Calcein acetoxymethyl ester ca am

Calcein acetoxymethyl ester (CA-AM) was obtained from Molecular Probes. The iron chelator, isonicotinoyl salicylaldehyde hydrazone (SIH) (a gift from Dr. P. Ponka, Lady Davis Institute for Medical Research, Montreal, Canada) was prepared as a 50 mM stock solution in dimethyl sulfoxide (DMSO). Briefly, 25,000 – 50,000 cells were cultured in 96-well plates (Black with Clear Bottom purchased from Coring) overnight. Cells were loaded with 2 μM CA-AM for 15 to 30 minutes at 37°C, and then washed with PBS. 100 μM starch-conjugated desferrioxamine (DFO; a generous gift of Biomedical Frontiers, Inc., Minneapolis, MN) was added to cells to remove extracellular iron. Fluorescence was measured at 485 nm excitation and 535 nm emission with a fluorescence plate reader (BioTek Synergy 2). After the fluorescence signal was stabilized, SIH was added at a final concentration of 10 μM to remove iron from calcein, causing dequenching. The change in fluorescence following the addition of SIH (ΔF) was used as an indirect measure of the labile iron pool.