Methods In Cell Biology[JOURNAL]
Sun
200 papers
Experimental models and methods for infected wound modeling with Staphylococcus aureus.
Pérez E, Landa G, Bescós I … +2 more , Ochoa I, Tapia E
Methods Cell Biol · 2026 · PMID 41724560 · Publisher ↗
Acute or chronic non-healing wounds place nowadays a massive financial burden on healthcare systems. Although we have gained much knowledge about the fundamental molecular and cellular mechanisms that promotes healthy, a...
Acute or chronic non-healing wounds place nowadays a massive financial burden on healthcare systems. Although we have gained much knowledge about the fundamental molecular and cellular mechanisms that promotes healthy, acute wound healing from various animal models, further preclinical research is necessary to explore the pathology and role of infection in the development and persistence of both acute and chronic non-healing wounds. In this study we present a comprehensive protocol for the generation of an infected acute or chronic non-healing wound on a mouse model that could serve as a useful tool for uncovering pathological mechanisms and potential therapeutic targets for eventual translation to the clinic.
Establishment single lung tumors through orthotopic injection.
Picabea B, Clémenson C, Echepare M … +5 more , Viu-Idocin C, Álava A, Rodríguez E, Mondini M, Valencia K
Methods Cell Biol · 2026 · PMID 41724559 · Publisher ↗
Lung cancer, the leading cause of cancer-related deaths, is often diagnosed at advanced stages, resulting in low survival rates. Despite the widespread use of subcutaneous murine models in preclinical research, they fail...
Lung cancer, the leading cause of cancer-related deaths, is often diagnosed at advanced stages, resulting in low survival rates. Despite the widespread use of subcutaneous murine models in preclinical research, they fail to replicate the complexity of the tumor microenvironment. Recent studies highlight the importance of understanding the tumor microenvironment in non-small cell lung cancer (NSCLC), particularly in relation to therapies like radiotherapy and immunotherapy. The orthotopic lung cancer model, which more accurately mirrors human lung cancer, is gaining traction as it better reflects tumor progression, metastasis, and microenvironment interactions. Tumor volume can be measured using Luciferase Bioluminescence (BLI) or Computerized Tomography (CT). The orthotopic model is crucial for developing new diagnostic and therapeutic strategies for lung cancer. This chapter outlines a protocol for orthotopic tumor implantation in mice.
Utilisation of spatial methodologies for the investigation of the tumour microenvironment.
Gardet M, Ginhoux F, Mulder K
Methods Cell Biol · 2026 · PMID 41688158 · Publisher ↗
The tumour microenvironment (TME) is a complex and dynamic component of cancer progression, significantly influencing tumour development and response to therapy. Recent advancements in spatial-omics, such as Spatial Tran...
The tumour microenvironment (TME) is a complex and dynamic component of cancer progression, significantly influencing tumour development and response to therapy. Recent advancements in spatial-omics, such as Spatial Transcriptomics and Multiplex Imaging, have provided critical insights into TME organisation. For instance, the spatial distribution, abundance, and proximity of macrophages and cytotoxic T lymphocytes (CTLs) have been identified as key biomarkers of immune function, prognosis, and treatment response. Additionally, spatial studies have shown that phenotypic changes in tumour cells often correlate with shifts in immune cell populations, promoting tumour progression and immune evasion. This chapter serves as a practical guide for researchers working with spatial transcriptomic technologies. We focus on three leading platforms: Merscope, CosMx, and Xenium, explaining their principles and highlighting their key differences. An overview of each technology is provided to help researchers understand their applications and potential advantages. Beyond theoretical insights, we detail step-by-step experimental protocols, covering all aspects from sample preparation to data analysis. To support reproducibility and troubleshooting, we also include practical tips and lessons learned from our own experience, helping researchers optimise workflows and enhance data quality. By offering clear guidance and detailed methodologies, this chapter aims to assist researchers in successfully implementing spatial transcriptomics in their studies. Whether you are new to these technologies or seeking to refine your techniques, this resource provides essential information to advance your research in TME characterisation and precision oncology.
