Exo flow kits

To analyze for selective subpopulations of EVs surface markers, Evs were incubated with Exo-Flow™ kits (System Biosciences, Mountain View, CA, USA) and then subjected to FACS analysis (FACSCalibur, BD Biosciences, San Jose, CA, USA). Briefly, Evs were incubated with commercially available magnetic beads of 9.1 nm diameter that incorporated different Evs markers including tetraspanins (#EXOFLOW150A-1, CD9, CD63, and CD81). EVs and the magnetic beads were incubated for 12 h at 4 °C according to the manufacturer’s manual. Two negative controls were also carried out, with negative #1 (all reagents without antibodies and no EVs) and negative #2 (all the reagents and beads but without EVs). In the FACS, 30,000 events were acquired and then analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR, USA). The average of MFI for negative #1 was used to normalize the samples with and without EVs.

Purified exosomes were incubated with flow cytometry (Exo-Flow kits; System Biosciences, Mountain View, CA) and analyzed using FACS analysis (FACSCalibur; BD Biosciences, San Jose, CA, USA). Different exosome markers were used based on their functions: tetraspanins (#EXOFLOW150A-1; CD9, CD63 and CD81), targeting/adhesion (#EXOFLOW200A-1; CD31), and membrane transport and fusion (#EXOFLOW500A-1; Rab5a). Negative controls were carried out in the absence of exosomes. Labeled exosomes markers were analyzed on a flow cytometer (FACSCanto II, FACSCalibur; BD Biosciences, San Jose, CA, USA) using FACSDiva software 2.56 (BD Biosciences, San Jose, CA, USA). Data analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA).