Fitc fluorescent nanobeads

All analyses were performed after 1:1 secretome dilution with PBS.
Nanoparticle tracking analysis (NTA): secretomes were run by Nanosight NS-300 system (NanoSight Ltd., Amesbury, UK) (5 recordings of 60 s) and EVs visualized with NTA software v3.4 providing both high-resolution particle size distribution profiles and concentration measurements.
Flow cytometry: 3 aliquots were analyzed. (i) Unstained, (ii) 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, Sigma-Aldrich, St. Louis, MO, USA) stained (1 µM final concentration, 30 min at 37 °C) to visualize EVs after transformation into FITC-channel fluorescent carboxyfluorescein succinimidyl ester (CFSE), iii) after CFDA-SE supplementation, stained (30 min at 4 °C) with CD9-APC clone HI9A, CD63-APC clone H5C6, CD81-APC clone 5A6, CD44-APC clone BJ18, CD73-APC clone AD2, CD90-APC clone 5E10 (Biolegend, San Die-go, CA, USA). Samples were analyzed with a CytoFlex flow cytometer collecting at least 30,000 events. FITC-fluorescent nanobeads (160, 200, 240, and 500 nm, Biocytex, Marseille, France) were used as internal control for efficient detection in the nanometric range.

Flow cytometry: cleared secretomes were 1:1 diluted with PBS and divided into 3 aliquots: (i) unstained, (ii) 5(6)-carboxyfluorescein-diacetate-succinimidyl-ester (CFDA-SE, Sigma-Aldrich, St. Louis, MO, USA)-stained (1 µM final concentration, 30 min at 37 °C), (iii) after CFDA-SE supplementation and incorporation leading to FITC-fluorescent carboxyfluorescein succinimidyl ester (CFSE), CD9-APC clone HI9A, CD63-APC clone H5C6, CD81-APC clone 5A6, CD44-APC clone BJ18, CD73-APC clone AD2, CD90-APC clone 5E10 (Biolegend, San Diego, CA, USA) stained (30 min at 4 °C). After a further 1:3 dilution with PBS, samples were analyzed with a CytoFlex flow cytometer. At least 30,000 events were collected. FITC-fluorescent nanobeads of 160, 200, 240, and 500 nm (Biocytex, Marseille, France) were used as internal control.
Nanoparticle tracking analysis (NTA): cleared secretomes were 1:1 diluted in PBS and visualized by Nanosight NS-300 system (NanoSight Ltd., Amesbury, UK) (5 recordings of 60 s). NTA software v3.4 provided both concentration measurements and high-resolution particle size distribution profiles.