Calcein acetoxymethyl ester ca am calcein am

The intracellular labile iron was quantified by monitoring the recovering of calcein fluorescence induced by iron chelators after the calcein fluorescence was quenched by intracellular labile iron^{57 (link)}. Briefly, ARPE-19 cells in a 96-well plate were loaded with 0.25 μM calcein acetoxymethyl ester (CA-AM; calcein-AM) (Sigma-Aldrich, Missouri, USA) for 30 min at 37 °C. After three times washing with KRPH buffer (Hyclone, Utah, USA), 200 μM membrane-permeable iron chelator PIH (APExBIO, Houston, USA) was added. Fluorescence was recorded at an excitation wavelength of 485 nm and an emission wavelength of 530 nm with a Varioskan Flash multimode reader (Thermo Fisher Scientific, USA) before (baseline) and 30 min after PIH addition. Cells were imaged using a fluorescence microscope (Leica DMi 8, Leica Microsystems, Germany) and counted using the Image J Cell Counter plugin. The increase in fluorescence intensity (ΔF) was calculated and normalized to the cell number.

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.