magnetic beads equilibrated in PBS. After incubating EVs with magnetic beads and washing with PBS, CD81+ EVs were eluted with 100 mM Glycine at pH 2.0 for 30 min at room temperature. The EVs were then added to an equal volume of 100 mM Tris-HCl at pH 7.5 in 1X PBS.