To assess the uptake of plasma sEV into RASM, they were treated with WsEV or SsEV, which
were labeled with PKH67 green fluorescence dye (Sigma Aldrich). WsEV or SsEV were reacted
with PKH67 (4 µM, RT, 3 min), which was stopped with the same volume of
sterilized PBS containing 10% bovine serum albumin (Nacalai Tesque). To wash the excess
dye, the sEV-PKH67 solution was taken into a tube containing a sterilized 0.971 M sucrose
solution in the bottom and ultracentrifuged (164,071 × g, 4°C, 35 min). The supernatants
were removed by pipetting and the pellets were resuspended in sterilized PBS. RASM were
treated for 2 hr with PKH67-labeled sEV (1.0 × 108particles/ml) or PKH67-reacted PBS (Cont) and fixed with 4%
paraformaldehyde (FUJIFILM Wako Pure Chemical) (4°C, 10 min). Nuclei were stained with 4’,
6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories, Kumamoto, Japan) (RT, 10 min) and
photographed by using a fluorescence microscope (BX-51) with a digital camera (DP74) and
CellSens standard dimension ver. 1.18 software (Olympus). Fluorescence density in a single
cell was measured using an ImageJ software (National Institutes of Health, Bethesda, MD,
USA).
were labeled with PKH67 green fluorescence dye (Sigma Aldrich). WsEV or SsEV were reacted
with PKH67 (4 µM, RT, 3 min), which was stopped with the same volume of
sterilized PBS containing 10% bovine serum albumin (Nacalai Tesque). To wash the excess
dye, the sEV-PKH67 solution was taken into a tube containing a sterilized 0.971 M sucrose
solution in the bottom and ultracentrifuged (164,071 × g, 4°C, 35 min). The supernatants
were removed by pipetting and the pellets were resuspended in sterilized PBS. RASM were
treated for 2 hr with PKH67-labeled sEV (1.0 × 108particles/ml) or PKH67-reacted PBS (Cont) and fixed with 4%
paraformaldehyde (FUJIFILM Wako Pure Chemical) (4°C, 10 min). Nuclei were stained with 4’,
6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories, Kumamoto, Japan) (RT, 10 min) and
photographed by using a fluorescence microscope (BX-51) with a digital camera (DP74) and
CellSens standard dimension ver. 1.18 software (Olympus). Fluorescence density in a single
cell was measured using an ImageJ software (National Institutes of Health, Bethesda, MD,
USA).