Qev10 70 nm

EVs produced by HEK293T/17 cells transfected with different GFP plasmids were purified by size exclusion chromatography (SEC) using qEVoriginal/70 nm or qEV10/70 nm (IZON). Cleared conditioned cell culture medium was first concentrated with 100 kD Amicon ultrafiltration tubes (Merck Millipore) and filtrated through a 0.8 μm membrane, and then loaded onto a PBS‐equilibrated SEC column. For EV separation efficiency analysis, qEVoriginal/70 nm column was used, and 20 fractions of 0.5 ml were collected. For other purposes, qEV10/70 nm column was used and only the EV fractions were collected and combined. The collected fractions were then concentrated by 100 kD Amicon ultrafiltration tubes, aliquoted and either directly used for downstream analyses or stored at −80°C.

Sepharose CL-6B (GE Healthcare, Uppsala, Sweden), Sephacryl S-400 HR (Cytiva, Uppsala, Sweden) and Superose 6 Prep Grade (Cytiva, Uppsala, Sweden) were packed into separate 1.5 × 50 cm glass columns fitted with a 30 µm bottom frit and equipped with flow adaptors (Bio-Rad Laboratories, Hercules, CA, USA). Approximately 75 mL of each resin was packed by applying distilled water under gravity flow. Columns were washed with at least 3 bed volumes of distilled water and equilibrated with 2 bed volumes of sterile PBS (Gibco, Grand Island, NY, USA) as running buffer prior to the first SEC run. Commercial qEV10/70 nm (Izon Science, Christchurch, New Zealand) column was washed and equilibrated in PBS as per the manufacturer’s instructions. The CSF-pool for SEC was created by combining equal volumes of 9 CSF samples from 3 severe TBI patients. The CSF-pool of 2.8 mL was loaded by a 5 mL sterile plastic syringe on each column and 50 fractions of 1.5 mL were collected in low-protein-binding tubes (Eppendorf, Hamburg, Germany) for each run. SEC was performed at room temperature and by gravity flow with the running buffer positioned 20 cm above the column. Columns were washed with at least two bed volumes of PBS between SEC runs.