For the quantification of intracellular labile iron concentration, we used a modified Calcein-AM staining protocol as described previously in which intracellular labile iron concentration is determined by the difference between Calcein-AM fluorescence values of a sample incubated in the presence and absence of an iron chelating agent 20 (link). Briefly, samples of BMDM lysate (lysed for 10 minutes in PBS containing 0.05% of saponin) were incubated for 15 minutes at room temperature with 250μM of deferoxamine mesylate (DFO) or PBS, followed by staining with Calcein-AM (ThermoFisher Scientific, Waltham, MA) at 125nM for 30 minutes at 37°C, in opaque 96 well plates (Corning, Corning, NY). Relative fluorescence units (RFU) were measured and labile iron levels are represented as ΔRFU (DFO-treated sample RFU – PBS treated sample RFU). Perls staining followed by DAB enhancement was performed by Histoserv, Inc. (Germantown, MD, USA) in histological sections from Mtb-infected mouse lungs previously fixed in PBS buffer containing 10% formalin, as described elsewhere 58 (link). Images were acquired in an Aperio Digital Pathology Slide Scanner (Leica, Buffalo Grove, IL, USA) and analyzed using ImageScope (Leica, Buffalo Grove, IL, USA) and ImageJ 59 (link) softwares.