Despite the significant expansion of the studies aimed at generating sophisticated in vitro systems to recreate 3D tissues and their natural perfusion, the use of animal models is still crucial to preclinically test the...Despite the significant expansion of the studies aimed at generating sophisticated in vitro systems to recreate 3D tissues and their natural perfusion, the use of animal models is still crucial to preclinically test the potential of immune-based anti-cancer therapies. Considering heterogeneity and complexity of the tumor systems, new models are continuously generated, to reproduce specific types of primary or metastatic malignancies, in the presence of murine or human immune environment. These models are adapted to answer peculiar questions regarding: the effectiveness of specific human immune cell subsets in controlling primary tumor growth or metastasis diffusion, the immune infiltration and the effects of tumor microenvironment, and, also, the relevance of key molecules involved in tumor cell recognition or immune suppression. In this chapter we present a model of human Non-small cell lung cancer (NSCLC) in NSG mice, which lack mature B, T, and NK cells, and are optimal recipient for the infusion and the study of human immune cells, including NK cells. In particular we describe analytical approaches of digital pathology and flow cytometry to characterize and quantify the dissemination of the tumor formations, and the activity of exogenous NK cells in terms of infiltration of the tumor tissue and control of tumor growth and spread.
Dendritic cells (DCs) are crucial in processing and cross-presenting tumor-associated antigens via MHC I molecules to prime CD8 T lymphocytes (CTLs) for antitumor immunity. However, the moderate regulation of antigen end...Dendritic cells (DCs) are crucial in processing and cross-presenting tumor-associated antigens via MHC I molecules to prime CD8 T lymphocytes (CTLs) for antitumor immunity. However, the moderate regulation of antigen endocytic escape from lysosomes into the cytoplasm hinders this cross-presentation process due to the lack of finely tunable tools at the organelle-specific level. Magneto-mechanical modulation technology provides a promising approach to regulate lysosomal membrane permeability (LMP) and promote antigen leakage for efficient cross-presentation. This approach relies on magnetic nanorobots within lysosomes that can exert adjustable intracellular forces in response to external rotating magnetic fields. This chapter presents a comprehensive protocol for evaluating the antitumor response of DCs induced by magneto-mechanical regulation. This protocol includes a step-by-step assessment of cytotoxicity on DCs caused by magneto-mechanical regulation, analysis of nanorobot colocalization with lysosomes, and their responses (such as LMP, cytoskeleton morphology, and mitochondria function) through fluorescence imaging. Additionally, we quantify DC mechano-activation and cross-presentation efficiency using flow cytometry.
Cytotoxic T Lymphocytes (CTLs) mediate tumor clearance and anti-tumoral responses through the elimination of target cancerous cells upon antigen recognition and TCR engagement. The killing of cancerous cells mediated by...Cytotoxic T Lymphocytes (CTLs) mediate tumor clearance and anti-tumoral responses through the elimination of target cancerous cells upon antigen recognition and TCR engagement. The killing of cancerous cells mediated by CTLs can be modulated by the strength of the TCR engagement, notably through the antigen affinity to the TCR, the quality of the immune synapse, the time of contact between CTLs and targets, among other parameters. Unveiling the mechanisms, kinetics and factors influencing cancer cell elimination by CTLs is essential for the amelioration of lymphocyte-based immunotherapies, such as CAR-T cells, and the understanding of tumoral clearance in vivo. Here, we provide a method to precisely monitor the cytotoxic capacity of murine CTLs in an antigen-specific manner, using ovalbumin (OVA) specific CTLs expressing the modified OT1 TCR and four different tumoral B16-F10 cell lines expressing OVA peptides with variable affinity for the OT1 TCR. We present step-by-step the implementation of a high throughput image-based pipeline allowing, on one hand, the time-lapse monitoring of CTL-mediated cytotoxicity and, on the other hand, the measurement of morphological parameters of CTLs and target cells based on fluorescent labeling. This method is suitable for ex-vivo exploration of lymphocyte cytotoxicity and related parameters, and can be adapted for mechanistic studies or screening approaches.
