4.7. Labile Iron Pool Assay and Calcein-AM Assay

The cellular LIP was measured as described elsewhere, with minor modification [37]. Briefly, HA22T/VGH cells (3 × 10⁴) were seeded on 96-well plates and treated with 100 µM DFO, DFP or didox for 4 h. The cells were incubated with 0.25 µM calcein-AM in MEM with 1 mg/mL BSA for 30 min at 37 °C. After washing with 1X phosphate-buffered saline (PBS), 100 μL of 1X Hank’s Balanced Salt Solution (HBSS) was added to the cells and the fluorescence was monitored at an excitation of 488 nm and an emission of 517 nm using an EnSight Multimode plate reader (Perkin Elmer). Cells were then fixed in 4% PFA, stained with crystal violet solution (0.1% crystal violet, 20% methanol) for 15 min. After washings, 100 μL of 10% acetic acid was added and absorbance was detected at 540 nm using a Multiskan^©EX plate reader (Thermo Scientific, Waltham, MA, USA). The data were expressed as fold change over the not treated cells (ratio of fluorescence of calcein-AM/absorbance at 540 of crystal violet). The quenching of calcein-AM is inversely proportional to the concentration of intracellular iron.