2.6. Prussian blue staining

Prussian blue can stain iron atoms in cells. First, 143B cells were seeded in 12-well plates at 1 × 10⁵ cells/well. After the cells became adherent, SPCIONPs/pIGFBP₅ were added, and a magnetic field was applied for 30 min. After 4 h, the cells were washed three times with phosphate-buffered saline (PBS) and fixed in paraformaldehyde for 20 min. Perls solution was prepared at a 1 : 1 ratio, and 200 µl of the solution was added to each well, followed by incubation at 37°C for 40 min. Cells were washed with ddH₂O, and 200 µl of neutral red solution was added and incubated with the cells at room temperature for 2 min. After the staining solution was rinsed with ddH₂O, the cells were observed under a light microscope.