The therapeutic success of adoptive T cell therapy (ACT) is closely tied to the in vivo persistence and differentiation state of transferred T cells. Clinical evidence consistently shows that less differentiated T cells,...The therapeutic success of adoptive T cell therapy (ACT) is closely tied to the in vivo persistence and differentiation state of transferred T cells. Clinical evidence consistently shows that less differentiated T cells, particularly memory T cells with stem-like properties, offer superior antitumor activity and long-lasting immune responses. However, these stem-like memory T cells are challenging to expand efficiently using conventional in vitro culture protocols. Existing strategies, such as brief CD3/CD28 antibodies co-stimulation combined with cytokines like IL-7, IL-15, and IL-21, have shown limited success but remain costly and operationally complex, hindering scalability for clinical use. There is a critical need for a rapid, efficient, cost-effective, and clinically viable method to induce and expand this T cell subset. Here, we present an optimized mannose-based culture approach that enables robust in vitro expansion of stem-like memory T cells, offering a practical solution to current limitations and paving the way for improved ACT applications.
Chimeric Antigen Receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, yet its success in solid tumors remains limited due to the complexity of the tumor microenvironment (TME) and limi...Chimeric Antigen Receptor (CAR) T cell therapy has shown remarkable efficacy in hematologic malignancies, yet its success in solid tumors remains limited due to the complexity of the tumor microenvironment (TME) and limited penetration. To address this, we developed a versatile two-step staining assay to assess CAR T cell-mediated cytotoxicity both in vitro and ex vivo on viable tumor tissues. The method combines detection of caspase-3/7 activity with phenotypic characterization of the tumor cells, offering a reliable readout of CAR T cell efficacy. The assay is compatible with live tissue slices and tumor cell lines, and can be customized for various tumor types and antigens. This model provides a robust tool for preclinical screening of CAR T cell function in physiologically relevant contexts.
Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation. Although its biological and clinical relevance is increasingly clear, routine and specific detection of ferroptosis remains chall...Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation. Although its biological and clinical relevance is increasingly clear, routine and specific detection of ferroptosis remains challenging, especially in tissues and clinical specimens. Indirect measurements of ferroptosis such as lipophilic fluorescent probes (C11-BODIPY581/591), reactive aldehydes (MDA/4-HNE), and pharmacological rescue either lack specificity for ferroptosis, or are impractical for in vivo analysis. Recent work has identified hyperoxidized peroxiredoxin 3 (PRDX3), also known as SO-PRDX3, as a ferroptosis-specific biomarker: Under ferroptotic stress, mitochondrial PRDX3 undergoes selective and irreversible hyperoxidation at its catalytic cysteine residue to generate sulfinic/sulfonic derivatives, a reaction facilitating ferroptosis. Building on this biology, here we outline a method that detects ferroptosis in cells and tissues using 5H7c, a rabbit monoclonal antibody recognizing hyperoxidized but not unmodified PRDX3. The approach offers high specificity, broad sample compatibility (cell lines, animal tissues), and platform flexibility (Western blotting, immunohistochemistry, immunofluorescence).
Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation, with emerging relevance in cancer biology, particularly in therapy-resistant tumors such as colorectal cancer. Accurate and repro...Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation, with emerging relevance in cancer biology, particularly in therapy-resistant tumors such as colorectal cancer. Accurate and reproducible methods to monitor ferroptosis are essential for understanding its mechanisms and therapeutic potential. In this methodological paper, we present an optimized protocol for the evaluation of lipid peroxidation as a surrogate marker of ferroptosis in colon cancer cell models. Specifically, we describe an approach for quantifying malonylaldehyde and hydroxyalkenals (MDA+ 4-HDA), as key byproducts of lipid oxidation, using a colorimetric assay adapted for cell culture. This methodological framework provides a reliable basis for dissecting ferroptotic responses and for evaluating the activity of ferroptosis-inducing compounds in colorectal cancer research.
