Exoquick

EVs were isolated using Exoquick™ (SBI) according to the manufacturer’s protocol described previously [18 (link)]. Briefly, 150 μl EDTA plasma was centrifuged for 15 min at 3000×g. The supernatant was filtered over a 0.45 μm Spin-X filter (Corning), which was flushed with preheated PBS (37°C) and 38 μl Exoquick™ solution was added to the filtrate. After vortexing, the sample was stored overnight at 4°C. The following day, the sample was centrifuged at 1500 × g for 30 min at room temperature, and the pelletwas lysed in 100 μl Roche Complete Lysis‒M with protease inhibitors (EDTA free). The sample was filtered over a 0.22 μm Spin-X filter (Corning) and protein concentration was determined using a Pierce® BCA Protein Assay Kit (Pierce Biotechnology, Rockford, USA), in order to correct the amount of measured EV-marker for the total amount of protein present in the EVs. Samples were stored at −80°C. After thawing, the lysed sample was diluted 20x with Roche complete Lysis-M buffer, and 50 μl was analysed in a multiplex immunoassay on levels of cystatin C, serpin G1, serpin F2 and CD14 using a Biorad Bioplex 200 system as described before [29 (link)]. Capture antibody, biotinylated detection antibody and antigen of all 4 proteins were purchased from R& D systems. A full description of preceding biomarkers proteomics discovery work is provided previously [18 (link)].