Measurement of intracellular iron

Quantitation of intracellular iron concentration in dermal fibroblasts was performed as described by Reimer et al. [44]. Briefly, cells were lysed using 50 mM NaOH for 2 h, neutralized by addition of equal volume of 10 mM HCl and sonicated. Protein concentration was determined using the DC protein assay, and lysates were then treated with freshly prepared acidic KMnO₄ solution (1.4M HCl and 4.5% (w/v) KMnO₄ in H₂O) at 60°C for 2 h with shaking. The total iron content of the lysates was determined by the addition of an iron detection reagent (6.5 mM ferrozine, 1 M ascorbic acid, 2.5 M ammonium acetate) followed by measurement of absorbance at 550 nm. To measure the expression of ferritin in fibroblasts, a rabbit polyclonal anti-ferritin heavy chain antibody (Santa-Cruz Biotechnology, sc-25617) was used.