Exosome Uptake Quantification Protocol

For the uptake of labelled exosomes, the recipient cells were seeded in a 24-well plate (3 × 10⁴ cells/well) for 24 h at 37 °C, with 5% CO₂. For FTS treatment, SW480 cells were treated with 50 μM FTS at 3 h after seeding. PKH26-labelled exosomes were added for 24 h of incubation at 37 °C, with 5% CO₂. The cells were harvested and washed twice with PBS before the analysis using imaging flow cytometry (ImageStream X Mark II, Merck Millipore, Jaffrey, NH, USA). A minimum of 10,000 cells was analyzed for each sample and the images were captured at 40× magnification. PKH26 fluorescence were excited at the wavelength of 488 nm. The fluorescence images were collected on channel 3 (560 nm to 595 nm) while bright field images were collected on channel 4. The data collected were then analyzed using the IDEAS software (Amnis Corporation, Washington, DC, USA).