Exo fitc exosome stain

Isolated exosomes were further purified using the immune-affinity Exo-Flow Exosome Capture kit (System Biosciences). Briefly, 40 µL of biotinylated capture antibodies (Rab5b, CD9, CD31, and CD44) were conjugated to 10 µL of Exo-Flow 9.1 µm streptavidin-conjugated magnetic beads on ice for 2 h to allow for the efficient capture of exosomes expressing these surface markers. Next, the samples were incubated on a rotating rack at 4°C overnight. To validate the isolation procedure, 200 µg of exosome-coated beads were stained with Exo-FITC exosome stain (System Bioscience) on ice for 2 h and then analyzed with a BD LSR II flow cytometer (BD Biosciences). Beads without the biotinylated capture antibodies were used as negative controls.

Platelet- or EV-specific surface marker staining for EVs was performed using the Exo-Flow exosome capture kit (System Biosciences, USA) according to the manufacturer’s instructions. Briefly, purified EVs were mixed with immune-magnetic beads coated with anti-human CD63 monoclonal antibody (BioLegend, USA) and incubated overnight at 4 °C. After incubation, the beads were washed using Bead Wash buffer (System Biosciences, USA). The EVs were incubated with the Exo-FITC exosome stain (System Biosciences, USA) for exosome detection, AF647-conjugated anti-CD41 monoclonal antibody for platelet-surface marker detection (BD Biosciences, USA), respectively; then, they were examined by flow cytometry (FACSCanto II, BD Biosciences, USA). AF647-conjugated IgG1 (BD Biosciences, USA) was used as an isotype control. All data were analyzed using the CellQuest (BD Biosciences) or FlowJo software.