Dulbecco phosphate buffered saline (dpbs)

EGFP-positive pectoral fin cells were isolated from zebrafish embryos using FACS. Tg(Mmu:Prx1-EGFP) embryos were collected at 24 hpf (CUT&RUN) and 48 hpf (RNA-seq) and digested in Pronase (1 mg/ml) (Roche) for 5-6 min to remove the chorion. Embryos were pooled together, washed in DPBS (Gibco), and dissociated in Accumax (Innovative Cell Technologies) and DNase I (50 U/100 embryos) (Roche) at 31°C for 1.5 h. Cells were homogenized every 8 min by pipetting up and down using pipette tips decreasing in size. Cells were washed in solution (300 U DNase I in 4 ml DPBS) before filtering through a 70 μm nylon mesh cell strainer (Thermo Fisher Scientific) into 50 ml conical tubes pre-coated with 5% fetal bovine serum (FBS; Invitrogen). Cells were spun down, resuspended in basic sorting buffer [1 mM EDTA, 25 mM HEPES (pH 7.0), 1% FBS in DPBS], stained with DAPI (1:1000), and sorted using FACS at the University of Colorado Cancer Center Flow Cytometry Shared Resource (Aurora, CO, USA) on the MoFlo XDP100 sorter (Beckman Coulter) with a 100 μm nozzle tip (Beckman Coulter).

sEVs derived from H226 cells (4 × 10⁶ cells) were labeled with PKH26 (cat. no. PKH26GL) according to the manufacturer’s protocol (Sigma-Aldrich). Briefly, PKH26 dye was diluted in 100 μL diluent C to a final concentration of 8 μM (dye solution). Then, 10⁷ particles of sEVs (NTA analysis) in 20 μL PBS were diluted with 80 μL diluent C, added to the dye solution, and incubated for 5 min while mixed with gentle pipetting. Excess dye was bound with 100 μL 10% BSA in PBS. Then the sEVs were diluted to 1 mL with DPBS and pelleted by ultracentrifugation at 120,000 × g for 1 h 10 min at 4 °C (Beckman Coulter). The pellet was gently resuspended in 50 μL PBS, and A549 and HUVECs (5 × 10⁴ cells/mL) were incubated with 10 μL of PKH26-labeled sEVs (2 × 10⁶ particles) at 37 °C with 5% CO₂. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed twice with PBS, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) were added, and the cells were incubated for 15 min at room temperature. Cellular uptake of cells-derived sEVs was observed under a confocal laser microscope.

The exosomes were isolated using serial differential centrifugations. Briefly, cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS, Biowest, Nuaillé, France) and a serum-free medium was added. After 48 hours the conditioned medium was collected and centrifuged for 10 minutes at 800 x g and then for 20 minutes at 3000 x g at 4 °C to eliminate larger vesicles and cellular debris. The supernatant was collected and stored at -80 °C until further use. After thawing, approximately 500 ml conditioned medium was filtered through a 0.2 μm membrane and concentrated using 100 kDa cut-off centrifugal filters (Merck KGa, Darmstadt, Germany). The concentrated conditioned medium was diluted 1:1 with DPBS and centrifuged at 120,000 x g for 90 minutes at 4 °C (Ti 70.1 rotor, ultracentrifuge Beckman coulter L7-65 both provided by Beckman Coulter, Munich, Germany). The obtained exosomal pellet was resuspended in DPBS or RIPA buffer according to further analysis. The concentration of exosomes was determined using Pierce™ BCA Protein Assay Kit (Thermofisher, San Jose, CA, USA) according to the manufacturer's instructions.