Slide 6 0.4 slides

The EVs were isolated by SEC as described above and labelled using the PKH67 Green Fluorescent Cell Linker Kit (Merck). In short, 3*10¹⁰ EVs were diluted in Diluent C (Merck) to a volume of 1 ml and 10 µl PKH67 dye was then gently mixed with the EVs for 5 min at room temperature. Next, dPBS (Lonza) was added to a final volume of 10 ml and the EVs were separated from free dye by SEC using a qEV10 column (Izon bioscience). For the control staining, the exact same labelling steps were carried out while omitting EVs. For the uptake analysis, MuSCs and FAPs were plated at 10.000 cells/well in µ-Slide VI 0.4 slides (Ibidi) and maintained as described above. The labelled EVs were mixed 1:1 with Opti-MEM and incubated with MuSCs and FAPs for 24 hours. The cells were then washed in dPBS (Lonza) and fixed in 4% paraformaldehyde. DAPI was added to stain the nuclei. The labelled EVs and nuclei were imaged on a Leica fluorescent microscope (DM6000 B, Leica) and an Olympus DP72 digital colour camera (12.8 megapixel, Olympus, Denmark).

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).