The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular �labile iron pool� (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at <pH 5. In the present study we show that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron.
Keywords
autophagy,
calcein,
iron,
labile iron pool,
lysosomes,
oxidative stress