Pkh26 red fluorescence kit

The extracted exosomes were labeled using a PKH26 red fluorescence kit (Sigma-Aldrich Chemical Company, St Louis, MO, USA) in accordance with the kit’s instructions. Next, the Exo were resuspended and incubated with the mouse cardiomyocytes at 37 °C for 24 h. After that, the cells were washed twice in PBS, fixed in 4% paraformaldehyde for 10 min, and stained with 4', 6-diamidino-2-phenylindole. The PKH26-labeled exosomes internalized by cardiomyocytes were observed under a confocal laser scanning microscope (Leica, Solms, Germany).

Purified BMSCs-EVs were labeled with PKH26 red fluorescence kit (Sigma-Aldrich). Next, the EVs were resuspended in 1 mL diluent C solution, and 4 μL of PKH26 ethanol staining solution was added to prepare a 4 × 10⁻⁶ M dye solution. A total of 1 mL of the EV suspension was mixed with dye solution for 5 min, and 2 mL of 1% EV-free FBS added for incubation for 1 min to terminate staining. The labeled EVs were ultracentrifuged at 1,000 r/min for 2 h, and the EVs in the samples were enriched in sucrose at a density of 1.13-1.19 g/mL, followed by the collection of the EVs. PKH26-labeled EVs were cocultured with chondrocytes at 37°C for 12 h. Next, the cells were fixed with 4% paraformaldehyde, and the nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI, D9542, Sigma-Aldrich). Finally, the uptake of EVs by chondrocytes was observed under a fluorescence microscope (ECLIPSE E800, Nikon, Tokyo, Japan).
To explore the transfer of NEAT1, FITC-NEAT1 (green) was transfected into BMSCs utilizing Lipofectamine 2000 reagent, and BMSC-EVs were then collected as the previous method and added with Dil tag (red). Afterward, the BMSC-EVs were cocultured with chondrocytes for 48 h, and the uptake of BMSC-EVs carrying FITC-NEAT1 by chondrocytes was observed under a fluorescence microscope (ECLIPSE E800, Nikon, Japan).