Exoquick ultra ev isolation kit for serum and plasma

Samples of plasma or CSF were mixed with ExoQuick Exosome Precipitation Solution (System Biosciences) or ExoQuick ULTRA EV Isolation Kit for Serum and Plasma (System Biosciences), and protocols were performed according to the manufacturer’s instructions. For ExoQuick, 0.5 ml of plasma or CSF was mixed with 126 µl of ExoQuick and incubated at 4°C for 30 min, followed by centrifugation at 1500×g for 30 min. Supernatant was removed, and samples were centrifuged at 1500×g for an additional 5 min. Residual supernatant was removed, and pellets were resuspended in 500 µl PBS. For ExoQuick ULTRA, 250 µl of plasma or CSF was used in accordance with instructions, and Simoa values were corrected by multiplying by 2 to match the 0.5 ml volume used for other samples. For each sample, 500 µl of EVs was eluted per column. For all precipitations, isolation was performed on 2 separate days and then resulting Simoa values were averaged.

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.

Serum exosomes were isolated using an ExoQuick Ultra EV Isolation Kit for Serum and Plasma (System Biosciences, Cat: EQULTRA-20A-1) according to the manufacturer’s User Manual. Then, the morphology of the exosomes was observed by transmission electron microscope (TEM), and the size distribution was determined by nanoparticle tracking analysis (NTA) as described before (Zhang et al., 2019 (link)). Specific protein markers of exosomes were detected by western blot.

Plasma samples were collected in EDTA-treated tubes and processed within 30 minutes. To eliminate the risk of bias related to hemolysis, samples were visually assessed and hemolyzed, icteric, or lipemic samples were excluded. Hemolysis was further assessed using miR-23a/miR-451 ratio, as described in the RNA isolation section. Three out of 19 patient samples of the discovery cohort did not satisfy the criteria and were excluded from the analysis. Two-hundred fifty microliters (µl) of plasma were precleared with thrombin (Cat#TMEXO-1, System Biosciences SBI, Palo Alto, CA) and then used for EV precipitation by ExoQuick Ultra EV Isolation Kit for Serum and Plasma according to the manufacturer’s protocol (Cat # EQULTRA-20A-1 System Biosciences SBI, Palo Alto, CA). The quality of isolated pEV was assessed using transmission electron microscopy (TEM) and the number of particles was quantitated by Tunable Resistive Pulse Sensing (TRPS) measurement. Common microvesicle markers were detected by western blot.