Measurement of the intracellular labile iron pool

RAW 264.7 cells were seeded into a 6-well plate at 2.5 × 10⁵ cells/well and cultured overnight to allow cells to adhere. The following day, cells were treated with 0.5 µM calcein-AM and cultured for 30 minutes at 37°C. The fluorescence of calcein-AM is quenched by intracellular iron.²² Cells were then washed with room temperature PBS and treated with complete medium alone or complete medium containing 200 µM DIBI or 1.28 mg/mL PVP. Cells that were not exposed to calcein-AM served as an additional control. After 4, 24, and 48 hours of culture cells were harvested and analyzed (1 × 10⁴ cell counts/sample) using the FL1 channel of a BD FACSCanto^TM flow cytometer (BD Biosciences, Mississauga, ON). Data were processed using FCS Express software (version 3.0, De Novo Software, Thornhill, ON).