Arvo x light luminometer

Direct addition system: Host cells were seeded in white-walled 96-well plates at a density of 2.5 × 10⁴ cells/well, and culture for 24 hours. Exosomes isolated from HT29/CD63Nluc and HCT116/CD63Nluc were added into each well, and the cells were incubated for 2 hours. After washing twice with PBS, they were lysed with Nano-Glo substrate diluted in provided buffer, and then luminescence was measured using ARVO X Light luminometer (PerkinElmer).
Co-culture system: Cellular uptake assay in co-culture condition was performed using a 24 multiwell insert system with 1.0 μm-pore PET membrane (Corning, Corning, NY, USA). As recipient cells, MEFs, A431, A549, HT29 and HCT116 cells (1 × 10⁵ cells in 1 mL medium) were seeded into the bottom chamber and engineered-donor HT29/CD63Nluc or HCT116/CD63Nluc cells (2 × 10⁴ cells in 600 μL medium) were seeded in the upper chamber. After 48 hours culture, recipient cells were washed with PBS three times, and then were lysed with Nano-Glo substrate diluted in provided buffer. The lysate was transferred to white-walled 96-well plate, and luminescence was measured using ARVO X Light luminometer (PerkinElmer).

To remove the cells and cellular debris, collected culture mediums were centrifuged at 2,000 g for 10 min at 4 °C, and transferred 50 μL supernatants into white-walled 96-well plates. 50 μL Nano-Glo substrate diluted 1:50 in provided buffer (Nano-Glo Luciferase Assay System: Promega) was added, and the luciferase intensity in each well was immediately measured using an ARVO X Light luminometer (PerkinElmer, Waltham, MA, USA).