Phosphate buffered saline (pbs)

Labelling of EVs was performed as previously described (Roberts-Dalton et al., 2017) . Briefly, Alexa Fluor 633 C5-maleimide (200μg/mL; Invitrogen, Carlsbad, California, CA, USA) was added to an aliquot of EVs purified after ultracentrifugation containing 60-100µg of total protein for a final volume of 50μL in 1x PBS (PAN Biotech, Aidenbach, Germany). Samples were incubated for 1h with no agitation in the dark at RT. Non-incorporated and excess of dye was removed by washing the sample in 1x PBS (PAN Biotech, Aidenbach, Germany) and centrifugation during 90min at 20 000xg (4°C) for ectosomes and at 100 000xg for 90min for exosomes (4°C) in a swing rotor (TH-641 Sorvall;
Thermo Fisher Scientific, MA, USA). As a control, 1x PBS (PAN Biotech, Aidenbach, Germany)
without EVs was also incubated with the dye and performed in parallel to confirm dye removal by washing with 1x PBS (PAN Biotech, Aidenbach, Germany) and ultracentrifugation. For microscopy analysis, labelled EVs were gently mixed with mowiol and applied into PLO-coated coverslips and glass slides.

After treatment, primary and cell lines cultures were first washed with 1x PBS (PAN Biotech, Aidenbach, Germany) and fixed with 4% of paraformaldehyde solution (PFA) for 20min at room temperature (RT). In order to quench PFA autofluorescence, samples were incubated with 50 mM of ammonium chloride (NH4Cl) solution for 30min. In order to label the plasma membrane, cells were incubated during 10 min with wheat germ agglutinin (WGA) Alexa FluorTM 633 conjugate in 1x HBSS at RT (5.0 µg/mL; Invitrogen, CA, USA). Cells were washed with 1x PBS (PAN Biotech, Aidenbach, Germany). Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, MO, USA) for 10min.
Following permeabilization, cells were blocked with 2% bovine serum albumin (BSA) in 1x PBS (Sigma-Aldrich, MO, USA) for 1h at RT and then incubated with the primary antibodies overnight at 4°C. Afterwards, the cells were washed with 1x PBS (PAN Biotech, Aidenbach, Germany) and then with fluorescence conjugated secondary antibodies for 2h at RT. Finally, nuclei were counter-stained with DAPI (Carl Roth, Karlsruhe, Germany) and mounted with mowiol for microscopy.