Qev original 70 nm sec columns

Patients were fasted from midnight the previous day according to the protocol for patients attending ERCP with sedation. These peripheral fasting blood samples were drawn via a freshly inserted 18-22G peripheral cannula or 21-23G butterfly syringe into BD K2 Ethylenediaminetetraacetic acid (EDTA) lined tubes and processed within 4 h. Blood was centrifuged at 2,500 x g for 10 min at room temperature to remove cells and aliquot the resulting platelet-free supernatant (plasma). The latter was snap-frozen at -80 °C until required (see reporting form in Additional File 2: MIBlood-EV Standardized Reporting Tool for Blood EV Research Human). For EV isolation, 1 mL of plasma was thawed on ice, after which SEC was performed using commercially available qEVoriginal 70 nm SEC columns (iZON Science) according to the manufacturer's protocol. Plasma samples were loaded onto the column and eluted with PBS (pH 7.4; Sterile-filtered, Sigma Aldrich). The eluate was collected in 30 sequential fractions of 0.5 mL, which were used immediately for EV characterisation and RNA extraction, or frozen at -80 °C until required. For the purposes of analysis, we deemed fractions 7-10 as EV-enriched (EV) and 22-24 as free protein-enriched (PROT) after initial characterisation.