Visualizing EV Uptake in HBMECs

For EV uptake by HBMECs, SEC-purified EVs were labelled using the PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich), as previously described [32 (link)]. After SEC separation, the sample was mixed 1:1 with ECM (ScienCell™ Research Laboratories). The no-EV control underwent the same procedure of labeling and SEC separation. HBMECs were seeded at 10,000 cells/well in µ-Slide VI 0.4 slides (ibidi, Gräfelfing, Germany) and incubated for 24 h before adding labelled EVs (10^9 EVs/mL), followed by an additional 24 h incubation. The cells were washed before fixation in 4% paraformaldehyde (PFA) and DAPI was used to stain nuclei. Images were obtained on a Leica fluorescent microscope (DM6000 B, Leica Microsystems, Wetzlar, Germany).