Optimized phospho-flow cytometry for quantitative analysis of early TCR-proximal signaling: Implications for T cell function in onco-immunology.
Porciello N, Tocci A, Campo G … +2 more , Palermo B, Nisticò P
Methods Cell Biol · 2026 · PMID 41688157 · Publisher ↗
T cells are central mediators of anti-tumor immune responses, with their activation critically depending on finely tuned, and kinetically controlled signaling through the T cell receptor (TCR)-CD3 complex. Early TCR-prox...
T cells are central mediators of anti-tumor immune responses, with their activation critically depending on finely tuned, and kinetically controlled signaling through the T cell receptor (TCR)-CD3 complex. Early TCR-proximal phosphorylation events shape downstream pathways that govern T-cell proliferation, differentiation, and effector functions, including responses to cancer immunotherapies, thus playing a pivotal role for effective tumor surveillance and eradication. Despite their importance, accurately capturing these early signaling events, at the single-cell level, remains technically challenging. Here, we present an optimized flow cytometry-based phospho-profiling protocol to assess early TCR signaling dynamics, using ERK phosphorylation as a sensitive readout. By overcoming the challenges of traditional approaches, this protocol provides an easy, yet powerful tool to evaluate T cell functionality both systemically and within the tumor immune microenvironment (TIME). Its application holds significant promise for advancing our understanding of T cell biology and for guiding the development and likely refinement of next-generation cancer immunotherapies.
Immunophenotyping of conventional and non-conventional T cell populations by surface staining of peripheral blood mononuclear cells.
Leufgen A, Steitz A, Rauber C … +1 more , Roberti MP
Methods Cell Biol · 2026 · PMID 41688156 · Publisher ↗
Here, we present a comprehensive and reproducible protocol for the isolation, cryopreservation, and immunophenotyping of human peripheral blood mononuclear cells (PBMCs) using a 28-color flow cytometry panel. This high-d...
Here, we present a comprehensive and reproducible protocol for the isolation, cryopreservation, and immunophenotyping of human peripheral blood mononuclear cells (PBMCs) using a 28-color flow cytometry panel. This high-dimensional panel enables the simultaneous assessment of conventional and unconventional T cells and their activation or functional states in a single sample, making it a valuable tool for translational and clinical immunology research. This protocol outlines detailed procedures for blood processing, density gradient separation, and optimized washing steps to minimise platelet contamination. Standardized cryopreservation methods are provided to facilitate longitudinal studies or multicentre trials. The staining strategy is specifically optimized for PBMCs and is compatible with both conventional and spectral flow cytometry systems, allowing robust and reproducible identification of lymphocyte populations with a focus on T lymphocytes. This method is applicable to both fresh and cryopreserved PBMCs and has been validated on material from healthy donors and patients. It provides a reliable framework for immune monitoring in contexts such as infection, cancer, or immunotherapy. Additionally, this protocol includes key troubleshooting steps, guidance on panel design, and practical advice for data acquisition and analysis. The workflow is adaptable to other staining panels and can seamlessly be integrated into standardized immune profiling pipelines.
Flow-cytometry-based assay to study CD8 T cell cytotoxicity against tumor cells.
Sarapulov AV, Rodriguez MA, Zappasodi R
Methods Cell Biol · 2026 · PMID 41688155 · Publisher ↗
Cancer immunotherapy approaches aim to instruct the immune system to eliminate tumor cells. Immune checkpoint blockade (ICB) therapies, such as anti-CTLA-4 and anti-PD-1, have significantly advanced the field, and other...
