MCF-7 breast adenocarcinoma cells and HCT-15 colon adenocarcinoma cells were cultured on a 96-well plate on glass coverslips in the amount of 20,000 cells per well and cultured for 24 h. Vesicles were obtained from MSCs and SNB-19 cells were preliminary stained with DiD vital dye (Vibrant Multicolor Cell-Labeling Kit, Invitrogen, Waltham, MA, USA) according to the previously described protocol.
Vesicles were added to the cells at a concentration of 2 µg per 100 µL (20 µg/mL) and cultured for 4 h. After washing three times for 5 min in PBS, cells were stained with DAPI fluorescent dye (4′,6-diamidino-2-phenylindole; dilution 1:50,000 in TBS; Invitrogen, Waltham, MA, USA) for 7 min, and washed again. Coverslips were mounted on the slides with a mounting medium (ImmunoHistoMount, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The samples were investigated under a LSM 780 confocal microscope (Carl Zeiss, Jena, Germany) using Zen black 2012 software (Carl Zeiss, Jena, Germany). All samples were imaged in the z-plane using identical confocal settings (laser intensity, gain, and offset).
Vesicles were added to the cells at a concentration of 2 µg per 100 µL (20 µg/mL) and cultured for 4 h. After washing three times for 5 min in PBS, cells were stained with DAPI fluorescent dye (4′,6-diamidino-2-phenylindole; dilution 1:50,000 in TBS; Invitrogen, Waltham, MA, USA) for 7 min, and washed again. Coverslips were mounted on the slides with a mounting medium (ImmunoHistoMount, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The samples were investigated under a LSM 780 confocal microscope (Carl Zeiss, Jena, Germany) using Zen black 2012 software (Carl Zeiss, Jena, Germany). All samples were imaged in the z-plane using identical confocal settings (laser intensity, gain, and offset).