60 nm polystyrene beads

Aliquots of blood plasma 16,000g and 160,000g pellets (fractions III and IV) were stained with mouse anti-human CD3-FITC, CD79a-PerCP-Cy5.5, CD41a-FITC, CD34-APC, and CD63-PE according to the manufacturer's recommendations (eBioscience, USA). Pellets were washed twice with dfPBS and analysed by FACSCanto II (Becton Dickinson, USA). Forward scatter and side scatter (FSC and SSC) PMT voltage settings were adjusted for the detection of blood vesicles/extracellular particles (60–1000 nm) using CST beads (Becton Dickinson, USA) and 60 nm polystyrene beads (Thermo Scientific, USA). PMT voltage settings for the detection of FITC, PE, PerCP-Cy5.5, and APC fluorescence were adjusted using Anti-Mouse Ig, κ/Negative Control Compensation Particles Set beads, according to manufacturer's recommendations (Becton Dickinson, USA). The following settings were used for flow cytometry analysis: FSC, 615; SSC, 310; FITC, 548; PE, 466; PerCP-Cy5.5, 505; APC, 300; threshold FSC/SSC, 200/200; compensation PE-FITC, 18; and compensation PerCP-Cy5.5/PE, 15. Gates were set according to unstained samples. Flow cytometry data were analysed with BD FACSDiva software v6.1.3 (Becton Dickinson, USA). Overlaid histograms were created using Flowing software v2.5.1 (Turku University, Finland).

Preparations of EVs obtained at 160,000 g were stained with mouse anti-human CD3-FITC, CD79a-PerCP Cy5.5, CD41a-FITC, CD34-APC, and CD63-PE according to the manufacturer’s protocol (eBioscience, San Diego, CA, USA). Pellets were twice washed with dfPBS and analyzed using FACS Canto II (Becton Dickinson, Franklin Lakes, NJ, USA). Forward scatter and side scatter (FSC and SSC) PMT voltage settings were adjusted for the detection of extracellular particles (60–1000 nm) using CST beads (Becton Dickinson, USA) and 60 nm polystyrene beads (Thermo Scientific, Waltham, MA, USA). PMT voltage settings for the detection of FITC, PE, PerCP-Cy5.5, and APC fluorescence were adjusted using “Anti-Mouse Ig, κ/Negative Control Compensation Particles Set beads”, according to the manufacturer’s protocol (Becton Dickinson, Franklin Lakes, NJ, USA). The following settings were used for the flow cytometry analysis: FSC, 615; SSC, 310; FITC, 548; PE, 466; PerCP-Cy5.5, 505; APC, 300; threshold FSC/SSC, 200/200; compensation PE-FITC, 18; and compensation PerCP-Cy5.5/PE, 15. Gates were set according to unstained samples. Flow cytometry data were analyzed with BD FACSDiva software v6.1.3 (Becton Dickinson, USA). Overlaid histograms were created using Flowing software v2.5.1 (Turku University, Turku, Finland).