Protocols in Current Issue
0 Q&A 131 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 214 Views Sep 20, 2023

Immune cell trafficking in steady-state conditions and inflammatory cell recruitment into injured tissues is crucial for the surveillance of the immune system and the maintenance of body homeostasis. Tracking the cell journey from the infection site in the skin to lymphoid tissues has been challenging, and is typically determined using fluorescent cell tracers, antibodies, or photoconvertible models. Here, we describe the detailed method to track Leishmania-infected myeloid cells migrating from the skin to lymphatic tissues by multiparametric flow cytometry. These methods involve labeling of infective Leishmania donovani parasites with fluorescent cell tracers and phenotyping of myeloid cells with fluorescent antibodies, to determine the infection status of migratory myeloid cells. We also describe the detailed protocol to trace donor monocytes transferred intradermally into recipient mice in Leishmania donovani infection. These protocols can be adapted to study skin-lymphoid tissue migration of dendritic cells, inflammatory monocytes, neutrophils, and other phagocytic myeloid cells in response to vaccine antigens and infection.

Key features

• Cell-tracking of cell-trace-labeled parasites and monocytes from the skin to lymphatic tissues after transference into donor mice.

• Identification of migratory cells labeled with fluorescent cell tracers and antibodies by flow cytometry.

• Isolation, labeling, and transference of bone marrow monocytes from donor mice into the skin of recipient mice.

• Description of a double-staining technique with fluorescent cell tracers to determine cell and parasite dissemination from the skin to lymphoid tissues.

Graphical overview

Overview of the methods to trace the migration of Leishmania and monocytes from the skin to lymphatic tissues by flow cytometry. Infective metacyclic promastigotes (from axenic culture) and monocytes (isolated from the bone marrow of donor mice) are labeled with fluorescent cell tracers. After intradermal injection into the test mouse (1, 2), migratory cells and infected cells are isolated from the skin and lymphoid tissues of the test mouse. These cells are then labeled with fluorescent antibodies against myeloid cells and recognized according to the differential excitation/emission wavelengths of the fluorochromes by flow cytometry.

0 Q&A 118 Views Sep 20, 2023

During life, the embryonic alveolar macrophage (AM) population undergoes successive waves of depletion and replenishment in response to infectious and inflammatory episodes. While resident AMs are traditionally described as from embryonic origin, their ontogeny following inflammation or infection is much more complex. Indeed, it appears that the contribution of monocytes (MOs) to the AM pool is variable and depends on the type of inflammation, its severity, and the signals released in the microenvironment of the pulmonary niche (peripheral imprinting) and/or in the bone marrow (central imprinting). Deciphering the cellular and molecular mechanisms regulating the differentiation of MOs into AMs remains an area of intense investigation, as this could potentially explain part of the inter-individual susceptibility to respiratory immunopathologies. Here, we detail a relevant ex vivo co-culture model to investigate how lung epithelial cells (ECs) and group 2 lung innate lymphoid cells (ILC2s) contribute to the differentiation of recruited MOs into AMs. Interestingly, the presence of lung ILC2s and ECs provides the necessary niche signals to ensure the differentiation of bone marrow MOs into AMs, thus establishing an accessible model to study the underlying mechanisms following different infection or inflammation processes.

Key features

• Ex vivo co-culture model of the alveolar niche.

• Deciphering the particular niche signals underlying the differentiation of MO into AMs and their functional polarization.

Graphical overview
This protocol described the isolation of bone marrow monocytes (MOs), lung epithelial cells (ECs), and lung group 2 lung innate lymphoid cells (ILC2s) and the ex vivo co-culture of these cells to drive the differentiation of bone marrow MOs into alveolar macrophages (AMs).

This co-culture experiment is composed of three steps (Graphical overview):
1. Identification and FACS-sorting of ECs and MOs isolated from the lung and the bone marrow of naive mice, respectively.
2. Culture of these ECs and bone marrow MOs for three days.
3. Addition of ILC2s isolated from the lung of naïve mice or mice subjected to a treatment/infection of interest.

Protocols in Past Issues
0 Q&A 218 Views Sep 5, 2023

Surface Plasmon Resonance (SPR) is a label-free optical technique to assess protein–protein interaction kinetics and affinities in a real-time setting. Traditionally, Biacore SPR employs a continuous film of gold to detect any change in the angle of re-emitted light when the refractive index of a ligand conjugated to the flat gold surface is altered by its interaction with a local analyte. In contrast, the Nicoya Lifesciences’ OpenSPR technology uses gold nanoparticles to detect small changes in the absorbance peak wavelength of a conjugated ligand after its engagement by an analyte. Specifically, when broadband white light is shone onto the gold nanoparticles, it produces a strong resonance absorbance peak corresponding to the refractive index of a ligand conjugated to the surface of gold nanoparticles. Upon its interaction with an analyte, however, the absorbance wavelength peak of the conjugated ligand will be changed and timely recorded as sensorgrams of dynamic ligand–analyte interactions. Thus, the improvement in the detection method (from traditional detection of changes in the angle of re-emitted light to the contemporary detection of changes in the wavelength of the absorbance peak) features OpenSPR as a cost-effective and user-friendly technique for in-depth characterization of protein–protein interactions. Here, we describe the detailed method that we used to characterize procathepsin L (pCTS-L) interactions with two putative pattern recognition receptors (TLR4 and RAGE) using the 1st generation of Nicoya Lifesciences’ OpenSPR instrument with a 1-channel detection.

