Accurate assessment of cell viability is fundamental in biomedical research, with applications ranging from cancer biology to drug discovery. Traditional assays based on metabolic activity or membrane integrity are cost-...Accurate assessment of cell viability is fundamental in biomedical research, with applications ranging from cancer biology to drug discovery. Traditional assays based on metabolic activity or membrane integrity are cost-effective but limited to endpoint measurements, often overlooking the dynamic nature of cell survival and death. Here, we present a workflow that combines live cell imaging with automated image analysis to provide continuous, unbiased quantification of cell viability. Using a fluorescent marker of membrane integrity and nuclear staining, time-lapse microscopy captures cell fate dynamics under diverse experimental conditions. Automated segmentation and classification, implemented through the open-source DIPlib library, enable reproducible distinction between viable and non-viable cells while minimizing observer bias. The method is scalable, adaptable to different imaging platforms and suitable for high-throughput applications.
Adoptive cell therapies based on immune effector cells - Natural Killer (NK) cells, Cytotoxic T Lymphocytes (CTL) - represent a promising approach for the treatment of resistant tumors, especially for the management of h...Adoptive cell therapies based on immune effector cells - Natural Killer (NK) cells, Cytotoxic T Lymphocytes (CTL) - represent a promising approach for the treatment of resistant tumors, especially for the management of hematological malignancies. The rapid growth of this field emphasises the need for reliable in vitro methods to evaluate the cytotoxic potential of the cell products developed against the desired target cells, many of which are cells in suspension and consequently pose additional technical challenges. Here, we present a flow-cytometry-based protocol for the quantification of tumor cell death after co-culture with immune effector cells. Compared to earlier techniques, this approach allows for the identification of target cells and quantification of both their basal and effector cell-induced apoptotic death. The protocol described is highly adaptable, making it suitable for the study of fragile or limited patient samples, the evaluation of combination therapies with drugs or antibodies, and for the analysis of additional cellular markers or cell death mechanisms. Altogether, the protocol provides an assay setup and gating strategy that generate precise and reproducible data, essential for adoptive cell therapy optimization.
Apoptotic cells undergo various morphological changes as they die, including membrane blebbing, apoptopodia formation, and the generation of apoptotic bodies. Recently, a novel step in the disassembly of apoptotic adhere...Apoptotic cells undergo various morphological changes as they die, including membrane blebbing, apoptopodia formation, and the generation of apoptotic bodies. Recently, a novel step in the disassembly of apoptotic adherent cells was described, wherein cells retract from the substrate leaving behind phosphatidylserine and F-actin rich membrane remnants that are tightly anchored to the substrate. Over time, this membrane, coined the 'FOotprint Of Death' or 'FOOD' rounds into extracellular vesicles known as 'FOOD-derived Apoptotic cell derived extracellular vesicles' or 'F-ApoEVs'. Here, we detail an imaging approach to monitor the morphological changes of apoptotic cells as they retract from the extracellular substrate and generate FOOD and F-ApoEVs, using fluorescent conjugated annexin V and actin cytoskeletal stains. With some variations, we expect this approach to be compatible with a range of fluorescent dyes, stains, cell types, and methods of apoptosis induction to detect the formation of FOOD/ F-ApoEV during apoptosis.
Cell death is a fundamental biological process with critical roles in both normal physiology and pathological conditions, especially programmed cell death, such as apoptosis, necroptosis and pyroptosis. Programmed cell d...Cell death is a fundamental biological process with critical roles in both normal physiology and pathological conditions, especially programmed cell death, such as apoptosis, necroptosis and pyroptosis. Programmed cell death is mediated by cascade signaling transduction rely on protein-protein interaction. Necroptosis mediator RIPK1, RIPK3 and MLKL have been shown to be regulated by different types of post-translational modifications (PTMs), suggesting that additional factors must associate with them during necroptosis. Proximity labeling (PL) has been used to tag and identify proteins, RNAs, or other biomolecules in close proximity (∼10-20 nm) to a target protein of interest. Therefore, utilizing proximity labeling coupled mass spectrometry to identify weak and transient interactors of necroptosis mediators, will be helpful for the further understanding of cell death mechanisms and functions. Here, in this chapter, we provide a step-by-step protocol for using TurboID-based proximity labeling to map interactors and regulators of key necroptotic proteins (RIPK1, RIPK3, MLKL and ZBP1).
