In the case of time-dependency studies, PANC-1 cells were seeded in 6-well plates (Corning B.V., Netherlands) at a density of 300,000 cells per well overnight. The three types of labelled exosomes were then added at a dose of 2.0 × 1010 particles per well and incubated with the cells for 1, 4, 12, and 24 h at 37°C. Cells were then washed with warm PBS, fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) in PBS for 15 min at RT, and rinsed with PBS for three times. The cells were resuspended in 100 µL of PBS. Cell images (≥2,000 cells; bright field, dark field and fluorescence images) were acquired with an Amnis ImageStreamX MARK II (ISX) flow cytometer (Merck Millipore, Seattle, WA); A 488 nm wavelength excitation laser, at a power of 100 mW was used for excitation of the FITC fluorophore. Untreated cells were imaged as control. All image analysis was done using the Ideas software environment (Merck Millipore, Seattle, WA).
For dose-dependency studies, PANC-1 cells were seeded in Costar® 24-well flat-bottom plates at a density of 30,000 cells per well one day in advance. Doses of 4 × 109, 1.2 × 1010, 2.0 × 1010, 2.8 × 1010, 3.6 × 1010 labelled exosomes derived from B16-F10, PANC-1 and HEK-293 cells were added to cultured PANC-1 cells and incubated for 24 h. The experiments were performed in triplicate. Cells were collected after incubation and analysed by flow cytometry.
For dose-dependency studies, PANC-1 cells were seeded in Costar® 24-well flat-bottom plates at a density of 30,000 cells per well one day in advance. Doses of 4 × 109, 1.2 × 1010, 2.0 × 1010, 2.8 × 1010, 3.6 × 1010 labelled exosomes derived from B16-F10, PANC-1 and HEK-293 cells were added to cultured PANC-1 cells and incubated for 24 h. The experiments were performed in triplicate. Cells were collected after incubation and analysed by flow cytometry.