A myriad of diverse developmental and pro-death signals converge on the mitochondrial pathway of apoptosis, which is governed by the BCL‑2 family of proteins. Comprised of both pro- and anti-apoptotic family members, the...A myriad of diverse developmental and pro-death signals converge on the mitochondrial pathway of apoptosis, which is governed by the BCL‑2 family of proteins. Comprised of both pro- and anti-apoptotic family members, the BCL‑2 family functions to regulate mitochondrial outer membrane permeabilization (MOMP), often considered the "point of no return" in which a cell commits to an apoptotic outcome. Specifically, the effector BCL‑2 family proteins, BAX and BAK, are responsible for inducing MOMP and therefore investigations into their structural, cellular, and pharmacological regulation are critical to understanding the cellular commitment to apoptosis. A gold standard methodology for studying activation of BAX or BAK is the permeabilization of large unilamellar vesicles (LUVs), which are biochemically-defined model liposomes that mimic the major lipid composition of the outer mitochondrial membrane (OMM). Here, we provide a detailed protocol for generating LUVs containing a fluorescent dye/quencher pair to monitor real-time BAX activation and membrane permeabilization using a standard plate reader. Additionally, we detail example assay strategies to model interactions within the BCL‑2 family and provide a robust mathematical model for fitting and parameterizing kinetic LUV permeabilization data.
Cell death complexes, such as the complex II and necrosome, play pivotal roles in executing programmed cell death pathways. Understanding the composition and dynamics of these complexes is essential for elucidating the m...Cell death complexes, such as the complex II and necrosome, play pivotal roles in executing programmed cell death pathways. Understanding the composition and dynamics of these complexes is essential for elucidating the molecular mechanisms underlying apoptosis and necroptosis. This protocol describes a robust method for the affinity purification of endogenous or tagged cell death complexes from mammalian cells, followed by analysis through western blotting and optional mass spectrometry. This approach enables investigation of protein-protein interactions and the dynamic assembly of complexes under various stimuli or genetic conditions, providing insights into their mechanisms in health and disease.
Apoptosis, a tightly regulated form of programmed cell death, eliminates damaged or malignant cells and is triggered by internal or external stress signals. A critical decision point is mitochondrial outer membrane perme...Apoptosis, a tightly regulated form of programmed cell death, eliminates damaged or malignant cells and is triggered by internal or external stress signals. A critical decision point is mitochondrial outer membrane permeabilization (MOMP), governed by BCL-2 family proteins. Pro-apoptotic members such as BAX and BAK form pores in the mitochondrial outer membrane, releasing intermembrane space proteins like cytochrome c into the cytoplasm. Once cytosolic, cytochrome c binds APAF-1 to form the apoptosome, which activates caspase-9 and subsequently caspase-3, driving apoptosis through cleavage of key cellular substrates. Cytochrome c release serves as a hallmark and point of no return in the apoptotic cascade. However, cytochrome c release can be variable, occurring at submaximal levels or from only a subset of mitochondria, which complicates detection in heterogeneous cell populations. To address this, we developed a semi-automated imaging-based method to quantify cytochrome c release at the single-cell level using immunofluorescence microscopy. Our approach uses CellProfiler, an open-source image analysis platform, to implement a pipeline that segments adherent cells into nuclear, mitochondrial, and cytoplasmic compartments based on compartment-specific reference stains. The pipeline quantifies cytochrome c distribution across these compartments, calculating the ratio of mitochondrially retained to cytoplasmic cytochrome c for each cell. Automation of segmentation and measurement ensures rapid, robust, and reproducible analysis, with only image acquisition and data interpretation performed manually. This method provides a quantitative readout of MOMP and can be readily adapted to any immunofluorescence-detectable protein given an appropriate compartmental marker, expanding its utility for broader cellular studies.
