2.4.2. HCS Assay: Incubation of Fluorescent Staining and Imaging

The Mitochondrial Health Kit (Invitrogen, Thermo Fisher, Houthalen, Belgium) was used to stain the nuclei (Hoechst 33342), the cell membrane (Image-iT^® DEAD Green™ viability stain) and the mitochondrial membrane (MitoHealth stain). Briefly, cells were exposed to NPs at the concentrations of 10, 20, 40 or 80 µg/mL and compared to negative and positive controls. After staining and cell fixation according to the manufacturer’s instructions, the IN Cell analyzer 2200 (GE Healthcare, Boston, MA, USA) was used to acquire images in high-throughput mode. Nine fields per well were imaged with a 10× objective, and an average of 2000 objects/field was counted for the analysis.