Experimental approaches used for online fluorescence assessment of labile iron chelation in cell compartments
Approach A: pretreatment by chelators . | Approach B: online assessment of chelation . | Approach B′: online assessment of chelation . |
|---|---|---|
| 1. Treat cells with chelator | 1. Load cells with CAL (± Fe) | 1′. Load cells with CDCF or DHR |
| 2. Analyze residual LIP: | 2. Analyze: add chelator and follow fluorescence recovery in cytosol and/or endosomes | 2′. Analyze: add H2O2 and chelator, and follow ROS formation by fluorescence in cytosol and/or in mitochondria |
| a. CAL-SIH cytosol | ||
| b. CDCF (ROS) cytosol | ||
| c. DHR (ROS) | ||
| mitochondria |
Approach A: pretreatment by chelators . | Approach B: online assessment of chelation . | Approach B′: online assessment of chelation . |
|---|---|---|
| 1. Treat cells with chelator | 1. Load cells with CAL (± Fe) | 1′. Load cells with CDCF or DHR |
| 2. Analyze residual LIP: | 2. Analyze: add chelator and follow fluorescence recovery in cytosol and/or endosomes | 2′. Analyze: add H2O2 and chelator, and follow ROS formation by fluorescence in cytosol and/or in mitochondria |
| a. CAL-SIH cytosol | ||
| b. CDCF (ROS) cytosol | ||
| c. DHR (ROS) | ||
| mitochondria |
In approach A, cells are treated first with chelators and subsequently assessed for residual LIP (a) in the cytosol by the calcein (CAL)–based method and/or by (b) the CDDHCF-DA-based method (Fe-dependent H2O2-prompted ROS formation) and (c) in mitochondria by the DHR-based method equivalent to that of CDCF.
In approach B, cells are first loaded with probes for LIP ([1] metallosensor or [1′] redox-reporter). In no. 1, one of the calcein (CAL) probes (supplemented or not with Fe), which is targeted to cytosol or endosomes (as CAL-Fe complexes), is used. The cell LIPs comprise resident forms of labile Fe, those induced by adding permeant sources of labile Fe, or those created in endosomes by endocytosis of CAL-Fe complexes. The various LIPs are revealed by chelating the iron complexed to CAL with excess permeant chelators that release the iron-free fluorescent probe in situ. In no. 2′, CDDHCF-DA is loaded into cytosol and DHR into mitochondria, and the labile (redox-active) Fe is revealed in the respective compartment by its involvement in H2O2-prompted ROS formation and susceptibility to added chelators.