Quantifying Nintedanib Internalization in Cells

Internalization of nintedanib was determined by exploiting the fluorescent emission of nintedanib by means of a cytofluorimetric assay [35 (link)]. Briefly, A549 cells were seeded in standard medium (2 × 10⁶ cells/100 mm Petri dishes, Sarstedt), and after adhesion, cells were grown in the presence or in the absence of TGFβ (10 ng/mL) for 48 h. Next cells were exposed to nintedanib or conjugated compounds at different concentrations. After 24 h, cells were gently detached using Accutase (ECB3056D, Euroclone, Pero, Milano, Italy) or trypsin and the fluorescence, associated with different cell populations, was evaluated using the BV480 channel of the FACSCanto BD instrument (using 405 nm laser). Untreated cells were used as the negative control. The same procedure was applied to evaluate the internalization in K562, SSM2c, and L929 cells.