Cell Proliferation Assay with EdU Staining

After 22 h ZnSO₄, ZnO NPs, CuO NPs and SiO₂ NPs treatment, a Click-iT 5-ethynyl-2′-deoxyuridine (EdU) kit (Molecular Probes, Carlsbad, CA, USA) was used to measure cell proliferation according to the manufacturer's procedures [124 (link)]. Cells were labeled with EdU (10 μmol/L) for 2 h. After fixation (4% paraformaldehyde, 60 minutes) and transparency (0.5% Triton X-l00, 30 minutes) treatment, the cells were incubated with Click-iT^® reaction cocktail, followed by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. After rinsing three times, cells were observed under an inverted fluorescence microscope with three random fields of view. The stained sections were visualized with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc., Melville, NY), and the captured fluorescent images were analyzed using MetaMorph software [118 (link)].