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Hema-CRISPR: A clone-based protocol for effective genetic editing of hematopoietic stem cells. Hema-CRISPR：一种基于克隆的有效造血干细胞基因编辑方案。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-09-29 DOI: 10.1016/bs.mcb.2025.09.002
Christoforos Vaxevanis, Mario Udinotti, D Kokoretsis, Andreas Wilfer, Marcus Bauer, Claudia Wickenhauser, Barbara Seliger
{"title":"Hema-CRISPR: A clone-based protocol for effective genetic editing of hematopoietic stem cells.","authors":"Christoforos Vaxevanis, Mario Udinotti, D Kokoretsis, Andreas Wilfer, Marcus Bauer, Claudia Wickenhauser, Barbara Seliger","doi":"10.1016/bs.mcb.2025.09.002","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.09.002","url":null,"abstract":"","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"201 ","pages":"185-194"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146097141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Experimental models and methods for infected wound modeling with Staphylococcus aureus. 金黄色葡萄球菌感染创面建模的实验模型及方法。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-07 DOI: 10.1016/bs.mcb.2025.12.009
Estela Pérez, Guillermo Landa, Isabel Bescós, Ignacio Ochoa, Elena Tapia
{"title":"Experimental models and methods for infected wound modeling with Staphylococcus aureus.","authors":"Estela Pérez, Guillermo Landa, Isabel Bescós, Ignacio Ochoa, Elena Tapia","doi":"10.1016/bs.mcb.2025.12.009","DOIUrl":"10.1016/bs.mcb.2025.12.009","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"203 ","pages":"111-127"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147271493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Establishment single lung tumors through orthotopic injection. 原位注射建立单肺肿瘤。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-09 DOI: 10.1016/bs.mcb.2025.12.004
Beñat Picabea, Céline Clémenson, Mirari Echepare, Cristina Viu-Idocin, Ane Álava, Estefanía Rodríguez, Michele Mondini, Karmele Valencia
{"title":"Establishment single lung tumors through orthotopic injection.","authors":"Beñat Picabea, Céline Clémenson, Mirari Echepare, Cristina Viu-Idocin, Ane Álava, Estefanía Rodríguez, Michele Mondini, Karmele Valencia","doi":"10.1016/bs.mcb.2025.12.004","DOIUrl":"10.1016/bs.mcb.2025.12.004","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"203 ","pages":"1-13"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147271551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Analyzing the effect of NK cells on in-vivo models of tumor dissemination by digital pathology and flow cytometry. 应用数字病理和流式细胞术分析NK细胞对肿瘤播散模型的影响。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-20 DOI: 10.1016/bs.mcb.2026.01.027
Paola Orecchia, Simonetta Astigiano, Valentina Delli Zotti, Filippo Piombo, Stefania Martini, Simona Pigozzi, Paolo Carrega, Guido Ferlazzo, Massimo Vitale, Monica Parodi
{"title":"Analyzing the effect of NK cells on in-vivo models of tumor dissemination by digital pathology and flow cytometry.","authors":"Paola Orecchia, Simonetta Astigiano, Valentina Delli Zotti, Filippo Piombo, Stefania Martini, Simona Pigozzi, Paolo Carrega, Guido Ferlazzo, Massimo Vitale, Monica Parodi","doi":"10.1016/bs.mcb.2026.01.027","DOIUrl":"10.1016/bs.mcb.2026.01.027","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"207 ","pages":"155-178"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A preclinical organotypic model for evaluating immunotherapy-related toxicities. 用于评估免疫治疗相关毒性的临床前器官型模型。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-10-03 DOI: 10.1016/bs.mcb.2025.09.005
Alice Machado, Lene Vimeux, David Espié, Barbara Burroni, Audrey Lupo-Mansuet, Marco Alifano, Diane Damotte, Emmanuel Donnadieu
{"title":"A preclinical organotypic model for evaluating immunotherapy-related toxicities.","authors":"Alice Machado, Lene Vimeux, David Espié, Barbara Burroni, Audrey Lupo-Mansuet, Marco Alifano, Diane Damotte, Emmanuel Donnadieu","doi":"10.1016/bs.mcb.2025.09.005","DOIUrl":"10.1016/bs.mcb.2025.09.005","url":null,"abstract":"<p><p>Immunotherapy has revolutionized cancer treatment by enhancing the immune system's ability to target malignant cells. Despite its success, significant challenges remain, particularly treatment-induced toxicities such as \"on-target, off-tumor\" (OTOT) effects and immune-related adverse events (irAEs). CAR-T cells, bispecific T-cell engagers (BiTEs), and monoclonal antibodies can target antigens shared by both tumors and normal tissues, leading to severe organ-specific toxicities. Similarly, immune checkpoint inhibitors (ICIs) disrupt immune self-tolerance, causing autoimmune-like complications in various organs. Current preclinical models fail to fully predict these toxicities due to their limited ability to replicate human immune-tissue interactions. To address this, we developed an ex vivo human tissue slice model that preserves native immune microenvironments and enables the study of T-cell-mediated toxicity. Using a superantigen to induce broad T-cell activation, our model assesses immunotherapy-induced cytotoxicity through cytokine secretion assays and apoptosis detection (cleaved caspase-3). This approach provides a powerful tool for predicting immunotherapy-induced toxicities and optimizing treatment strategies.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"193-203"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assessing spheroid viability in high throughput. 高通量评估球体活力。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-09-27 DOI: 10.1016/bs.mcb.2025.09.004
Sabrina Forveille, Flora Doffe, Marion Leduc, Guido Kroemer, Oliver Kepp
