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Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium ( S. tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl 3 ) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe 2+ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe 2+ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress. Key features • Easy quantification of Fe 2+ in cultured macrophages with a fluorescent probe. • Analysis of iron in living cells without the need for fixation. • Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria., Competing Interests: Competing interestsThe authors declare no conflicts of interest., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Macrophages are at the center of innate immunity and are the main target cells of the intracellular pathogen Salmonella enterica serovar Typhi. The production of reactive oxygen and nitrogen species (ROS/RNS) is the host's early response to invading microbes, as oxidative stress is highly toxic for bacteria. Adequate ROS/RNS production in infected macrophages is critical for the clearance of intracellular pathogens; this is achieved by several enzymes, including inducible NADPH phagocyte oxidase (NOX) and nitric oxide synthase (iNOS), respectively. The pro-inflammatory cytokine interferon gamma (IFNγ), primarily produced by activated natural killer cells and T-helper cells type 1, is a potent inducer of iNOS. Therefore, it is crucial for infection control through oxidative microbicidal activity. To characterize the early oxidative stress response via ROS formation, which is critical for the reduction of Salmonella proliferation within macrophages, we established an in vitro model of murine macrophages infected with Salmonella enterica serovar Typhimurium ( S .tm). This serovar induces a systemic infection in mice that is frequently used as a model for typhoid fever, which, in human subjects, is caused by Salmonella Typhi. We generated bone marrow-derived macrophages (BMDM) from C57BL/6N wildtype mice using macrophage colony-stimulating factor (M-CSF) stimulation for six days. Thereafter, we infected BMDM with S. tm for one hour. Shortly before infection, cells were stained with CellROX TM Deep Red reagent. In its reduced form, CellROX TM is non-fluorescent. As a result of oxidation by ROS, this reagent exhibits strong fluorescence and persists within the cells. Subsequently, changes as a result of the oxidative stress response can be measured with a TECAN Spark microplate reader over time. We designed this protocol to measure oxidative stress in macrophages through the course of an infection with an intracellular bacterium. The protocol has several advantages over established techniques. First, it allows to continuously monitor and quantify ROS production in living cells from the very start of the infection to the final clearance of the intracellular pathogen. Second, this protocol enables efficient ROS detection without stressing the cells by detaching or staining procedures. Graphical abstract., Competing Interests: Competing interests The authors declare no conflicts of interest., (Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC.)

Macrophages are important for host defense against intracellular pathogens like Salmonella and can be differentiated into two major subtypes. M1 macrophages, which are pro-inflammatory and induce antimicrobial immune effector mechanisms, including the expression of inducible nitric oxide synthase (iNOS), and M2 macrophages, which exert anti-inflammatory functions and express arginase 1 (ARG1). Through the process of phagocytosis, macrophages contain, engulf, and eliminate bacteria. Therefore, they are one of the first lines of defense against Salmonella. Infection with Salmonella leads to gastrointestinal disorders and systemic infection, termed typhoid fever. For further characterization of infection pathways, we established an in vitro model where macrophages are infected with the mouse Salmonella typhi correlate Salmonella enterica serovar Typhimurium ( S. tm), which additionally expresses red fluorescent protein (RFP). This allows us to clearly characterize macrophages that phagocytosed the bacteria, using multi-color flow cytometry. In this protocol, we focus on the in vitro characterization of pro- and anti-inflammatory macrophages displaying red fluorescent protein-expressing Salmonella enterica serovar Typhimurium, by multi-color flow cytometry., Competing Interests: Competing interestsThe authors declare no conflicts of interest., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative, facultative intracellular bacterium, which causes gastrointestinal disorders in humans, and systemic, typhoid fever-like infections in mice. Our current knowledge regarding the involvement of cellular and humoral immunity in the defense from S. Typhimurium infections is largely based on animal models with attenuated strains. Cells of the innate immune system act as one of the first barriers in the defense from bacteria. We established a robust experimental model for the characterization of these cell types and their response during host-pathogen interactions. Therefore, this protocol focuses on the characterization of macrophages, monocytes, and neutrophils in the spleens of infected animals by employing multi-color flow cytometry., Competing Interests: Competing interestsThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)