High-content screening (HCS) is a powerful approach for rapidly and efficiently assessing the harmfulness of numerous compounds across a wide range of cultured cell types. We recently developed a fully automated, miniatu...High-content screening (HCS) is a powerful approach for rapidly and efficiently assessing the harmfulness of numerous compounds across a wide range of cultured cell types. We recently developed a fully automated, miniaturized HCS wet-plus-dry pipeline (MITOMATICS) which leverages mitochondrial morphology as a sensitive and dynamic biomarker of cellular health or damage. Mitochondria are indeed not only vital for energy production and homeostasis, but also serve as critical gatekeepers of apoptotic cell death. MITOMATICS incorporates a proprietary software tool (MitoRadar) designed in-house to perform fast, comprehensive and cost-effective analysis of mitochondrial morphology in live cells. Together, the pipeline and its associated big data analytics software provide a valuable framework for early detection of acute mitotoxic effects of chemicals agents or physical stressors. To illustrate this, we present here a complete protocol for quantifying the impact of the pesticide chlorpyrifos-methyl on mitochondrial morphology of human lung epithelial BEAS-2B cells. Our results show that chlorpyrifos-methyl, even as a single compound, induces profound disruptions in mitochondrial subcellular structure. Beyond this case study, MitoRadar opens up promising avenues for investigating mitotoxicity across diverse cell types and environmental exposures, paving the way for a new generation of cellular diagnostics that could be of interest to the cell death community.
Cell death is essential for the survival and homeostasis of nearly all organisms. Programmed cell death (PCD) originally described the reproducible elimination of specific cells during metazoan development. Apoptosis, th...Cell death is essential for the survival and homeostasis of nearly all organisms. Programmed cell death (PCD) originally described the reproducible elimination of specific cells during metazoan development. Apoptosis, the best-studied form of PCD, is characterized by distinct morphological, cellular, and biochemical hallmarks. However, the identification of alternative cell death pathways has highlighted the need to clearly distinguish among them, especially when exploring new paradigms of developmental alternative cell death pathways. Furthermore, the discovery of numerous non-lethal functions of apoptotic caspases, collectively termed non-lethal caspase-dependent processes (CDPs), has underscored the importance of differentiating apoptosis from CDPs within tissues in vivo. Morphological analysis remains essential for differentiating death pathways and for determining whether cells are dying or surviving, yet such analyses traditionally relied on electron microscopy, which is labor-intensive, costly, and technically demanding. Expansion microscopy (ExM), a technique that physically enlarges biological samples isotropically, has transformed light microscopy by enabling super- and ultra-resolution imaging using standard fluorescence or confocal platforms. Here, we describe two ExM-based protocols optimized for, but not limited to, Drosophila tissues: ExM, achieving ∼4× expansion, and pan-ExM, yielding ∼12-16× expansion. ExM supports super-resolution imaging of immunofluorescent labels, whereas pan-ExM provides access to ultrastructural features, albeit with limited capacity for specific protein detection. We also compare apoptotic and non-apoptotic cell death morphologies before and after ExM and pan-ExM. Given the growing diversity of ExM strategies, this chapter offers a comprehensive introduction to these rapidly advancing methodologies.
Efferocytosis is a critical process by which phagocytes identify, engulf and clear apoptotic cells, thereby preserving immune tolerance and tissue homeostasis. While in vitro assays have advanced our understanding of the...Efferocytosis is a critical process by which phagocytes identify, engulf and clear apoptotic cells, thereby preserving immune tolerance and tissue homeostasis. While in vitro assays have advanced our understanding of the molecular mechanisms underlying apoptotic cell clearance, they lack the complexity of the physiological environment. This chapter presents a detailed in vivo approach for assessing efferocytosis by mouse peritoneal macrophages using the intraperitoneal (IP) clearance assay. By administering fluorescently labelled apoptotic Jurkat T cells into the peritoneal cavity, this method enables the quantification of apoptotic cell uptake in a native tissue context, capturing the influence of local immune signals, stromal interactions and systemic factors. This protocol is compatible with genetic and pharmacological manipulations, allowing researchers to investigate how various interventions affect efferocytic capacity. Overall, this approach provides a robust and physiologically relevant framework for studying efferocytosis and its implications in both homeostatic and pathological settings.
