Cpc200 calibration particles

Extracellular vesicles were isolated from one mL of platelet‐poor plasma by using a previously described procedure [24 (link)]. Briefly, platelet‐poor plasma was obtained by centrifugation at 3000 g for 10 min at room temperature and diluted with PBS 1X to reduce the viscosity of the media (1 : 3 dilution). Then, positively charged beads (GE Healthcare, 17‐5268‐01) in suspension with NiSO₄ (Sigma‐Aldrich, Darmstadt, Germany) were gently poured on the upper solution of the plasma to capture EVs and the solution was placed in orbital shaking for 30 min. Bead‐bound EVs were then recovered by gentle centrifugation at 600 g and eluted with a buffer designed to allow pH shifting and EV‐beads dissociation. After purification, EVs were measured by Tunable Resistive Pulse Sensing (TRPS) technology using qNANO instrument (Izon Science, Christchurch, New Zealand) upon calibration with CPC200 calibration particles (Izon Science) and NP250 nanopores (Izon Science A61484, A61489, A61531, and A61534) were used to characterize particles with a range diameter of 110–630 nm.

Tunable Resistive Pulse Sensing (TRPS) technique, by Izon qNano instrument (Izon, Christchurch, New Zealand), was used to measure the size distribution and concentration of particles in isolated MV- and exosome-enriched fractions. A reagent kit from Izon (Izon EV reagent kit) was used for both pre-treating the pore and suspending EVs in order to prevent EV binding to the pore or spontaneous EV aggregation. MVs or exosomes produced by 1 × 10⁶ microglia in 1 h were re-suspended in a volume of 100 μl. NP300 nanopore (150–600 nm diameter range; Izon) was used for MV sample analysis, while NP150 nanopore (85–300 nm diameter range; Izon) was used for exosome sample analysis. In each experiment, the same applied voltage, pressure and pore stretch values were set for all MV/exosome sample recordings and relative calibration. CPC200 calibration particles (carboxylated polystyrene particles, supplied by Izon and diluted following manufacturer’s instructions) were used as standards. They were measured immediately before or after the experimental samples under identical conditions. Data acquisition and analysis were performed using Izon Control Suite software (version V3.2). MV and exosome concentration values were normalized on protein concentration relative to donor cell sample, determined through bicinchoninic acid assay (BCA, Fisher Scientific, Waltham MA, USA).