Exosomes were labeled with the green fluorescent dye PKH-67 using the PKH67 Green Fluorescent Cell Linker kit for General Cell Membrane Labeling (Sigma-Aldrich; Merck KGaA) as previously described (28 (link)). Briefly, 6 µg 0 and 5 Gy-Exo were labeled with 2 µM PKH-67 for 5 min at 25°C. Then, free PKH-67 was removed by centrifugation 14,000 × g for 2 min at 25°C using the VIVACON 500 ultracentrifugation device (100,000 MWCO; Sartorius Stedim Biotech; Sartorius AG). MIAPaCa-2 cells were cultured for 24 h at 37°C, after which the culture media was replaced with that containing the labeled exosomes. Following incubation at 37°C overnight, cells were gently washed twice with PBS and fixed by 4% paraformaldehyde solution (Nacalai Tesque) for 20 min at 25°C. After washing with Hanks' Balanced Salt Solution (Gibco; Thermo Fisher Scientific, Inc.), cells were incubated with 10 µg/ml WGA, Alexa Fluor 594 conjugate (Invitrogen, Thermo Fisher Scientific, Inc.) for 10 min at 25°C. Finally, samples were incubated with Hoechst 33342 (1:2,000; Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min at 37°C.
Images were captured using a confocal microscope (LSM700; Carl Zeiss AG) equipped with an oil immersion objective lens (magnification, ×40). Images were analyzed with ZEN 2012 (Carl Zeiss AG) and processed using ImageJ software ver.1.51 (National Institutes of Health) (29 ).
Images were captured using a confocal microscope (LSM700; Carl Zeiss AG) equipped with an oil immersion objective lens (magnification, ×40). Images were analyzed with ZEN 2012 (Carl Zeiss AG) and processed using ImageJ software ver.1.51 (National Institutes of Health) (29 ).