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).