Tracking Extracellular Vesicle Internalization

To track the internalization of EVs, EVs were labelled with PKH67 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described [33 (link), 34 (link)]: EVs resuspended in a buffer provided in the kits were mixed with the PKH67 dyes and were incubated for 5 min at room temperature. Next, the samples were added to PBS supplemented with 5% bovine serum albumin and were ultracentrifuged at 100,000 g for 1 h to remove free dyes. The labelled EVs were co-cultured with HUVECs for 6 h, which were then fixed with 4% paraformaldehyde at 25 °C for 20 min. The internalization of labelled EVs by the HUVECs was analysed using a fluorescence confocal microscope. All the fluorescence images were captured.