2.4. EV uptake
To examine whether the BMSCs internalize EVs, the BMSCs were seeded on chamber slides (Millipore, Billerica, MA) at a density of 20,000 cells/cm2 and cultured overnight. All six EV groups were labeled with a PKH67 Green Fluorescent Cell Linker Kit. PKH67-labeled EVs were diluted in the culture medium and added to the BMSCs in the culture at a concentration of 10 µg/mL media based on reported therapeutic doses of EVs [33]. After 48 h, the cells were washed with PBS to remove non-internalized EVs, and then BMSCs were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific), which is blue fluorescent nuclei stain. Cells were then fixed with 2% formaldehyde for 15 min and washed again. The chamber slides were then mounted with Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, CA) and visualized using confocal microscopy (Leica TCS LSI Macro Confocal).