Vybrant dio cell labelling

After the washing step, each EV pellet was labelled with Vybrant® DiO cell-labelling diluted 1:1000 in serum free-medium ( V22886, www.thermofisher.com) for 20 min at 37 °C, then washed three times for 10 min each at 37 °C with serum-free medium. After centrifugation, each pellet was resupended in 200 μL of PBS and analysed with an epifluorescence microscope for size analysis or used in uptake assay.

The amount of 5 × 10⁴ cells were plated on glass coverslips placed in 12-well plates. Cells were left to expand for 3–4 consecutive days to form a confluent monolayer (unless stated otherwise) before fixation with 4% paraformaldehyde (PFA) was performed. Immunofluorescence staining was performed as described in [125 (link)]. In brief, cells were permeabilized in 0.5% Triton-X-100 for 15 min at room temperature; after blocking for 1 h with a mixture of 3% w/v BSA and 5% v/v normal goat serum in PBS, cells were stained sequentially by using the indicated primary antibodies. Primary antibody binding was detected using sequential labelling with Alexa Fluor488 or 595 conjugated secondary antibodies (Invitrogen) for 1 h at room temperature. Phalloidin-Alexa594 or Phalloidin-Alexa680 was purchased from Invitrogen and used according to manufacturer instructions. DRAQ5 (Thermo Fisher) or 4′,6-diamidino-2-phenylindole (DAPI; Carl Roth) were used for nuclear counterstaining. Membrane staining of fixed adherent cells using Vybrant DiO cell labelling (Thermo Fisher Scientific) was performed according to manufacturer instructions.