Induced pluripotent stem cells (iPSCs) represent an innovative tool to model neurodegenerative disorders, providing access to disease-relevant cell types such as neurons that are otherwise inaccessible. In the context of...Induced pluripotent stem cells (iPSCs) represent an innovative tool to model neurodegenerative disorders, providing access to disease-relevant cell types such as neurons that are otherwise inaccessible. In the context of Alzheimer's disease (AD), iPSC-derived neural cultures offer a unique opportunity to investigate pathological mechanisms in a controlled environment, overcoming limitations of animal models. Central to AD pathogenesis is the amyloid cascade hypothesis, which is based on the concept that accumulation and aggregation of the toxic oligomeric species, initiates a cascade of events leading to synaptic dysfunction, neuronal loss, and cognitive decline. It is known that application of the Aβ oligomers to neurons reproduces key features of synaptic impairment, preceding overt neuronal death. In this study, we proposed an optimized protocol employing iPSC-derived neurons exposed to Aβ1-42 peptide. This approach provides a clear and reliable method to evaluate neurotoxic effects of Aβ peptide on neuronal morphology and viability. Indeed, on the one hand neuronal morphology, assessed through immunofluorescence using specific neuronal markers, allows precise monitoring of neurite length and synaptic connectivity, crucial parameters to evaluate neuronal health. On the other hand, cytotoxicity is directly quantified using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, which confirm Aβ-induced neuronal injury. Notably, this combined approach provides novel insights into early Aβ-driven neurodegenerative processes and offers a platform to identify therapeutic strategies targeting the initial phases of AD pathology.
This protocol provides detailed methods for the preparation, culture, and maintenance of two essential primary cell lines used in cell death studies: mouse embryonic fibroblasts (MEFs) and bone marrow-derived macrophages...This protocol provides detailed methods for the preparation, culture, and maintenance of two essential primary cell lines used in cell death studies: mouse embryonic fibroblasts (MEFs) and bone marrow-derived macrophages (BMDMs). These primary cells serve as valuable models for investigating molecular mechanisms of cell death pathways due to their physiological relevance compared to immortalized cell lines. The protocol covers isolation techniques from mouse embryos (for MEFs) and bone marrow (for BMDMs), differentiation protocols, culture conditions, cryopreservation methods. This comprehensive guide enables researchers to establish reliable primary cell culture systems for apoptosis, necroptosis, pyroptosis, and other cell death-related studies.
Type 1 conventional dendritic cells (cDC1s) play a central role in directing CD8 T-cell responses. Mechanistic studies on cDC1-CD8 T-cell communication are hindered by the scarcity of cDC1s and their inability to be prop...Type 1 conventional dendritic cells (cDC1s) play a central role in directing CD8 T-cell responses. Mechanistic studies on cDC1-CD8 T-cell communication are hindered by the scarcity of cDC1s and their inability to be propagated in vitro. Furthermore, dendritic cells derived from bone marrow or monocytes generally lack phenotypic and functional properties of ex vivo cDC1s. Here, we present a controlled in vitro CD8 T-cell priming platform based on the isolation of ex vivo primary cDCs from spleens of B16-FLT3L tumor-bearing mice. This system enables read out of CD8 T-cell proliferation and cytotoxic effector differentiation in response to debris from (tumor) cells that die by different modes of cell death. The set up can also be modified to include read outs for cDC status and phagocytic activity. Our approach establishes a reproducible and physiologically relevant method to study cDC-T-cell interactions in vitro, with broad applications in tumor immunology and vaccine development.
Autophagy-dependent cell death (ADCD) describes a form of regulated cell death (RCD) that is driven by autophagy machinery. While other modes of RCD such as apoptosis and necrosis can be defined by distinct cellular feat...Autophagy-dependent cell death (ADCD) describes a form of regulated cell death (RCD) that is driven by autophagy machinery. While other modes of RCD such as apoptosis and necrosis can be defined by distinct cellular features, the characterisation of ADCD relies primarily on genetic manipulation, with limited guidelines for assessing cellular morphology. Drosophila melanogaster larval midgut histolysis provides the strongest in vivo evidence of developmental ADCD, resulting in degradation of gastric caeca and the proventriculus during larval-to-pupal transition (metamorphosis). To establish morphological hallmarks of ADCD, in this protocol, we describe methods for dissecting and preparing larval midguts for morphological characterisation of ADCD using transmission electron microscopy (TEM).
Cancer research is a rapidly expanding field, requiring new methods and techniques to investigate the complex biology of the disease and its characteristics. A pivotal aspect of this research is the evaluation of the eff...Cancer research is a rapidly expanding field, requiring new methods and techniques to investigate the complex biology of the disease and its characteristics. A pivotal aspect of this research is the evaluation of the effects and efficacy of therapies. Here, we describe a simple protocol to generate mammospheres from MCF-7 cells, a useful model of breast cancer stem cells to evaluate cell death after therapy treatment. This subpopulation of cells, found in all types of tumors, is critically involved in therapeutic response as well as in disease recurrence and relapse, and therefore requires particular attention in oncology. The use of mammospheres represents a valuable approach to obtain preliminary in vitro results that closely resemble in vivo responses, providing an effective means to assess the efficacy of both conventional and next-generation therapeutic agents on breast cancer cell death.
Samra A, Radoua A, Tanaydin ES
… +12 more, Alrustom B, Bordessoules M, Fischer T, Callot M, Georgsson S, Rávai B, Mátravölgyi B, Bálint E, Eriksson LA, Chluba J, Plenchette S, Micheau O
Methylene blue is a versatile dye used in cell biology to quantify cell adhesion and indirectly assess cell death. Unlike viability assays that rely on metabolic activity or membrane integrity, methylene blue binds to ce...Methylene blue is a versatile dye used in cell biology to quantify cell adhesion and indirectly assess cell death. Unlike viability assays that rely on metabolic activity or membrane integrity, methylene blue binds to cellular proteins, making it particularly effective for staining adherent cells. This property is the basis of the assay we describe here, which allows direct measurement of cell detachment, a hallmark of various cell death processes, including apoptosis and anoikis. Using a simple staining and washing procedure, methylene blue selectively labels cells that remain attached to the culture surface after detached or dying cells have been removed. After washing, the retained dye can then be eluted and quantified spectrophotometrically, providing a reliable and cost-effective method for evaluating cytotoxicity, drug responses, and adhesion dynamics. This chapter outlines the principles, advantages, and practical applications of methylene blue staining in cell-based assays, highlighting its role as a powerful tool for studying cell survival, or detachment in both basic and applied research.
Macrophages are innate immune cells that are critical in the maintenance of tissue homeostasis and defense against pathogens. Programmed cell death is a critical tool macrophages use to clear pathogens and to alert surro...Macrophages are innate immune cells that are critical in the maintenance of tissue homeostasis and defense against pathogens. Programmed cell death is a critical tool macrophages use to clear pathogens and to alert surrounding cells to damage following induction of cell death. Diverse forms of cell death, including pyroptosis, necroptosis, ferroptosis, and secondary apoptosis following apoptosis, result in loss of plasma membrane integrity. Membrane disruption occurs following the oligomerization of pore-forming proteins into the cell membrane, releasing cytokines and damage associated molecular patterns that trigger the immune response. Thus, quantification of cell membrane permeability is an effective method for assessing cell death in macrophages. Many different factors, including macrophage polarization, stimulation, and inflammatory state, can impact macrophage predisposition to, and rate of, programmed cell death. Thus, it is important to have a method of accurately assessing macrophage cell death kinetically, rather than at a single endpoint. In this protocol, we describe a protocol for assessing cell death in macrophages by quantifying cell membrane permeability using kinetic microscopy. This method overcomes limitations of common single time point metrics for assessing cell death and is adaptable and scalable for use in assessing cell death across different cell types and treatment conditions.