Cancer immunotherapy approaches aim to instruct the immune system to eliminate tumor cells. Immune checkpoint blockade (ICB) therapies, such as anti-CTLA-4 and anti-PD-1, have significantly advanced the field, and other immunotherapeutic modalities are constantly being developed and tested in pre-clinical and clinical studies. One of the most important outcomes of cancer immunotherapy is efficient elimination of tumor cells by cytotoxic CD8+ T cells. Thus, accurate measurement of tumor cell sensitivity to T-cell-mediated killing is crucial for improving treatment development. This paper presents a flow cytometry-based protocol to assess antigen-specific CD8+ T-cell cytotoxicity using ovalbumin (OVA)-specific CD8+ T cells from OT-1 TCR transgenic mice and OVA-presenting tumor cell lines (either transduced with OVA or pulsed with a class-I-MHC-restricted OVA-derived peptide). This method minimizes contribution from non-antigen-mediated killing into readouts by incorporating non-target cells (not expressing/presenting-OVA cells) into co-cultures. The procedure also allows for the evaluation of T-cell-mediated cytotoxicity under various conditions and enables concurrent immunophenotyping of effector T cells and target tumor cells if desired. The protocol can be easily customized, offers advantages over methods that rely on luciferase-expressing tumor cell lines and provides versatility in detecting multiple parameters, making it a useful and valuable tool for advancing cancer immunotherapy research.
Assays to examine lymphocyte invasion.
Lekan AA, Maynard RE, Weiner LM
Methods Cell Biol · 2026 · PMID 41688154 · Publisher ↗
The immune system plays a critical role in a number of pathologic conditions, including infection, autoimmunity, and cancer. The migratory capacity of immune cells is essential to their ability to reach and invade into s...
The immune system plays a critical role in a number of pathologic conditions, including infection, autoimmunity, and cancer. The migratory capacity of immune cells is essential to their ability to reach and invade into sites of infection, tissue damage, or solid tumors. Notably, studying the migration and invasion of immune cells has become increasingly important in the treatment of cancer. Immunotherapy has revolutionized the treatment of cancer. One critical barrier to the efficacy of immunotherapy, especially in solid tumors, is the ability of immune cells, specifically T and Natural Killer (NK) cells, to infiltrate into the tumor microenvironment (TME). Because of this, it has become increasingly important to study the invasive and migratory capabilities of immune cells in the context of cancer. Additionally, in vitro models that better recapitulate the TME are necessary in order to examine the invasion of human immune cells. Here, we describe 2D and 3D methods that can be used to examine the migratory and invasive capabilities of immune cells. These techniques have been adapted from previously described techniques.
Methodological advances to analyze cancer-associated fibroblast-derived ECM effects on macrophage polarization.
Tocci A, Valenti L, Porciello N … +4 more , Palermo B, Di Carlo A, Di Modugno F, Nisticò P
Methods Cell Biol · 2026 · PMID 41688153 · Publisher ↗
The tumor microenvironment (TME) represents a complex ecosystem composed of tumor cells and various non-cancerous cell types, embedded within an altered extracellular matrix (ECM). In solid tumors, the ECM plays multiple...
The tumor microenvironment (TME) represents a complex ecosystem composed of tumor cells and various non-cancerous cell types, embedded within an altered extracellular matrix (ECM). In solid tumors, the ECM plays multiple roles: it provides mechanical support, delivers signaling molecules and transmits biophysical stimuli that influence cellular functions. Various cell types, primarily cancer-associated fibroblasts (CAFs) and immune cells such as macrophages, actively participate in the secretion and remodeling of ECM. However, whether the ECM directly instructs or educates immune cells, particularly macrophages within the TME, remains poorly understood. Here, we present a protocol to investigate the impact of ECM derived from non-small cell lung cancer (NSCLC) CAFs on macrophage state.
Phenotypic characterization and function of tumor infiltrating and peripheral B cells in cancer patients.
Rosado MM, Frisullo G, Campo G … +2 more , Petrella M, Nisticò P
Methods Cell Biol · 2026 · PMID 41688152 · Publisher ↗
Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context multiparametric flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identi...
Lymphocyte characterization is primarily based on the differential expression of surface markers. In this context multiparametric flow-cytometry analysis (FACS) is an exceptional technique that not only allows the identification of lymphocyte subsets but can also be used to evaluate cell function, activation and proliferation. Owed the recent observation that B cells play key roles in anti-tumour immune responses as well as on the outcome of immune checkpoint inhibitor therapy, herein we will describe the combination of polyclonal B cell stimulation, ELISpot, ELISA and flow cytometry tecniques that allow the identification of tumour infiltrating and peripheral B-cell subsets as well as B cell ability to proliferate and generate antibody producing cells, in solid tumor beharing patients (see graphic abstract Figure 1).
Quantitative assessment of arterial remodeling from confocal microscopy image analysis of the mid-gestation mouse decidua.
Alghabban D, Muller AJ
Methods Cell Biol · 2026 · PMID 41688151 · Publisher ↗
The pathophysiological process of tumorigenesis involves numerous parallels with the physiological process of placentation. One critical feature that both tumors and embryos share is the need to access the host blood sup...
The pathophysiological process of tumorigenesis involves numerous parallels with the physiological process of placentation. One critical feature that both tumors and embryos share is the need to access the host blood supply. During gestation, the uterine spiral arteries, from which maternal blood is accessed, undergo a remodeling process to become enlarged, dilated, low-resistance vessels capable of delivering an increased volume of blood through placental circulatory connections made by invasive, extravillous trophoblasts. Decidual immune cells are associated with the initial stages of this remodeling prior to the arrival of trophoblasts, and mouse studies have demonstrated that, in the absence of IFNγ produced by uterine natural killer cells, spiral artery remodeling is impaired. To better understand the role of inflammatory decidual immune cells in spiral artery remodeling, it is important to have an assay that quantifiably assesses the remodeling process. Here we describe a confocal microcopy-based methodology for digitally evaluating individual vessels within the decidua basalis of mid-gestation mouse concepti by immunofluorescence staining using DAPI for general cellularity and antibodies to CAV1 (caveolin 1) and SMA (smooth muscle alpha actin) for ECs (endothelial cells) and VSMCs (vascular smooth muscle cells), respectively. Reflecting the loss of VSMCs during normal remodeling, SMA within the vascular media is markedly reduced between gd 7.5 and 9.5, while retention of SMA is a characteristic feature of impaired remodeling. Through quantitative analysis of the relative percentage of SMA within vessel walls, the methodology described here provides an objective means to assess impairment of the spiral artery remodeling process.
Immunofluorescent microscopy to quantify and characterize chromosomal instability (CIN).
Jones KM, Choi Y, Leelasukseree R … +4 more , Messick NN, Bryan A, Bice S, Cosper PF
Methods Cell Biol · 2026 · PMID 41688150 · Full text
Chromosomal instability (CIN) is the continuous missegregation of chromosomes or chromosome fragments over multiple cell divisions and is present to some degree in nearly all cancer types. Chromosome missegregation cause...
Chromosomal instability (CIN) is the continuous missegregation of chromosomes or chromosome fragments over multiple cell divisions and is present to some degree in nearly all cancer types. Chromosome missegregation causes aneuploidy, or daughter cells with an abnormal chromosome content, which is a hallmark of cancer. CIN can be caused by altered expression of mitotic checkpoint genes, defects in chromosome congression to the metaphase plate, hyperstable kinetochore-microtubule attachments, defective error correction, replication stress, defects in sister chromatid cohesion, and telomere crisis, among other intrinsic factors. CIN can also be induced by exogenous factors such as DNA tumor viruses, ionizing radiation, and microtubule stabilizing drugs. CIN has been shown to promote or suppress tumors or have no effect depending on the rate of chromosome missegregation. However, increasing CIN above a maximally tolerated threshold of chromosome loss leads to cell death, implying that altering the rates of CIN could be a potential therapeutic strategy. Furthermore, CIN leads to the formation of micronuclei, which can release cytosolic DNA and activate the cGAS-STING pathway, leading to increased innate and adaptive immune responses depending on the chronicity of CIN. Here, we detail a simple method to quantify and characterize CIN and micronuclei in cells by immunofluorescent microscopy. The technique described here can be implemented and optimized for human or murine cell lines or tissues.
Multicentric harmonization of a 6-color flow cytometry panel for immunological monitoring of human whole blood and PBMC samples.
La Sorsa V, Macchia I, Urbani F
Methods Cell Biol · 2026 · PMID 41688149 · Publisher ↗
Personalized medicine in oncology needs standardized immunological assays. Multiparametric flow-cytometry (MFC) methods represent an essential tool for immunomonitoring, and their harmonization is crucial to obtain compa...
Personalized medicine in oncology needs standardized immunological assays. Multiparametric flow-cytometry (MFC) methods represent an essential tool for immunomonitoring, and their harmonization is crucial to obtain comparable data in multicenter clinical trials. Here we report a real-life harmonization protocol of a flow cytometric assay, able to address the most effective issues contributing to intra- and inter-operator variability in the context of a multicenter project. Studies have shown that certain characteristics of the T cell compartment, including the ratio of naïve to memory T cells, the presence of specific memory T cell subsets, and the degree of T cell exhaustion, can impact patient responses to immunotherapy. Naïve/memory status of T lymphocytes is thus recognized as a potential prognostic/predictive immunological biomarker, and various MFC panels are currently applied in both mono- and multi-center clinical studies. Here, we introduce the protocol inspired by a project conducted at our institute, aimed at harmonizing a six-color MFC panel focused on determining the naïve/memory maturation status of CD3+ T lymphocytes, intended for use with both peripheral blood mononuclear cell (PBMC) and whole blood (WB) human samples. To offer a comprehensive overview of the methodological approach, we outline specifics concerning the pre-analytical and experimental analytical phases, emphasizing the final statistical analysis processing. The goal is to promote standardization and reproducibility by addressing variability in sample handling and preparation prior to applying the protocol to patient samples in clinical or translational multicenter trials. At the same time, the protocol design enables proficiency testing for the participating operators, instruments and centers.
Assessment of cytoplasmic dsDNA for cellular immunogenicity.
Mondal I, Das O, Sun R … +1 more , Lu RO
Methods Cell Biol · 2026 · PMID 41688148 · Publisher ↗
Detection of cytosolic double-stranded DNA (dsDNA) is critical for understanding its role in innate immunity, infection, cancer, and autoimmune diseases. Various methods have been developed to identify and quantify cytos...
Detection of cytosolic double-stranded DNA (dsDNA) is critical for understanding its role in innate immunity, infection, cancer, and autoimmune diseases. Various methods have been developed to identify and quantify cytosolic dsDNA, each offering unique strengths in terms of sensitivity, specificity, and throughput. Immunofluorescence microscopy enables direct visualization of cytosolic dsDNA, providing insights into its localization and dynamics within cells. Enzyme-linked immunosorbent assays (ELISAs) and immunoblotting techniques detect dsDNA indirectly through associated proteins like cGAS. Fluorescent spectroscopy-based methods allow for rapid and specific quantification of dsDNA. Additionally, biosensors and nanotechnology-based approaches are emerging as novel tools for dsDNA detection with enhanced sensitivity. This methodological compendium provides an overview of the common lab bench methodologies, highlighting their applications, limitations, and potential advancements in the detection of cytosolic dsDNA in various sample forms, such as cells, sectioned tissue, and cellular cytosol extracts.
Large-scale CRISPR-Cas9 screens to define regulators of immune checkpoints.
Mu M, Melms JC, Ho P … +1 more , Izar B
Methods Cell Biol · 2026 · PMID 41688147 · Publisher ↗
Immune checkpoints, which have emerged as potent target for the treatment of a variety of cancers, are central to tumor immunobiology and deciphering their dynamic regulation will continue to enable therapeutic developme...
Immune checkpoints, which have emerged as potent target for the treatment of a variety of cancers, are central to tumor immunobiology and deciphering their dynamic regulation will continue to enable therapeutic development. CRISPR-Cas9 screening has recently been leveraged as a powerful tool to systematically interrogate regulators of immune checkpoints. Here, we describe a framework for such screens coupled with fluorescence-activated cell sorting (FACS) as a reliable and direct method of isolating and comparing how specific CRISPR perturbations impact the expression and maintenance of immune checkpoints. This approach has provided critical insights into immune checkpoint regulation and interactions in melanoma models and can feasibly be expanded to other systems.
Cytometric assessment of antigen-specific T cell signaling.
