Exoquick precipitation solution

Whole blood from human or mice was collected in acid-citrate-dextrose buffer and centrifuged to obtain platelet poor plasma (PPP). PPP underwent an additional centrifugation at 3000g for 15 minutes. Small PEVs were isolated using the ExoQuick™ Precipitation Solution (System Biosciences) according to the manufacturer’s protocol. In separate experiments, Size Exclusion Chromatography (iZON Science) was performed according to the manufacture’s protocol to isolate small PEV and nanoparticle tracking analysis was performed (Nanosight, Malvern Instruments) to confirm purity. To further isolate platelet-specific small EVs, biotinylated antihuman CD42b (Biorbyt, orb114027) was added to the suspended EVs for 90 minutes at room temperature. Streptavidin Plus UltraLink resin was added for 30 minutes. The incubation sample was centrifuged at 400g for 5 minutes and the supernatant containing platelet specific small EVs was removed. 0.05mM acetic acid was then added to release the immune complexes followed by a final centrifugation at 4000g for 10 minutes and removal of the supernatant which contained the platelet-derived exosomes. Small PEV were analyzed by western blot for CD9 (BioWorld Cat. No. BS3022).

For proteomic analysis, mouse serum EVs were isolated by sucrose gradient centrifugation as previously described (22 (link)) with modifications. One mL of serum was layered on top of a 10–70% sucrose gradient in SW55 tubes (Beckman Coulter Inc., Brea, CA, USA) with subsequent centrifugation at 100,000 g overnight. Fractions three to five, which contained EV markers CD63 and CD81 (Supplementary Figure 1A and B), were collected. Each fraction was diluted in phosphate-buffered saline (PBS) and EV pellets obtained by centrifugation at 100,000 g for 70 min. The pellets from each fraction were resuspended in PBS and pooled. Sucrose was removed by a final centrifugation of the pooled suspension at 100,000 g for 70 min.
For all other procedures, ExoQuick precipitation solution (System Biosciences, Mountain View, CA, USA) was used to isolate EVs. From 200–250 µL of mouse serum, cells and cellular debris were pelleted by centrifugation at 3,000 g for 15 min, and 50–63 µL of ExoQuick solution were added to the supernatants. The mixture was incubated at 4 °C for 30 min, and the EV pellet was obtained by centrifugation at 1,500 g for 30 min. The supernatant was aspirated, and the residual ExoQuick solution was removed without disturbing the pellets after centrifugation at 1,500 g for 5 min. EV pellets were resuspended in sterile water or PBS and stored at −80 °C.