Alterations in programmed cell death pathways play a critical role in cancer development and maintenance. Yet the detailed mechanisms contributing to tumor initiation, progression, and therapeutic response in lung cancer...Alterations in programmed cell death pathways play a critical role in cancer development and maintenance. Yet the detailed mechanisms contributing to tumor initiation, progression, and therapeutic response in lung cancer remain incompletely understood. Also, models to study how changes in the cell death machinery impact tumor-immune interactions are limited. To address this, we describe two complementary murine models of lung adenocarcinoma that enable functional interrogation of cell death pathways in vivo. The first model is a clinically relevant, genetically engineered mouse model (GEMM) driven by Kras activation and Tp53 loss, in which somatic CRISPR-Cas9-mediated gene editing permits tumor cell-specific knockout of candidate genes, facilitates in-depth studies of programmed cell death and immune signaling within 19 weeks. The second approach illustrates how a syngeneic orthotopic transplantation model can be used to study target genes and pathways that influence the tumor microenvironment and immunogenic cell death in a two-week timeframe. Together, these methods provide reproducible and versatile tools to investigate how modulation of cell death pathways impacts lung cancer development and progression and affects the tumor immune microenvironment, thus providing important information to guide the development of novel therapeutic strategies in lung cancer.
The sarco-endoplasmic reticulum (SR/ER) forms an extensive network within muscle fibers, playing a central role in calcium (Ca2 +) homeostasis, protein synthesis, folding, sorting and quality control. ER functional distu...The sarco-endoplasmic reticulum (SR/ER) forms an extensive network within muscle fibers, playing a central role in calcium (Ca2 +) homeostasis, protein synthesis, folding, sorting and quality control. ER functional disturbance causes endoplasmic reticulum (ER) stress and leads to the accumulation of unfolded or misfolded proteins, thereby triggering the unfolded protein response (UPR) to alleviate cellular stress and re-establish homeostasis. ER stress can be triggered by defects in the balance between protein folding and degradation, altered calcium and lipid levels and changes in ER-mitochondria contacts. Accumulating evidence suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders characterized by myofiber degeneration and replacement with fibrotic tissue. In particular, myopathy could be due to chronic ER stress and a maladaptive response leading to defective muscle force and atrophy. Considering this evidence, the characterization of degenerated muscle fibers could be conducted through the analysis of proteins involved in endoplasmic reticulum stress. Here we propose a protocol, intended as a practical and reproducible workflow, for evaluating ER-stress associated proteins in muscle tissue.
The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effec...The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.
Copper is an essential trace element involved in key biological processes, including cellular respiration, antioxidant defense, and enzymatic activity. Intracellular copper homeostasis is regulated through coordinated up...Copper is an essential trace element involved in key biological processes, including cellular respiration, antioxidant defense, and enzymatic activity. Intracellular copper homeostasis is regulated through coordinated uptake, trafficking, storage, and excretion mechanisms. Disruption of this balance can result in copper overload, leading to cuproptosis-a recently characterized, copper-dependent form of regulated cell death marked by aggregation of lipoylated mitochondrial proteins, disruption of the tricarboxylic acid cycle, and proteotoxic stress. Cuproptosis has been implicated in the pathogenesis of neurodegenerative diseases, cancer, and metabolic disorders. Accurate monitoring of intracellular copper dynamics is therefore critical for understanding disease mechanisms. In this chapter, we describe the use of fluorogenic probes for detecting intracellular copper ions in cultured cells, providing practical guidelines for their application.
Antigen-specific T cell responses are critical for effective cancer cell elimination and serve as the basis for T cell-based immunotherapies. Here, we present an in vitro system that models key steps of antigen-specific...Antigen-specific T cell responses are critical for effective cancer cell elimination and serve as the basis for T cell-based immunotherapies. Here, we present an in vitro system that models key steps of antigen-specific T cell activation and cancer cell killing. Using OT-1 and P14 T cell receptor (TCR) transgenic mouse strains, we generate cytotoxic T lymphocytes (CTLs) through in vitro priming with cognate peptides. Cancer cells are engineered to present antigens via lentiviral transduction or peptide pulsing, facilitating the formation of MHC class I-antigen complexes recognized by the TCRs. These antigen-expressing cancer cells are then co-cultured with CTLs to assess antigen-specific cytotoxicity. Cancer cell viability is quantified by assessing the metabolic activity of living cells, providing a precise measure of CTL killing efficiency. As both lentiviral transduction and peptide pulsing are broadly applicable to various cancer cell models, this platform offers a versatile tool for dissecting antigen-specific cancer cell-T cell interactions and evaluating immunotherapeutic strategies.
