Phalloidin ifluor 680 conjugate

To investigate the co-localization of MVs and mitochondria, MVs labeled with the ExoSparkler Exosome Membrane Labeling Kit-Green (Dojindo) were added to iCECs seeded on 4-well chamber slides. The cells were incubated at 37°C for 4 hours. After 3 PBS washes, the cells were incubated with 500 nmol/L MitoTracke Red CMXRos at 37°C for 15 minutes and washed 3 times with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.1% Triton-X in 1× PBS for 20 minutes at room temperature. Subsequently, the cytoskeleton was fluorescently labeled using the Phalloidin-iFluor 680 conjugate (AAT Bioquest, Sunnyvale, CA), following the manufacturer’s protocol, and the nuclei were stained with DAPI. Images were captured using a spinSR10 confocal microscope imaging system (Evident, Japan).