*2.2. Intracellular Fe Concentration Determination*

A colorimetric test based on ferrozine was used to assess the quantity of intracellular ionic Fe accumulated under different circumstances, as described before [29]. Briefly, suspensions containing 10<sup>8</sup> parasites were obtained from various cultures and washed three times with PBS pretreated with 5 g/100 mL Chelex resin (Sigma-Aldrich). The cells were lysed with 100 μL of 50 mM NaOH, and then 100 μL of 10 mM HCl was added; the release of ionic Fe bound to intracellular structures was induced by adding 100 μL of a mixture containing 1.4 M HCl and 4.5% (*w*/*v*) KMnO4 (1:1) to the cell lysate, followed by incubation at 60 ◦C for 2 h. Then, 30 μL Fe detection reagent was added (6.5 mM ferrozine, 6.5 mM neocuproin, 2.5 M ammonium acetate, and 1 M ascorbic acid). The sample's absorbance at 550 nm was measured after 30 min of incubation at room temperature. A standard curve with known FeCl3 concentrations (0–75 μM; [36]) (Merck, Darmstadt, Germany) was used to calculate the Fe content.
