Rheumatoid arthritis (RA) is a chronic disease involving inflammation of the joints. While the etiology of RA remains unknown, evidence exists for a significant contribution of the major histocompatibility complex (MHC). Environmental factors are also indicated as playing an etiological role. Since it is impossible to define the mechanisms contributing to disease onset and progression in humans, mouse models have been used widely. While many animal models have been generated by immunization with self-proteins to induce arthritis, type II collagen (CII)-induced arthritis is one of the most commonly used models utilized to understand the immunopathology of RA. CII constitutes 80% to 90% of total collagen content of hyaline cartilage found in joints and is a genetically conserved sequestered protein. Immunization with heterologous CII with an adjuvant in mice leads to cellular and humoral responses to heterologous and autoreactive CII-specific responses and collagen-induced arthritis (CIA). Mice immunized with CII develop inflammatory arthritis that shares many similarities in clinical, serological, and radiological features with RA in humans. However, selecting an antigen for inducing arthritis and the mouse strain are important, as not all strains are susceptible to CIA. A critical difference between RA and CIA is that in mice that lack the expression of human MHC II, the development of CIA is linked to the H2A locus, which is the homologue of HLA-DQ, while most human studies have linked RA susceptibility with HLA-DR alleles. Mice expressing HLA-DQ and HLA-DR molecules have been used to understand the role of MHC genes in susceptibility to RA. The protocols for inducing CIA in the HLA expressing transgenic mice described in this article can be used to understand how the different HLA molecules confer susceptibility to RA. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Use of mouse strains and humanized mice for modeling rheumatoid arthritis Basic Protocol 2: Use of humanized mice for therapeutic protocols.
An estimated 40 million people are infected with human immunodeficiency virus type 1 (HIV-1) and there is no effective cure. Humanized mice are a useful model to study transmission, pathogenesis, prevention, and treatment of HIV-1, which only infects human cells. Here we provide detailed protocols to humanize immunodeficient mice, e.g., NSG or MISTRG6 strains, using human hematopoietic stem cells isolated from cord blood and a flow cytometry panel of antibodies to assess human immune cell reconstitution. Furthermore, we describe how to produce, concentrate, and titer HIV-1 in vitro, which can be used to challenge humanized mice either systemically or mucosally. Finally, we adapted a sensitive quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay with internal control to detect HIV-1 RNA in longitudinal plasma samples to measure viremia over time in the mice. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Isolation of CD34+ cells from human cord blood Basic Protocol 2: Injection of human CD34+ cells in immunodeficient mice Basic Protocol 3: Flow cytometry of peripheral blood mononuclear cells from mouse blood Basic Protocol 4: Production and titration of HIV-1 Basic Protocol 5: Systemic HIV-1 challenge of humanized mice Alternate Protocol: Mucosal HIV-1 challenge of humanized mice Basic Protocol 6: Single-copy HIV-1 RNA detection in mouse plasma Support Protocol 1: Retrovirus internal control for plasma virus isolation and RT-qPCR Support Protocol 2: Production of HIV-1 standard for RT-qPCR.
The oncogenic Epstein Barr virus (EBV) is an exclusively human pathogen with related lymphocryptoviruses (γ1-herpesviruses) only present in monkeys. Therefore, experimentation with EBV infection in a small animal model requires reconstitution or adoptive transfer of human lymphocyte populations, primarily EBV's main host cell, the human B cell. In this protocol we describe human immune system reconstitution after neonatal transfer of CD34+ hematopoietic progenitor cells in lymphodeplete immune compromised mouse strains, using NOD-scid γc -/- (NSG) mice as a commonly used example. Such reconstituted humanized mice allow intraperitoneal and intranasal infection with EBV and we describe injection of 105 infectious particles of the prototypic EBV strain B95-8 that can be produced from a recombinant bacmid (p2089) in HEK293 cells. Infection with this dose mimics symptomatic primary EBV infection, infectious mononucleosis (IM), with high viral loads plateauing 4 weeks after infection, with CD8+ T-cell lymphocytosis at week 5 and 6 after infection. This IM-like primary EBV infection in humanized mice leads to clonal EBV-induced B-cell lymphoproliferations that resemble large B-cell lymphomas with the latency III program of EBV infection. We describe Basic Protocols to monitor viral loads, immunohistochemistry of infected tissues and spectral flow cytometry to characterize protective T-cell expansion. The described mouse model has been used by us and others to characterize mutant EBV infections, cell-mediated immune control of EBV, modulation of EBV pathogenesis by co-infections with human immunodeficiency virus (HIV) and Kaposi sarcoma associated herpesvirus (KSHV), as well as passive transfer of vaccine elicited antibodies to test their protection against EBV infection. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CD34+ human hematopoietic progenitor cell isolation and characterization Basic Protocol 2: Human immune system reconstitution and characterization Basic Protocol 3: Recombinant EBV production and humanized mouse infection Basic Protocol 4: Viral load quantification, lymphoma assessment, and immunohistochemistry after EBV infection of humanized mice Basic Protocol 5: Human T-cell response analysis after EBV infection of humanized mice.
