Cell Labeling and Prussian Blue Staining

Prussian blue staining assay was used to determine the location of Mn-IONPs within cells and the rate of uptake. Approximately 1×10⁴ cells per well were seeded overnight on tissue-culture treated glass coverslips (Solarbio, Beijing, China) in 24-well plates. Cells were then challenged with Mn-IONPs suspensions (Fe concentrations of 45, 90, 180 and 360 μM) for 24 hours. To ensure the accuracy of the labeling rate, the treated cells were washed three times with PBS to remove the free Mn-IONPs and the weakly attached NPs on the cytomembranes. The labeled cells were then fixed with 4% paraformaldehyde for 15 mins, incubated with Prussian blue staining solution (containing equal volumes of 2% hydrochloric acid and 2% potassium ferrocyanide) for 30 mins, and subsequently in 1% eosin solution for 15 mins in sequence. The labeling rate was acquired by calculating the percentage of prussian blue stained cells in total cells. The images were obtained using an inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany).