Dil fluorescent dye

Clec9A-targeting tailored nanoemulsions (TNEs) loaded with a pool of neoepitopes were prepared as previously described [14 (link)]. Briefly, peptides were dissolved in ultrapure water to a final protein concentration of 1 mg/mL and emulsified with Span 80 (1%, w/v) (Sigma-Aldrich by Merk, Bayswater, VIC, Australia). The resulting emulsion (1 mL) was frozen rapidly in dry ice for 2 h before being lyophilized for 24 h. The resulting pellet was dissolved in Miglyol 812 (20 µL) (Cremer Oleo, Hamburg, Germany) and used as an oil phase for preparing Clec9A-targeting tailored nanoemulsions (TNEs) loaded with peptides as described in [14 (link)]. In some experiments, TNEs were labelled using the Dil fluorescent dye (Invitrogen^TM, Scoresby, Australia) as previously described [14 (link)].

To obtain Dil (1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate)‐labelled exosomes, purified urinary exosome from patients with CKD or conditioned media‐derived exosomes were incubated in the presence of 5 μl/ml Dil fluorescent dye (V22885, Invitrogen) for 30 min at 37 °C, then resuspended in 30 ml of PBS and ultracentrifuged at 200,000 × g for 2 h to remove Dil dye. After being washed twice, the labelled urinary exosomes were resuspended in PBS; active‐β‐catenin‐ exosomes were resuspended in serum‐free medium and then add to fibroblasts and incubate cells for 12 h.

The purified extracellular vesicles were suspended in phosphoric acid buffer. The samples were dropped onto carbon-coated copper grids, adsorbed for 90 s and then stained with uranium acetate solution for 30 s. Transmission electron microscopy was adopted to observe the morphology of the extracellular vesicles. The size distribution of the extracellular vesicles was assessed by nanoparticle tracking analysis. CD9 (1:1000, 13174, CST, USA), CD63 (1:1000, 25682-1-AP, Proteintech, China), CD81 (1:1000, SAB3500454, Sigma, USA) and Cav-1 (1:1000, ab2910, Abcam, UK) are extracellular vesicles-specific biomarkers, and their expression can be visualized by western blotting. Extracellular vesicles labeled with Dil fluorescent dye (Invitrogen, USA) and excess fluorescent dye was removed by ultracentrifugation at 100,000 × g for 1 h then washed twice. The labeled extracellular vesicles were incubated with the cells and the cell fluorescence was observed by confocal microscopy to judge whether the extracellular vesicles could be ingested by the target cells.