Saliva plays a central role in maintaining oral homeostasis by supporting tooth integrity, providing lubrication, and functioning as an antimicrobial wash. It also serves as a transport medium, carrying byproducts and signaling metabolites across oral niches and through the gastrointestinal tract. Because of its biological relevance and ease of collection, saliva is increasingly used as a noninvasive biospecimen for measuring cortisol, cytokines, and metabolites. However, the validity and reliability of saliva as an indicator of local and systemic biomarkers remain under investigation across diverse populations and research applications. Specific patient populations (e.g., individuals with alcohol use disorder) are particularly vulnerable to oral health problems, periodontal disease, and high rates of nicotine use. In addition to behavioral factors (e.g., food, drink, toothbrushing, and mouthwash), patient-specific variables can introduce contaminants such as nicotine and blood into saliva, potentially compromising the accurate measurement of analytes of interest. Protocols that account for possible contaminants are essential to ensure rigorous and reproducible biomarker research. Assessing factors such as pH, flow rate, and visible discoloration helps reduce limitations in analysis and improves interpretation in studies that include heterogeneous populations and health behaviors. Yet, the literature provides limited guidance on standardized methods for saliva collection, processing, and measurement of patient-specific confounders alongside analytes of interest. This protocol addresses these gaps by presenting detailed methodologies for saliva collection and processing, assessment of quantitative and qualitative salivary properties, and quantification of patient-specific modifiers. These approaches support reproducible diagnostics and have applications in populations with high rates of smoking, periodontal disease, and alcohol use. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Saliva collection by the passive drool method Basic Protocol 2: Processing, storage, and characterization of saliva (visual assessment scale, pH, and flow rate) Basic Protocol 3: Quantification of cotinine in salivary supernatant Basic Protocol 4: Quantification of transferrin in salivary supernatant.
Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by tense blisters and itchy erythema, predominantly affecting elderly individuals. The pathogenic autoantibodies mainly target collagen XVII (COL17), leading to subepidermal blister formation and infiltrations of immune cells, including eosinophils and neutrophils. Although systemic oral corticosteroids remain the mainstay of treatment, their use in elderly patients is often limited by serious complications and adverse effects, highlighting the unmet need for novel therapeutic targets. Several mouse models for BP have been reported; however, the inconsistency of disease induction has hindered therapeutic investigations. The active BP mouse model has emerged as a reliable system that recapitulates key disease features, making it valuable for both preclinical therapeutic studies and elucidation of BP pathophysiology. Furthermore, autoantigen-humanized mouse models, including the active BP model and the neonatal passive IgG transfer model, provide significant advantages for the development of antigen-specific therapies. Here, we describe detailed materials and methods for mouse models for BP using the COL17-humanized mouse, including the active BP mouse model and the neonatal passive IgG transfer model. Protocol modifications may be necessary when using different donor mice and antibodies. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Active BP mouse model Basic Protocol 2: Passive IgG transfer mouse model using neonatal mice.
T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.
Antigen‐specific memory B cell (MBC) populations mediate the rapid, strong, and high‐affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome‐labeled antigens together with sequencing methods such as Drop‐seq and CITE‐seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen‐reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long‐established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody‐secreting cells (ASCs). Post‐stimulation antigen‐specific measurement of the MBC‐derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen‐reactive MBC populations is provided by analysis of MBC‐derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post‐stimulation readout to determine the size of antigen‐specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC
T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor‐reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen‐specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single‐cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor‐reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC.
Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8+ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone-marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona-fide cross-presenting DCs. We also describe methods to assess cross-presentation by DCs using the activation of primary CD8+ T cells as a readout. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of bone marrow progenitor cells Basic Protocol 2: In vitro differentiation of dendritic cells with GM-CSF Support Protocol 1: Preparation of conditioned medium from GM-CSF producing J558L cells Basic Protocol 3: In vitro differentiation of dendritic cells with Flt3L Support Protocol 2: Preparation of Flt3L containing medium from B16-Flt3L cells Basic Protocol 4: Expansion of cDC1s in vivo for use in ex vivo experiments Basic Protocol 5: Characterizing resting and activated dendritic cells Basic Protocol 6: Dendritic cell stimulation, antigenic cargo, and fixation Support Protocol 3: Preparation of model antigen coated microbeads Support Protocol 4: Preparation of apoptotic cells Support Protocol 5: Preparation of recombinant bacteria Basic Protocol 7: Immunocytochemistry immunofluorescence (ICC/IF) Support Protocol 6: Preparation of Alcian blue-coated coverslips Basic Protocol 8: CD8+ T cell activation to assess cross-presentation Support Protocol 7: Isolation and labeling of CD8+ T cells with CFSE.
SARS‐CoV‐2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS‐CoV‐2, which causes severe respiratory illness or death. Defining the antibody response to SARS‐CoV‐2 will be essential for understanding disease progression, long‐term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS‐CoV‐2 antibodies. The basic protocol is a focus reduction neutralization test (FRNT), which involves immunostaining infected cells with a chromogen deposit readout. The alternate protocol is a modification of the FRNT that uses an infectious clone−derived SARS‐CoV‐2 virus expressing a fluorescent reporter. These protocols are adapted for use in a high‐throughput setting, and are compatible with large‐scale vaccine studies or clinical testing. © 2020 Wiley Periodicals LLC
Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR7 with immune-response inducing ligands in cultured immune cells. This review will focus on the TLR stimulation of mouse macrophages as an example; however, the technique is applicable to any immortalized immune cell and any soluble stimuli. The methodology includes protocols for metabolic labeling of immune cells (stable isotope labeling of amino acids in cell culture, i.e., SILAC); ligand-initiated stimulation of immune receptors followed by cell lysis; in-solution trypsin digestion of proteins and enrichment of the resulting peptide mix for collecting phosphopeptides, which are then analyzed by high-resolution LC-MS/MS (liquid-chromatography tandem mass spectrometry). © 2020 Wiley Periodicals LLC. Basic Protocol 1: SILAC labeling of mouse macrophages Basic Protocol 2: Stimulation, cell lysis and Western Blotting Basic Protocol 3: Trypsin digestion, fractionation and phosphopeptide enrichment Basic Protocol 4: Quantitative mass spectrometry Alternate Protocol: Culturing SILAC-labeled cells from frozen mouse macrophages cells.
Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intestine and colon biopsies. Additionally, this article provides an overview of several downstream applications of human intestinal organoids. © 2020 The Authors. Basic Protocol 1: Establishment of human small intestine and colon organoid cultures from fresh biopsies Basic Protocol 2: Mechanical splitting, passage, and expansion of human intestinal organoids Alternate Protocol: Differentiation of human intestinal organoids Basic Protocol 3: Cryopreservation and thawing of human intestinal organoids Basic Protocol 4: Immunofluorescence staining of human intestinal organoids Basic Protocol 5: Generation of single-cell clonal intestinal organoid cultures Support Protocol 1: Production of Wnt3A conditioned medium Support Protocol 2: Production of Rspo1 conditioned medium Support Protocol 3: Extraction of RNA from intestinal organoid cultures.
Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and biochemical responses similar to those seen in septic humans. Further, a transient numerical reduction of multiple immune cell types, followed by development of prolonged immunoparalysis, occurs in CLP-induced sepsis just as in humans. Use of the CLP model has led to a vast expansion in knowledge regarding the intricate physiological and cellular changes that occur during and after a septic event. This updated article details the steps necessary to perform this survival surgical technique, as well as some of the obstacles that may arise when evaluating the sepsis-induced changes within the immune system. It also provides representative monoclonal antibody (mAb) panels for multiparameter flow cytometric analysis of the murine immune system in the septic host. © 2020 Wiley Periodicals LLC. Basic Protocol: Cecal ligation and puncture in the mouse.
This article describes a procedure for isolating T cell subpopulations using various methods including indirect panning and immunopanning by microarray. In these methods, cells are selected by their capacity to bind to antibody-coated plates (or slides) on the basis of particular cell-surface markers. Such methods can be superior to the antibody/complement lysis method (Alternate Protocol), as they can select additional cell population for analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of T cell populations by indirect panning Basic Protocol 2: Immunopanning with microarray Alternate Protocol: Isolation of T cell populations by antibody/complement-mediated cytotoxicity.