Key features

• Nicoya OpenSPR is a benchtop small-size equipment that provides in-depth label-free binding kinetics and affinity measurement for protein–protein interactions in real-time fashion.

• This technology is relatively intuitive and user-friendly for scientists at any skill level.

• OpenSPR sensors employ nanotechnology to reduce the cost of manufacturing complex optical hardware and Sensor Chips, and similarly reduce the consumption of precious analyte samples.

• The manufacturer provides online training for OpenSPR (Catalog: TRAIN-REMOTE) and TraceDrawer (Catalog: TRAIN-TD) to customer scientists.

0 Q&A 115 Views Sep 5, 2023

Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity.

Key features

• This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT.

• Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies.

• Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist.

• For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.

0 Q&A 1058 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 449 Views Aug 20, 2023

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry–based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers.

Key features

• This protocol describes a flow cytometry–based method to analyze the myeloid cell response in retinopathy mouse models.

• The protocol can distinguish between microglia- and monocyte-derived macrophages.

• It can be modified to incorporate markers of interest.

We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.

0 Q&A 178 Views Aug 5, 2023

Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite’s pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.

0 Q&A 163 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 393 Views Jul 20, 2023

Innate lymphoid cells (ILCs) are a rare cell population subdivided into ILC1s, ILC2s, and ILC3s, based on transcription factor expression and cytokine production. In models of lung inflammation, the release of alarmins from the epithelium activates ILC2s and promotes the production of Th2-cytokines and the proliferation and migration of ILC2s within the lung. ILC2s are the innate counterpart to CD4+ Th2s and, as such, express Gata-3 and produce IL-4, IL-5, and IL-13. Due to the low number of ILCs and the lack of specific surface markers, flow cytometry is the most reliable technique for the identification and characterization of ILCs. In this protocol, multicolor flow cytometry is utilized to identify LineageThy1.2+ ILCs. Intracellular cytokine staining further identifies ILC2s within the lung. This protocol presents a reliable method for promoting ILC2-mediated lung inflammation and for monitoring ILC2 biology.

Key features

• In this protocol, ILC2s are expanded via intranasal challenges with Alternaria alternata, a fungal allergen, or recombinant IL-33.

• Bronchoalveolar lavage (BAL) and lung are collected and processed into single-cell suspension for multicolor flow cytometric analysis, including intracellular staining of transcription factors and cytokines.

• During lung inflammation, the percentage of ILC2s and eosinophils increases. ILC2s express greater levels of Gata-3 and Ki-67 and produce greater amounts of IL-5 and IL-13.

Graphical overview

0 Q&A 445 Views Jul 20, 2023

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

0 Q&A 437 Views Jul 20, 2023

The immune-inhibitory molecule programmed cell death ligand 1 (PD-L1) has been shown to play a role in pathologies such as autoimmunity, infections, and cancer. The expression of PD-L1 not only on cancer cells but also on non-transformed host cells is known to be associated with cancer progression. Generation of PD-L1 deficiency in the murine system enables us to specifically study the role of PD-L1 in physiological processes and diseases. One of the most versatile and easy to use site-specific gene editing tools is the CRISPR/Cas9 system, which is based on an RNA-guided nuclease system. Similar to its predecessors, the Zinc finger nucleases or transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 catalyzes double-strand DNA breaks, which can result in frameshift mutations due to random nucleotide insertions or deletions via non-homologous end joining (NHEJ). Furthermore, although less frequently, CRISPR/Cas9 can lead to insertion of defined sequences due to homology-directed repair (HDR) in the presence of a suitable template. Here, we describe a protocol for the knockout of PD-L1 in the murine C57BL/6 background using CRISPR/Cas9. Targeting of exon 3 coupled with the insertion of a HindIII restriction site leads to a premature stop codon and a loss-of-function phenotype. We describe the targeting strategy as well as founder screening, genotyping, and phenotyping. In comparison to NHEJ-based strategy, the presented approach results in a defined stop codon with comparable efficiency and timelines as NHEJ, generates convenient founder screening and genotyping options, and can be swiftly adapted to other targets.

0 Q&A 648 Views Jul 20, 2023

Current means to quantify cells, gene expression, and fibrosis of liver histological slides are not standardized in the research community and typically rely upon data acquired from a selection of random regions identified in each slide. As such, analyses are subject to selection bias as well as limited subsets of available data elements throughout the slide. A whole-slide analysis of cells and fibrosis would provide for a more accurate and complete quantitative analysis, along with minimization of intra- and inter-experimental variables. Herein, we present LiverQuant, a method for quantifying whole-slide scans of digitized histologic images to render a more comprehensive analysis of presented data elements. After loading images and preparing the project in the QuPath program, researchers are provided with one to two scripts per analysis that generate an average intensity threshold for their staining, automated tissue annotation, and downstream detection of their anticipated cellular matrices. When compared with two standard methodologies for histological quantification, LiverQuant had two significant advantages: increased speed and a 50-fold greater tissue area coverage. Using publicly available open-source code (GitHub), LiverQuant improves the reliability and reproducibility of experimental results while reducing the time scientists require to perform bulk analysis of liver histology. This analytical process is readily adaptable by most laboratories, requires minimal optimization, and its principles and code can be optimized for use in other organs.

Graphical overview

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