Obtained exosomes were stained with PE-anti-CD81 (clone SA6, Sony Biotechnology, USA), BV 421-anti-β3 integrin (clone VI-PL2, BD Biosciences, USA), BV 650-anti-αVβ5 (clone ALULA, BD Biosciences, USA) antibodies and left for 20 min at RT. Next, 900 μL of sterile PBS was added to the samples before the acquisition on the CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). The cytometer was calibrated using a mixture of non-fluorescent Flow Cytometry Sub-micron Size Reference Kit (Invitrogen, Waltham, MA, USA) with sizes ranging from 0.02 µm to 2.0 µm. This calibration step enabled the determination of the sensitivity and resolution of the flow cytometer and the size of extracellular vesicles. All samples were acquired at a high flow rate and high dilution to avoid a coincidence or swarm detection. The analysis of the data was performed with CytExpert software (Beckman Coulter, Brea, CA, USA).
Free full text: Click here