Sjögren's syndrome (SS) is a systemic autoimmune disease affecting multiple organ systems. Salivary and lacrimal gland involvement cause dry mouth and dry eye and are the most common clinical presentations of the disease. Patients with SS also have autoantibodies targeting multiple nuclear and cytoplasmic antigens. Innate immune activation plays a critical role in SS pathogenesis. This article describes the activation of specific innate immune pathways in mice to study SS salivary gland manifestations. Methodologies for evaluating salivary gland inflammation and salivary function are described. This article also describes protocols for in-house assays to measure autoantibody titers in serum. © 2020 Wiley Periodicals LLC Basic Protocol 1: Acceleration of Sjögren's syndrome by activating the toll-like receptor 3 pathway Basic Protocol 2: Induction of Sjögren's syndrome by activating the stimulator of interferon genes pathway Alternate Protocol: Acceleration of Sjögren's syndrome by the administration of Freund's incomplete adjuvant Support Protocol 1: Evaluating salivary gland function Support Protocol 2: Evaluating salivary gland inflammation Support Protocol 3: Measuring autoantibody titers by indirect immunofluorescence.
In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: LCMV infection routes in mice Support Protocol 1: Preparation of LCMV stocks ASSAYS TO MEASURE LCMV TITERS Support Protocol 2: Plaque assay Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC) Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody Support Protocol 4: Preparation of splenic lymphocytes Support Protocol 5: Making BHK21-LCMV lysate Basic Protocol 7: Challenge models TRANSGENIC MODELS Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge.
Tight junctions form a selectively permeable barrier that limits paracellular flux across epithelial-lined surfaces. Small molecules (less than ∼8 Å diameter) can traverse the junction via the size- and charge-selective, high-conductance pore pathway. In contrast, the low-conductance leak pathway accommodates larger macromolecules (up to ∼100 Å diameter) and is not charge-selective. Flux across the tight junction-independent, high-conductance, non-selective, unrestricted pathway occurs at sites of epithelial damage. Cytokines can regulate each of these pathways, but commonly used measures of barrier function cannot discriminate between tight junction regulation and epithelial damage. This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay.
Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Enteroids are generated from biopsies or resected tissue from any segment of the human gut and maintained in long-term cultures as three-dimensional structures through supplementation of stem cell growth factors. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC). Monocytes from PBMC are differentiated into macrophages by cytokine stimulation prior to co-culture. The methods are divided into the two main components of the model: (1) generating enteroid/colonoid monolayers and isolating immune cells and (2) assembly of enteroid/colonoid-immune cell co-cultures with separate apical and basolateral compartments. Co-cultures containing macrophages can be maintained for 48 hr while those involving neutrophils, due to their shorter life span, remain viable for 4 hr. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Seeding enteroid fragments onto Transwells for monolayer formation Alternate Protocol: Seeding enteroid fragments for monolayer formation using trituration Basic Protocol 2: Isolation of monocytes and derivation of immune cells from human peripheral blood Basic Protocol 3: Isolation of neutrophils from human peripheral blood Basic Protocol 4: Assembly of enteroid/macrophage or enteroid/neutrophil co-culture.
Cellular interactions are often essential to regulate immune cell activities during an immune response. To understand the details of this process, it is necessary to study individual receptor/ligand interactions in a quantitative fashion. However, this is often very difficult or even impossible when using real cells for stimulation. Here, we present a method to use cell-sized latex beads for such studies. These beads can be coated with agonistic antibodies or specific ligands in a defined and quantifiable fashion. This creates the possibility of titrating the strength of the stimulation for a specific receptor in a three-dimensional system. Using natural killer (NK) cells as an example, we demonstrate how these beads can be used to stimulate NK cell responses. © 2020 The Authors. Basic Protocol 1: Covalent coating of latex beads with antibodies Basic Protocol 2: Quantification of the amount of antibodies on the beads with the QIFIKIT® Alternate Protocol 1: Covalent coating of latex beads with streptavidin to bind biotinylated proteins Alternate Protocol 2: Quantification of the amount of protein on the beads with the QIFIKIT® Support Protocol: Functional testing of the beads in a natural killer cell degranulation assay.
Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role that NK cells play is analogous to that of cytotoxic T cells in that they provide rapid responses to virus-infected cells and responses to tumor formation. Unmodified NK cells have long been used in various immunotherapies to treat different tumors, with only marginal success. However, in the last few years, NK cells modified to express chimeric antigen receptors (CAR-NK cells) have emerged as particularly ideal cellular platforms for antigen-specific antitumor agents. Unlike CAR-T cells, they do not elicit allogeneic responses or graft-versus-host disease and therefore can be administered to recipients with differing MHC expression. This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells Support Protocol: Production of lentiviral supernatant.
