In total, 250 μL of plasma was used with the ExoQuick® Ultra EV Isolation Kit for Serum and Plasma (System Biosciences, CA, USA, EQULTRA-20A-1) for the isolation of EVs by precipitation per kit instructions. This includes an initial centrifugation of 3000× g for 15 min for further removal of platelets. We utilized ExoQuick® due to the small volumes of materials available as in [19 (link)], and we are aware of the issues involving its use, particularly from complex biofluids such as plasma [73 (link)]. Note below that we do use ultracentrifugation for secondary isolation of surface protein-stripped EVs. Total EV protein concentration was measured by a bicinchoninic acid (BCA) assay (Thermo Scientific, IL, USA, A53225).
Regarding the presence of platelets in the plasma, following the 780× g centrifugation of the whole blood to collect plasma, per ExoQuick® instructions the plasma was spun at 3000× g for 15 min; this step should remove platelets. Further, in studies where cytokines associated with platelet EVs were measured, the vesicles were from isolated platelets that were activated to release larger particles (frequently termed “microparticles” [26 (link),74 (link),75 (link)]). As we did not isolate platelets, nor activate them, and our EVs were of considerably smaller diameters (Figure S1 ), we rule out significant contributions to EV cytokine profiles from platelets.
Regarding the presence of platelets in the plasma, following the 780× g centrifugation of the whole blood to collect plasma, per ExoQuick® instructions the plasma was spun at 3000× g for 15 min; this step should remove platelets. Further, in studies where cytokines associated with platelet EVs were measured, the vesicles were from isolated platelets that were activated to release larger particles (frequently termed “microparticles” [26 (link),74 (link),75 (link)]). As we did not isolate platelets, nor activate them, and our EVs were of considerably smaller diameters (
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