Chimeric antigen receptor (CAR) T cell therapy has redefined cancer immunotherapy, offering remarkable efficacy against hematologic malignancies and solid tumors. However, CAR T cell therapy targeting T cell malignancies...Chimeric antigen receptor (CAR) T cell therapy has redefined cancer immunotherapy, offering remarkable efficacy against hematologic malignancies and solid tumors. However, CAR T cell therapy targeting T cell malignancies is limited by fratricide, self-directed cytotoxicity caused by shared antigen expression on both therapeutic and endogenous T cells. This process impairs CAR T cell expansion, viability, and overall efficacy. In this study, we present a live cell microscopy-based methodology to visualize and quantify CAR T cell fratricide and immune synapse. Through fluorescent labeling, time-lapse imaging and automated analysis, we track dynamic T cell interactions, cytotoxic events, and synaptic structures. This protocol enables precise measurement of fratricide events kinetics, serial killing behavior, and immune synapse morphology, thereby offering valuable insights into CAR design optimization and underlying mechanisms. This imaging-based approach complements conventional assays by providing temporally and spatially resolved data, thereby enhancing our understanding of CAR T cell function and cytotoxic regulation in the context of fratricide.
Chimeric Antigen Receptor T-cell (CAR-T) based immunotherapy represents a ground-breaking advancement in the treatment of certain haematological malignancies, such as acute lymphoblastic leukemia (ALL) and multiple myelo...Chimeric Antigen Receptor T-cell (CAR-T) based immunotherapy represents a ground-breaking advancement in the treatment of certain haematological malignancies, such as acute lymphoblastic leukemia (ALL) and multiple myeloma (MM), often leading to remarkable remission rates. Despite these successes, many patients relapse within the first-year post-treatment, highlighting a critical limitation of these therapies. Furthermore, translating the success of CAR-T therapies to solid tumors has been challenging, with limited clinical efficacy, highlighting the need for innovative designs and strategies to achieve durable responses. There is strong scientific interest in enhancing CAR-T antitumor efficacy in solid tumors. Current research efforts are focused primarily on two main strategies: first, the development of enhanced fourth- and fifth-generation CAR-T cells through genetic modification; and second, the optimization of the manufacturing protocols to improve cell quality and functionality. All these novel CAR-T cell designs must be rigorously characterized using standardized and robust protocols. In this review, we describe a protocol for generating murine CAR-T cells and, in detail, the methodology to assess their lytic capacity-both through a real-time impedance-based assay and a conventional endpoint cytotoxicity assay.
The recognition of antigens by B cells is the initial step in the humoral response leading to antibody production and antibody-mediated immunological memory. The majority of antigens encountered by B cells are presented...The recognition of antigens by B cells is the initial step in the humoral response leading to antibody production and antibody-mediated immunological memory. The majority of antigens encountered by B cells are presented in a membrane-tethered format on antigen presenting cells (APCs). Antigen-specific recognition by B cell receptors triggers the formation of an immunological synapse (IS) via a spreading and contraction response. This is critical in driving affinity discrimination and maturation by modulating the amount of antigen internalized and presented to acquire T cell help. The interaction between B cells and APCs is mimicked in vitro by planar lipid bilayers (PLBs), which have been used to reveal the importance of biophysical properties such as antigen mobility and density in triggering B cell activation. With the advent of DNA origami enabling precise control over antigen display with nanometer precision, further dissection of spatial requirements underlying IS formation is now possible. To this end, here we describe how to generate DNA origami nanogrids, attach them to PLBs in two different housing systems, and use them to stimulate B cell IS formation.
The ever more insightful dissection of the NK cell population in humans has produced a plethora of (more or less) defined subpopulations with quite differentiated functional properties. Considering the potential clinical...The ever more insightful dissection of the NK cell population in humans has produced a plethora of (more or less) defined subpopulations with quite differentiated functional properties. Considering the potential clinical perspectives offered by peripheral blood NK cells, it is crucial to design strategies to functionally assess a large number of cell subsets directly from the peripheral mononuclear cells (PBMCs). In this chapter, we describe a simple method to quantify cytotoxic degranulation and IFN-γ production in classical NK cell subsets, focusing, particularly on the NKG2C+ "adaptive" NK cells.
