The PKH67 green fluorescence kit (Sigma-Aldrich) was used to label the purified exosomes. The exosomes were suspended in 1 mL Diluent C solution, and 4 μL PKH-67 ethanol dye solution was added to 1 mL Diluent C to prepare 4 × 10−6 M dye solution. The staining was terminated with FBS or 1% BSA for 1 min. The labeled exosomes were ultracentrifuged for 70 min at 100,000 × g, washed with PBS, ultracentrifuged again, and resuspended in 50 μL PBS. The PKH67-labeled exosomes were incubated with CRC cells at 37°C for 12 h. The cells were then fixed with 4% paraformaldehyde and the nuclei were stained with the DAPI. Finally, the uptake of labeled exosomes by CRC cells was determined using a microscope.
To identify the transfer of exosomal miR-590-3p, cy3-labeled miR-590-3p was transfected to CAFs, which were then co-cultured with CRC cells for 48 h in a 24-well Transwell chamber. Immunofluorescent cells were prepared according to the methods described above. In addition, the cytoskeleton of CRC cells was stained selectively using fluorescein isothiocyanate (FITC) phalloidin (Yeasen, Shanghai, P.R. China). The internalization of exosomal miR-590-3p was measured by a laser scanning confocal microscope (LSCM).46 (link)
To identify the transfer of exosomal miR-590-3p, cy3-labeled miR-590-3p was transfected to CAFs, which were then co-cultured with CRC cells for 48 h in a 24-well Transwell chamber. Immunofluorescent cells were prepared according to the methods described above. In addition, the cytoskeleton of CRC cells was stained selectively using fluorescein isothiocyanate (FITC) phalloidin (Yeasen, Shanghai, P.R. China). The internalization of exosomal miR-590-3p was measured by a laser scanning confocal microscope (LSCM).46 (link)