After tracheal intubation of the mice, the lungs were gently lavaged thrice with 1 mL of ice cold, sterile phosphate-buffered saline (PBS). The fluids collected from mice were maintained at −80°C and immediately processed for exosome isolation.
Exosome isolation was performed through differential ultracentrifugation as previously described (28 (link), 29 (link)). The concentration of protein in exosome pellets was measured using the bicinchoninic acid assay (Beyotime, Shanghai, China). For the in vitro uptake experiment, exosomes were labeled using a PKH67 green fluorescent cell linker mini kit (Sigma Aldrich, St. Louis, MO, USA) according to the instructions provided by the manufacturer. Ten random fields were counted. For the in vivo uptake study, the labeling procedure was identical to that used in the in vitro analysis. The labeled exosomes were delivered into the mice, and 18 h later the lungs were removed and embedded in OCT compound, and stored at −80°C. Photographs were captured using an immunofluorescence microscope (Nikon, Tokyo, Japan).
Exosome isolation was performed through differential ultracentrifugation as previously described (28 (link), 29 (link)). The concentration of protein in exosome pellets was measured using the bicinchoninic acid assay (Beyotime, Shanghai, China). For the in vitro uptake experiment, exosomes were labeled using a PKH67 green fluorescent cell linker mini kit (Sigma Aldrich, St. Louis, MO, USA) according to the instructions provided by the manufacturer. Ten random fields were counted. For the in vivo uptake study, the labeling procedure was identical to that used in the in vitro analysis. The labeled exosomes were delivered into the mice, and 18 h later the lungs were removed and embedded in OCT compound, and stored at −80°C. Photographs were captured using an immunofluorescence microscope (Nikon, Tokyo, Japan).
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