Natural Killer (NK) cells are potent effectors of the innate immune system, whose cytotoxicity relies on the balance of activating and inhibitory signals they receive. The diverse array of NK cell receptors that integrat...Natural Killer (NK) cells are potent effectors of the innate immune system, whose cytotoxicity relies on the balance of activating and inhibitory signals they receive. The diverse array of NK cell receptors that integrate these signals plays a key role in defining the many functional and developmental NK cell subsets. However, identifying these subsets is challenging due to receptor diversity, necessitating large and complex flow cytometric panels. Designing such panels can be further complicated by the substantial co-expression of markers, making fluorophore selection difficult due to spectral overlap and compensation constraints. Moreover, simple NK cell phenotyping does not directly correlate with their function or response to specific stimuli, limiting insights into NK cell behavior. To address these challenges, we present a degranulation assay protocol using tumor cells, followed by a 31-marker spectral flow cytometry panel on purified human NK cells. This approach enables the simultaneous screening of activating and inhibitory receptor expression, exhaustion markers, cytokine production and degranulation (via CD107a). By providing a comprehensive framework, this method supports a variety of research applications.
Natural Killer (NK) cells are at the interface of the innate and adaptive immune system. While they are naturally able to kill pathogen-infected, malignant and compromised cells, they also drive the recruitment and activ...Natural Killer (NK) cells are at the interface of the innate and adaptive immune system. While they are naturally able to kill pathogen-infected, malignant and compromised cells, they also drive the recruitment and activation of other immune cells through the production of inflammatory cytokines and chemokines. Evaluation of NK cells cytotoxic activity is critical to determine accurately the anti-tumor potency of novel immunotherapies, in particular, when the later aim to overcome NK cells exhaustion driven by an immunosuppressive environment such as the tumor microenvironment. Here, we provide a detailed protocol for the assessment of primary NK cells cytotoxic functions against pancreatic cancer cells stimulated by a directed approach on NK cells based on the cytokine IL-15. This article describes the protocol of a flow cytometry-based assay and a calcein acetoxymethyl ester (AM) cell viability assay to quantify NK cell cytotoxic activity. Both methods are robust, fast and sensitive to be applicable in clinics and can readily be adapted for the assessment of other immunotherapies on NK cells cytotoxicity.
Mitochondria play a central role in cellular metabolism, ATP production, and redox homeostasis, all of which are essential for sustaining T lymphocyte and CAR-T cell effector functions. Mitochondrial dysfunction has been...Mitochondria play a central role in cellular metabolism, ATP production, and redox homeostasis, all of which are essential for sustaining T lymphocyte and CAR-T cell effector functions. Mitochondrial dysfunction has been linked to T cell and CAR-T cell exhaustion, reduced cell expansion capacity, and impaired tumor clearance. To investigate the contribution of mitochondria to T lymphocyte and CAR-T cell functionality, this protocol leverages advanced imaging analysis techniques to assess different parameters related to mitochondrial dynamics, structure and function, which may serve as indicators of both metabolic activity and the exhausted state of T cells. Specifically, the protocol focuses on measuring mitochondrial morphology, energization state, and mitochondrial translocation in the three-dimensional space. The image-based methodologies described in this protocol will contribute to a deeper understanding of mitochondrial regulation during T-cell and CAR-T cell responses. This knowledge will facilitate the identification of key metabolic vulnerabilities and support the refinement of therapeutic strategies to enhance T lymphocyte cytotoxic potential, ultimately improving clinical outcomes in T cell-based adoptive cancer immunotherapy.
Antibody dependent cellular cytotoxicity (ADCC) is an effector function performed by natural killer (NK) cells to target and clear viral infections and cancer. ADCC is a critical feature of several antibody and cellular...Antibody dependent cellular cytotoxicity (ADCC) is an effector function performed by natural killer (NK) cells to target and clear viral infections and cancer. ADCC is a critical feature of several antibody and cellular therapeutics as well as vaccination strategies. Using microscopy to understand the details of molecular events driving ADCC is essential to improving such therapeutics but has been limited by technologies that cannot practically provide the spatial resolution necessary to study protein function at the single molecule level in cells. In this chapter, we describe a model system using MINFLUX nanoscopy to study the molecular distribution of human FcγRIIIa (CD16a), the IgG receptor, in the NK cell immunological synapse during ADCC. The technique described here will enable further exploration of how CD16a drives NK cell ADCC and can also be applied to the study of other important protein receptors for which nanometer localization precision is needed.