Facsdiva software 2

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).

Human vein endothelial cells (HVEC) were incubated with PKH67-labeled EVs for 4 h at 37°C; subsequently, cell monolayers were washed with PBS 1×, detached using trypsin, and re-suspended in PBS 1×. Flow-cytometry analysis was performed on a FACSCanto II (BD Biosciences) using the FACSDiva software 2.56 (BD Biosciences). Gate was set on living cells based on forward/side scatter properties and a minimum of 10³ events within the gated live population were collected per sample. The intracellular PKH67-labeled EVs were measured by the peak fluorescence intensity shift of PKH67, calculated by the geometric mean of the population. HVECs were stained with mouse anti CD31-APC (allophycocyanin) primary antibody. Internalized EVs were stained with or without PKH67-labeled EVs (unlabeled EVs were used as cell auto-fluorescence control).