Protocols in Current Issue
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0 Q&A 122 Views Feb 5, 2024

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 (, the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl3) 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 Fe2+ 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 Fe2+ 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 Fe2+ 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.

Graphical overview

0 Q&A 1336 Views Dec 20, 2023

Clearance of dying cells, named efferocytosis, is a pivotal function of professional phagocytes that impedes the accumulation of cell debris. Efferocytosis can be experimentally assessed by differentially tagging the target cells and professional phagocytes and analyzing by cell imaging or flow cytometry. Here, we describe an assay to evaluate the uptake of apoptotic cells (ACs) by human macrophages in vitro by labeling the different cells with commercially available dyes and analysis by flow cytometry. We detail the methods to prepare and label human macrophages and apoptotic lymphocytes and the in vitro approach to determine AC uptake. This protocol is based on previously published literature and allows for in vitro modeling of the efficiency of AC engulfment during continual efferocytosis process. Also, it can be modified to evaluate the clearance of different cell types by diverse professional phagocytes.

Graphical overview

0 Q&A 1107 Views Nov 20, 2023

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes.

Key features

• This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I.

• Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

0 Q&A 741 Views Sep 20, 2023

Gammaherpesviruses such as Epstein-Barr virus (EBV) are major modulators of the immune responses of their hosts. In the related study (PMID: 35857578), we investigated the role for Ly6Chi monocytes in shaping the function of effector CD4+ T cells in the context of a murine gammaherpesvirus infection (Murid gammaherpesvirus 4) as a model of human EBV. In order to unravel the polyfunctional properties of CD4+ T-cell subsets, we used multiparametric flow cytometry to perform intracellular staining on lung cells. As such, we have developed herein an intracellular staining workflow to identify on the same samples the cytotoxic and/or regulatory properties of CD4+ lymphocytes at the single-cell level. Briefly, following perfusion, collection, digestion, and filtration of the lung to obtain a single-cell suspension, lung cells were cultured for 4 h with protein transport inhibitors and specific stimulation media to accumulate cytokines of interest and/or cytotoxic granules. After multicolor surface labeling, fixation, and mild permeabilization, lung cells were stained for intracytoplasmic antigens and analyzed with a Fortessa 4-laser cytometer. This method of quantifying cytotoxic mediators as well as pro- or anti-inflammatory cytokines by flow cytometry has allowed us to decipher at high resolution the functional heterogeneity of lung CD4+ T cells recruited after a viral infection. Therefore, this analysis provided a better understanding of the importance of CD4+ T-cell regulation to prevent the development of virus-induced immunopathologies in the lung.

Key features

• High-resolution profiling of the functional properties of lung-infiltrating CD4+ T cells after viral infection using conventional multiparametric flow cytometry.

• Detailed protocol for mouse lung dissection, preparation of single-cell suspension, and setup of multicolor surface/intracellular staining.

• Summary of optimal ex vivo restimulation conditions for investigating the functional polarization and cytokine production of lung-infiltrating CD4+ T cells.

• Comprehensive compilation of necessary biological and technical controls to ensure reliable data analysis and interpretation.

Graphical overview

Graphical abstract depicting the interactions between immune cells infiltrating the alveolar niche and the lung during respiratory infection with a gammaherpesvirus (Murid herpesvirus 4, MuHV-4). Two distinct situations are represented: the inflammatory response developed during viral replication in the lung, either in the presence (WT mice) or absence of regulatory monocytes (CCR2KO mice). Sequential process of the experiment is represented, starting from intratracheal instillation of MuHV-4 virions to tissue dissociation and multicolor staining for flow cytometry analysis.

0 Q&A 1747 Views Aug 20, 2023

T cells are endowed with T-cell antigen receptors (TCR) that give them the capacity to recognize specific antigens and mount antigen-specific adaptive immune responses. Because TCR sequences are distinct in each naïve T cell, they serve as molecular barcodes to track T cells with clonal relatedness and shared antigen specificity through proliferation, differentiation, and migration. Single-cell RNA sequencing provides coupled information of TCR sequence and transcriptional state in individual cells, enabling T-cell clonotype-specific analyses. In this protocol, we outline a computational workflow to perform T-cell states and clonal analysis from scRNA-seq data based on the R packages Seurat, ProjecTILs, and scRepertoire. Given a scRNA-seq T-cell dataset with TCR sequence information, cell states are automatically annotated by reference projection using the ProjecTILs method. TCR information is used to track individual clonotypes, assess their clonal expansion, proliferation rates, bias towards specific differentiation states, and the clonal overlap between T-cell subtypes. We provide fully reproducible R code to conduct these analyses and generate useful visualizations that can be adapted for the needs of the protocol user.

Key features

• Computational analysis of paired scRNA-seq and scTCR-seq data

• Characterizing T-cell functional state by reference-based analysis using ProjecTILs

• Exploring T-cell clonal structure using scRepertoire

• Linking T-cell clonality to transcriptomic state to study relationships between clonal expansion and functional phenotype

Graphical overview

0 Q&A 284 Views Aug 5, 2023

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations.

Graphical overview

0 Q&A 385 Views Jul 5, 2023

Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in histological sections of the gastrointestinal tract and is therefore accepted as a biomarker of neutrophil invasion in the gut. This protocol describes an easy, cost-effective kinetic colorimetric assay to quantify myeloperoxidase activity in intestinal tissue samples. It is explained using tissue collected in mice but can also be used for other laboratory animals. In a first step, tissue specimens are homogenized using a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which extracts MPO from neutrophils. The obtained supernatant is added to a reagent solution containing o-dianisidine dihydrochloride, which is a peroxidase substrate. Finally, the change in absorption is measured via spectrophotometry and converted to a standardized unit of enzyme activity. The assay is illustrated and compared to a commercially available enzyme-linked immunoassay (ELISA), demonstrating that MPO activity does not necessarily correlate with MPO protein expression in tissue samples.

Key features

• Optimized for use in mice and rats but can also be used for samples of other species.

• Measures enzymatic activity instead of mRNA or protein expression.

• Requires a spectrophotometer.

• Can be performed in duplo using 10 mg of (dry-blotted) gut tissue or more.

Graphical overview

1 Q&A 947 Views Jun 20, 2023

Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 μL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 μL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.

Graphical overview

0 Q&A 672 Views Feb 5, 2023

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 ( 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 for one hour. Shortly before infection, cells were stained with CellROXTM Deep Red reagent. In its reduced form, CellROXTM 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

0 Q&A 512 Views Jan 5, 2023

Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.

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