Quantifying Intracellular Iron Levels

The level of intracellular iron was measured as previously described [42 (link)]. After treatment with NPs, cells were trypsinized, washed with phosphate-buffered saline (PBS), centrifuged and counted. The cell pellet was digested in 5 N HCl for 24 h at 37 °C and then centrifuged at 2000× g rpm for 10 min. A volume of 50 μL of supernatant was mixed with 50 μL 1% ammonium persulfate solution (to convert the Fe²⁺ to Fe³⁺ ions) and 100 μL of 0.5 M potassium thiocyanate and shaken for 5 min in order to develop the red colored iron-thiocyanate. The formed complexes were spectrophotometrically detected at 450 nm. A standard curve with FeCl₃•6H₂O ranging from 0–250 μg Fe³⁺/mL was used to quantify the intracellular iron. The total amount of iron was related to the cell number.