Internalization by confocal microscope

For visualized internalization assay by microscopy, the ADCs (n501-SN38 and m603-SN38) were labeled DyLight 650 NHS ester (ThermoFisher) according to the product manual. BxPC-3 cells were seeded on coverslips at a density of 5 × 10⁴ cells/mL for overnight. Cells were incubated with 50 nM ADCs-DyLight 650 at 4°C for 1 h. The culture media was removed, and cells were washed twice with PBS and then continued to culture at 37°C for various times. After washing with PBS containing 0.2% FBS (PBS-FBS), cells were fixed in formalin (4% p-formaldehyde in PBS) for 30 min and incubated with 5 μg/mL DAPI for 30 min. Coverslips were mounted and cells were visualized using a Leica confocal microscope and processed with LAS AF Lite software.