Background

Owing to their central regulatory functions for systemic iron metabolism, macrophages in multiple tissues swiftly alter their intracellular iron levels upon encounter of diverse stimuli during microbial infections and diseases (Nairz et al., 2017). Of note, the majority of cellular iron ions is bound by the major iron storage protein Ferritin within the cytoplasm or is localized to compartments like mitochondria or lysosomes (Ma et al., 2015; Soares and Hamza, 2016). However, the labile iron pool represents an unbound fraction of cytoplasmic iron, which is metabolically accessible and pivotal for cellular iron homeostasis and metabolism (Cabantchik, 2014). Interestingly, subtle concentration changes within this macrophage iron pool can greatly impact the outcome of infections, especially with intracellular pathogens (Ganz and Nemeth, 2015).

However, these intracellular iron fluxes in the nanomolar to millimolar range are highly dynamic and are additionally regulated in a spatiotemporal manner, providing a significant challenge for accurate experimental quantification (Cabantchik, 2014; Ma et al., 2015). Methods for iron import/export studies, using radioactive ⁵⁹Fe isotopes, are highly sensitive and specific, but are quite limited in their application for the broad interested scientific community owing to safety issues and governmental regulations. Further, quantification of chelated iron by spectroscopy is hampered by either physical cell disruption, low sensitivity or the large amounts of required biological materials.

To address these experimental challenges, Epsztejn et al. described a Calcein-AM-based method to analyze the labile iron pool (Epsztejn et al., 1997). When Calcein-AM is taken up by metabolically active cells, this acetoxy-methyl-ester is cleaved by intracellular esterases, releasing the iron-specific fluorescent dye Calcein exclusively into the cytoplasm (Ma et al., 2015). Thereby, Calcein fluorescence is quenched upon binding to cytoplasmic iron ions (Figure 1). Further, this method was refined for analysis by flow cytometry (Prus and Fibach, 2008). In general, these protocols rely on the usage of strong iron chelators to sequester cytoplasmic iron ions for subsequent Calcein fluorescence rescue. However, some iron chelators are commercially unavailable (e.g., salicylaldehyde isonicotinoyl hydrazone = SIH) or can have diminished chelating effects under specific experimental conditions.


Figure 1. Quenching of cellular Calcein fluorescence upon binding of iron ions. Calcein fluoresces in the unbound state but gets quenched during binding by accessible iron ions within the cytoplasm. Addition of exogenous iron upon treatment with FeCl₂/8-hydroxyquinoline (FeHQ) complexes further quenches Calcein fluorescence.

Thus, in order to increase practical applicability, this Calcein-AM-based assay was modified by analyzing the quenchable iron pool (QIP) instead (Du et al., 2015; Siegert et al., 2015). Since Calcein shows reduced fluorescence in the iron-bound state, its fluorescence gets additionally quenched when exogenous iron is swiftly delivered into the cytoplasm by the addition of a FeCl₂/8-hydroxyquinoline (FeHQ) complex (Figure 1) (Prachayasittikul et al., 2013; Ma et al., 2015; Chobot et al., 2018). Hence, the difference in cellular Calcein fluorescence before and after FeHQ addition represents the QIP (Figure 2A). Of note, the size of the unbound, cytoplasmic iron pool inversely correlates with the ability of a given cell to take up iron ions. Thus, iron-starved cells can substantially take up more exogenous Fe (upon FeHQ addition) than iron-saturated cells (Figure 2B). Therefore, cells with minor cytoplasmic iron pools exhibit higher QIPs when compared to iron-loaded cells with reduced QIPs (Figure 2A).

In conclusion, QIP analysis by flow cytometry represents a powerful tool to accurately determine alterations in cytoplasmic iron levels upon diverse cellular stimuli. Further, this protocol can be established easily in almost every laboratory environment and can also be applied for the investigation of iron homeostasis in other immune cell types in vitro and in vivo.


Figure 2. Theoretical concept of QIP analysis. A. Schematic representation of QIP calculation. Primary bone marrow-derived macrophages (BMDMs) were left untreated or treated with 50 μM FeSO₄ for 17 h and the MFI (Calcein) was determined in naive and FeHQ-challenged BMDMs. The QIP at the respective condition is calculated as the MFI difference. B. The cytoplasmic levels of unbound iron inversely correlate with the cellular iron uptake ability. Thus, cells with low cytoplasmic iron levels can take up more exogenous iron (upon FeHQ treatment), than already iron-saturated cells. MFI, median fluorescence intensity.