Uptake of exosomes

Uptake of exosomes was observed by fluorescence. First, CD34⁺-Exos and miR-26a-CD34⁺-Exos were labeled with PKH26 red dye (Sigma Aldrich, St Louis, MO, USA) according to the manufacturer’s protocol. Then, exosomes were ultracentrifuged at 110,000×g for 70 min and the supernatant was discarded. The pellet was resuspended in 500 μL dilution C, and 4 μL PKH26 dye was dissolved in another 500 μL dilution C. The two tubes of dilution C were mixed and co-incubated for 5 min at room temperature to obtain exosomes-PKH26. The exosomes-PKH26 were then co-cultured with BMSCs and HUVECs at a concentration of 20 μg/mL. After 12 h, cells were fixed with 4% paraformaldehyde (Well, Shanghai, China) for 15 min, treated with 0.1% Triton X-100 (Beyotime) for 5 min for membrane penetration, stained with DAPI for 5 min, and washed with PBS three times. Photographic images were acquired using a fluorescence microscope (Olympus IX 70).