2.5. Iron Staining and Microscopy (Prussian Blue)

To qualitatively validate the cellular uptake and localization of MNPs in cells as well as their behavior in the respective culture media by light microscopy, Prussian blue staining of iron was implemented. For this, 1 × 10⁴ cells were seeded in eight-well chamber slides (BD, Franklin Lakes, NJ, USA) and grown overnight under standard culture conditions. Thereafter, MNPs (100 µg Fe/mL) were added and the cells were further incubated for 24 h at 37 °C. Then, the cells were washed three times to get rid of free MNPs and fixed with 3.7% formaldehyde (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) in phosphate-buffered saline (PBS). For staining, the slides were incubated in a solution consisting of equal volumes of 20% (v/v) HCl (Carl Roth GmbH & Co.) and 10% (w/v) potassium hexacyanoferrate (II) trihydrate (Sigma-Aldrich GmbH, Steinheim, Germany) for 30 min, and then rinsed with distilled water. The nuclei of the cells were subsequently stained with nuclear fast red aluminium sulphate solution (Carl Roth GmbH & Co.). The slides were separated from the chambers and the cells were mounted with Pertex (MEDITE GmbH, Burgdorf, Germany) and coverslipped for microscopy. Images were captured on an EVOS xl AMG microscope (PEQLAB, Erlangen, Germany) at 60× magnification.