Lipid droplets (LDs) are dynamic organelles that not only store energy and regulate metabolism but also serve as key modulators of cell death signaling and disease progression. LDs influence cell fate by buffering lipid...Lipid droplets (LDs) are dynamic organelles that not only store energy and regulate metabolism but also serve as key modulators of cell death signaling and disease progression. LDs influence cell fate by buffering lipid peroxidation or releasing fatty acids during ferroptosis, modulating apoptotic protein expression, and facilitating autophagic degradation. Their multifunctional roles are context-dependent and span multiple cell death pathways. In disease, aberrant LD accumulation is closely linked to metabolic disorders, neurodegeneration, cancer therapy resistance, and pathogen infection. Alterations in LD morphology and abundance have emerged as diagnostic indicators. Thus, precise detection and efficient isolation of LDs are critical for elucidating disease mechanisms, advancing targeted therapies, and translating LD biology into clinical applications. This chapter outlines key methodologies-including lipid staining, fluorescent probes, high-content microscopy, density gradient centrifugation, and immunoaffinity purification-for evaluating lipid droplet function and achieving their isolation in the context of cell death.
Intracellular iron is essential for numerous biological processes, yet its redox activity makes it potentially cytotoxic. Because of this, a tight regulation of its cellular compartmentalization is required. Lysosomes an...Intracellular iron is essential for numerous biological processes, yet its redox activity makes it potentially cytotoxic. Because of this, a tight regulation of its cellular compartmentalization is required. Lysosomes and mitochondria play central roles in iron metabolism. Lysosomes are crucial for iron redistribution after its endocytosis, while mitochondria utilize it for heme and Fe-S cluster synthesis. Disruption of the functional crosstalk between these two organelles can lead to iron dyshomeostasis and ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Recent evidence highlights the importance of mitochondria-lysosome contact sites (MLCs) in mediating iron trafficking, particularly under pathological conditions. However, studying these nanoscopic, dynamic structures poses significant technical challenges. Here, we describe a novel live-cell imaging protocol combining super-resolution structured illumination microscopy (SIM) with organelle-specific dyes and a selective mitochondrial Fe(II) probe to visualize MLC formation and track iron transfer in real time. This approach enables the precise investigation of subcellular iron dynamics and their implications for ferroptosis and disease.
Ferroptosis is an iron-dependent form of regulated cell death characterized by the uncontrolled accumulation of lipid peroxides, primarily within phospholipid membranes. This oxidative process, driven by a radical-mediat...Ferroptosis is an iron-dependent form of regulated cell death characterized by the uncontrolled accumulation of lipid peroxides, primarily within phospholipid membranes. This oxidative process, driven by a radical-mediated chain reaction known as autoxidation, plays a central role in numerous pathological conditions including neurodegeneration, ischemia-reperfusion injury, and cancer. Pharmacological inhibition of lipid peroxidation by radical-trapping antioxidants (RTAs) has thus emerged as a promising strategy for therapeutic intervention. However, conventional 'antioxidant assays' such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or oxygen radical absorbance capacity (ORAC) are inadequate for assessing reactivity toward lipid peroxyl radicals, limiting their utility in ferroptosis research. To address this gap, the Fluorescence-ENabled Inhibited autoXidation (FENIX) platform was developed as a physiologically relevant, high-throughput assay system for the mechanistic and kinetic characterization of lipid peroxidation inhibitors. FENIX-1 quantifies RTA activity by measuring rate constants for RTA-lipidperoxyl radical reactions initiated by a highly lipid-soluble radical initiator in liposomal systems wherein a fluorescent reporter is co-autoxidized along with the lipids. In FENIX-2 lipid peroxidation is initiated via iron-mediated decomposition of pre-formed phospholipid hydroperoxides, enabling the study of both RTA and non-RTA inhibitors, including iron chelators and peroxidase mimics. Together, these assays are complementary and mechanistically informative platforms for the identification and evaluation of inhibitors of lipid peroxidation and - by extension - ferroptosis. Here, we present a detailed overview and standardized protocols for both FENIX-1 and FENIX-2, with the aim of encouraging their application in lipid peroxidation research, ferroptosis modulation, and redox-targeted drug discovery.