Walsh ZH, Shah S, Melms JC … +1 more , Izar B
Methods Cell Biol · 2026 · PMID 41688146 · Publisher ↗
T cell receptor (TCR) signaling strength influences critical T cell characteristics including cytotoxic capacity, differentiation, memory formation, and exhaustion. Naturally occurring or engineered single-nucleotide var...
T cell receptor (TCR) signaling strength influences critical T cell characteristics including cytotoxic capacity, differentiation, memory formation, and exhaustion. Naturally occurring or engineered single-nucleotide variants (SNVs) and gene deletions which modulate T cell signaling pathways can significantly impact the potency of T cell cytolytic activity, including in the setting of T cell-based immunotherapies such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor T cell (CAR T) therapy. Thus, studying T cell signaling represents a valuable component of the engineering and preclinical testing process for cell therapies and immune checkpoint blockade (ICB). Flow cytometry is a powerful experimental and diagnostic tool which enables rapid, quantitative analysis of T cell signaling responses by phosphoprotein detection with fluorophore-labeled antibodies. However, many approaches that have been developed to induce and study T cell signaling rely on supraphysiological and non-specific stimulation methods, such as crosslinking of T cell CD3 and CD28 or treatment with chemical stimulating agents such as ionomycin and phorbol 12-myristate 13-acetate (PMA). These assays bypass the endogenous TCR machinery and limit the conclusions which can be drawn regarding the physiological relevance of the T cell responses measured. Here, we present a simple, efficient, and scalable workflow to assess physiological T cell signaling responses to antigen-specific stimulation with cognate peptide-MHC expressing target cells using flow cytometry. With minimal modifications, this approach can be successfully applied to the study of chimeric antigen receptor (CAR) signaling or signaling in other immune cell subtypes such as B cells, using analogous antigen-matched co-culture systems.
In situ imaging of pulmonary invariant Natural Killer T cells in precision cut lung slices.
Langelaar T, de Visser KE, Burrello C
Methods Cell Biol · 2026 · PMID 41688145 · Publisher ↗
Invariant Natural Killer T (iNKT) cells are key players in bridging innate and adaptive immunity, recognizing lipid antigens presented by CD1d molecules. Flow cytometry has tremendously contributed to the study of iNKT c...
Invariant Natural Killer T (iNKT) cells are key players in bridging innate and adaptive immunity, recognizing lipid antigens presented by CD1d molecules. Flow cytometry has tremendously contributed to the study of iNKT cell biology through the use of CD1d tetramers. While this technique was pivotal to uncover the anti-tumor potential of iNKT cells, spatial characteristics are difficult to assess as effective tools are lacking. Here, we describe a method for detecting iNKT cells in mouse lung tissue by employing Precision Cut Lung Slices (PCLS) alongside CD1d tetramer staining and fluorescence imaging. This technique maintains lung architecture while facilitating the spatial localization of iNKT cells within the pulmonary milieu. The accuracy of our imaging approach was validated with critical negative controls, including unloaded CD1d tetramer staining and iNKT cell-deficient (Traj18) mice. Co-staining with anti-CD3 confirmed the specificity of the CD1d tetramer signal and validated iNKT cell detection. This method provides a valuable tool to map iNKT cell localization and proximity to cells and histological structures, offering a novel angle to study their biology in lungs.
Mouse and human T cell Cas9-RNP/nucleofection-mediated gene-editing.
Sohn M, Pantsulaia G, Brody J
Methods Cell Biol · 2026 · PMID 41620285 · Publisher ↗
CRISPR/Cas9 technology has revolutionized genome editing, providing a precise and expeditious means of genetic modification. This breakthrough has enhanced our understanding of gene function, including T cell immunology....
