2.5. Intracellular iron measurement

The cellular iron uptake was validated by the quantification of the total iron content of cells using atomic absorption flame emission spectrophotometry (AAS). Cells were treated with 400 µmol/L FAC for 24 hours in the 6‐well plates (10 × 10⁵ cells/2 mL culture medium). Cells were then washed twice with PBS, harvested and counted. 30 × 10⁵ cells were suspended in 65% HNO₃ (1 mL) for 24 hours at room temperature. Finally, lysed cells were analyzed by AA‐6200 Atomic Absorption Spectrophotometer (Shimadzu Scientific Instruments, Inc). The concentration of iron was calculated per 10⁶ cells.