3 hydroxy 1 2 dimethyl 4 1h pyridone deferiprone or l1

HEY and MCF7 cells were seeded in 6-well plates at a density of 5.0 × 10⁵ cells/well and grown overnight, and treated at the concentration indicated for 24 h. Cells were loaded with 0.25 μM calcein acetoxymethyl ester (CA-AM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C, then washed with PBS1X and treated or not with 200 mM 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone or L1) (Sigma-Aldrich, St. Louis, MO, USA). Following staining, and washing with PBS, cells were analyzed using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The ∆ mean fluorescence intensity (∆MFI) between chelator-treated and untreated cells reflected the amount of LIP [54 (link)].

The intracellular labile iron pool (LIP) was quantified by monitoring the recovering of calcein fluorescence induced by iron chelators after the calcein fluorescence was quenched by intracellular LIP. Briefly, cancer cells were seeded in 6-well plates at a density of 4 × 10⁵ cells/well and grown overnight. Then cells were loaded with 0.25 μM calcein acetoxymethyl ester (CA-AM; calcein-AM) (Sigma-Aldrich, Missouri, USA) for 30 min at 37°C. After washing twice with PBS, cells were treated with 200 μM 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone or L1) (Sigma-Aldrich, Missouri, USA) or left untreated. Following staining, and washing PBS, cells were analyzed by a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The difference in the mean fluorescence index between chelator-treated and untreated cells (Δ mean fluorescence intensity, ΔMFI) reflects the amount of LIP. Each experiment was performed in triplicate.