CRISPR/Cas9 technology has revolutionized genome editing, providing a precise and expeditious means of genetic modification. This breakthrough has enhanced our understanding of gene function, including T cell immunology. Efficient gene editing in primary T cells not only offers a robust investigative tool for dissecting gene function but also holds promise for advancing T cell-based immunotherapies, including next-generation chimeric antigen receptor T cells. In this article, we introduce a highly efficient gene editing protocol for unstimulated human CD8 T cells and unstimulated and stimulated murine CD8 T cells, utilizing transient nucleofection of ribonucleoprotein complexes composed of synthesized modified single guide RNAs (sgRNAs) and purified Cas9 protein. This protocol, initially devised for primary CD8 T cells, can be readily adapted to other primary cell cultures through optimization of nucleofection conditions as well. In essence, our method provides a practical and powerful approach for achieving precise and swift gene knockout in primary CD8 T cells.
Quantification and functional assessment of antigen(-) tumor cell variants.
Odak I, Xie X, Pantsulaia G … +1 more , Brody J
Methods Cell Biol · 2026 · PMID 41620284 · Publisher ↗
T-cell redirecting therapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies (bsAbs) creating an immunological synapse between target and effector cells are at the forefront of cancer immunother...
T-cell redirecting therapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies (bsAbs) creating an immunological synapse between target and effector cells are at the forefront of cancer immunotherapies. Selective pressure by these therapies can cause the enrichment of antigen negative (Ag)/ tumor cell variants which are resistant to therapy and may drive clinical relapse, i.e. Ag escape. Ag cells generally exist in the tumor tissue prior to immunotherapies. Therefore, Ag escape is an integral part of tumor survival and poses a significant hurdle to overcome. Here we describe an approach used to quantify and functionally assess Ag tumor cells. This method enables high-throughput screening of single-cell biopsy or PBMC samples and allows for the assessment of their propensity to be targeted in a straightforward in vitro assay. Methods described here can be modulated and be incorporated to study samples derived from different anatomical locations and can be incorporated in a larger panel of flow cytometry for extensive phenotyping.
Measuring T-cell/CAR T-cell bystander tumor killing in vitro and in vivo.
Lin MJ, Charytonowicz D, Brody J
Methods Cell Biol · 2026 · PMID 41620283 · Publisher ↗
In the era of T cell-mediated immunotherapies, a central and growing problem is the recurrence of tumors lacking target antigen (Ag). Strategies that can prevent outgrowth of antigen-loss cells may improve response to th...
In the era of T cell-mediated immunotherapies, a central and growing problem is the recurrence of tumors lacking target antigen (Ag). Strategies that can prevent outgrowth of antigen-loss cells may improve response to therapy more effectively than those that rely on identifying multiple Ag targets after resistance arises. In addition to a T cell's direct killing response to binding cognate Ag, upregulation of death-receptor ligands and secretion of pro-inflammatory cytokines contribute to the indirect killing of surrounding, antigen-negative (bystander) cell populations, in a process termed "bystander killing". To investigate the mechanism and scope of T cell bystander killing, we describe methods of in vitro killing assays with flow cytometry and live microwell imaging, as well as in vivo tumor models with bioluminescent imaging and multiphoton live imaging, to observe this process in real time. The approaches can be easily adapted to investigate many other tumor types, T cell therapies, and targeting strategies.
High-parameter T-cell spectral phospho-flow-cytometry.
Pantsulaia G, Brody J
Methods Cell Biol · 2026 · PMID 41620282 · Publisher ↗
Phospho-flow is an invaluable tool for investigation into immunological signaling, enabling concurrent staining of cell lineage markers alongside sensitive intracellular phospho-proteins. Phospho-flow holds promise as a...
Phospho-flow is an invaluable tool for investigation into immunological signaling, enabling concurrent staining of cell lineage markers alongside sensitive intracellular phospho-proteins. Phospho-flow holds promise as a powerful diagnostic and therapeutic tool that can enable signal profiling, cell phenotyping, drug screening, pharmacodynamic profiling and assessment of drug efficacy across multiple cell types. When combining phospho-flow with fluorescent cell barcoding (FCB) multiplexing technique, high throughput flow cytometry can be achieved. FCB enhances experiment robustness while reducing variability in staining and decreasing antibody usage. Despite its utility, inter-operator technique variability persists, highlighting the need for protocol and analysis standardization. Here we describe an experimental mechanism for stimulating mouse and human T cells that demonstrates robust activation that can be adapted for various experimental designs.
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