{"title":"Assessing spheroid viability in high throughput.","authors":"Sabrina Forveille, Flora Doffe, Marion Leduc, Guido Kroemer, Oliver Kepp","doi":"10.1016/bs.mcb.2025.09.004","DOIUrl":"10.1016/bs.mcb.2025.09.004","url":null,"abstract":"<p><p>Phenotypic two-dimensional (2D) high-throughput screening (HTS) is a well-established approach extensively employed in oncological drug discovery by both Academia and the pharmaceutical industry. This methodology has played a pivotal role in the development of a wide range of systemic and targeted therapeutic anticancer agents for clinical use. Recent advances in automation, imaging technologies, and labware design have paved the way for image-based HTS in three-dimensional (3D) cell culture systems. These 3D systems enable the analysis of more physiologically relevant models that closely replicate the characteristics of tumors and their microenvironment. In this study, we present an image-based phenotypic 3D HTS assay utilizing imaging-compatible labware specifically designed to support spheroid formation.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"13-25"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581906","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
High-content microscopy quantification of mitochondrial membrane potential. 线粒体膜电位的高含量显微镜定量。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-04-10 DOI: 10.1016/bs.mcb.2025.03.019
Camilla Bean, Federica Dal Bello, Caterina Vianello, Luca Persano, Elena Rampazzo, Tomáš Knedlík, Emad Norouzi Esfahani, Saradha Ramesh, Alessio Gianelle, Marta Giacomello
{"title":"High-content microscopy quantification of mitochondrial membrane potential.","authors":"Camilla Bean, Federica Dal Bello, Caterina Vianello, Luca Persano, Elena Rampazzo, Tomáš Knedlík, Emad Norouzi Esfahani, Saradha Ramesh, Alessio Gianelle, Marta Giacomello","doi":"10.1016/bs.mcb.2025.03.019","DOIUrl":"10.1016/bs.mcb.2025.03.019","url":null,"abstract":"<p><p>In nearly all pathophysiological processes, mitochondrial membrane potential serves as a crucial indicator of mitochondrial function and activity. However, there remains a need for high-content imaging techniques that incorporate multiparametric measurements for comprehensive mitochondrial assessment. This paper introduces a novel unbiased approach for quantifying mitochondrial membrane potential in vitro, applicable to both two-dimensional and three-dimensional experimental systems. Furthermore, the incorporation of automated image analysis with machine learning algorithms enabled precise identification and segregation of distinct cell types within complex co-culture systems, allowing for targeted evaluation of individual subpopulations. Here, we provide a protocol for large-scale profiling of mitochondrial activity across various experimental contexts.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"141-153"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Three-dimensional organoid screening using a Leica Thunder microscope. 使用徕卡雷显微镜进行三维类器官筛选。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-24 DOI: 10.1016/bs.mcb.2025.09.007
Tudor Manoliu, Benoît Maury
{"title":"Three-dimensional organoid screening using a Leica Thunder microscope.","authors":"Tudor Manoliu, Benoît Maury","doi":"10.1016/bs.mcb.2025.09.007","DOIUrl":"10.1016/bs.mcb.2025.09.007","url":null,"abstract":"<p><p>3D organoids are vital for biomedical research, offering complex tissue models superior to 2D cultures for studying development, disease, and drug responses. High-throughput imaging of these structures is challenging. This report details a method for screening 3D organoids using the Leica Thunder microscope, featuring Computational Clearing for enhanced deep imaging. The protocol covers sample preparation, optimized image acquisition using LAS X, and analysis with FiJi/ImageJ for quantification. Applications include drug discovery and disease modeling. The Leica Thunder facilitates efficient 3D organoid screening.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"205-236"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assessing the cleavage of gasdermin-family cell death effectors by microbial proteases. 微生物蛋白酶对气皮蛋白家族细胞死亡效应物的裂解作用。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-03 DOI: 10.1016/bs.mcb.2026.01.008
Doris L LaRock, Christopher N LaRock
{"title":"Assessing the cleavage of gasdermin-family cell death effectors by microbial proteases.","authors":"Doris L LaRock, Christopher N LaRock","doi":"10.1016/bs.mcb.2026.01.008","DOIUrl":"10.1016/bs.mcb.2026.01.008","url":null,"abstract":"<p><p>Gasdermin-family proteins initiate the lytic cell death program pyroptosis upon proteolytic removal of an autoinhibitory domain. For each gasdermin, proteases capable of mediating an activating cleavage event have been clearly defined. However, for new proteases, there are unique challenges in evaluating whether a specific cleavage event results in an active or inactive gasdermin. This chapter outlines methods and key controls suitable for the initial discovery and evaluation of protease activation of gasdermins that are particularly suitable for the study of alternative pathways for activation, such as by the proteases of bacteria and viruses.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"205 ","pages":"37-45"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147662993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Optimized strategies for necroptosis induction and evaluation. 坏死下垂诱导和评估的优化策略。
4区 生物学
Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-05-02 DOI: 10.1016/bs.mcb.2026.04.004
Jie Lu, Haohao Lu, Sudan He
{"title":"Optimized strategies for necroptosis induction and evaluation.","authors":"Jie Lu, Haohao Lu, Sudan He","doi":"10.1016/bs.mcb.2026.04.004","DOIUrl":"10.1016/bs.mcb.2026.04.004","url":null,"abstract":"<p><p>Necroptosis is a regulated form of cell death that relies on receptor-interacting serine/threonine-protein kinase 3 (RIPK3) -mediated phosphorylation of mixed-lineage kinase domain-like pseudokinase (MLKL). This phosphorylation drives MLKL oligomerization and membrane translocation, eventually leading to plasma membrane rupture and the release of damage-associated molecular patterns (DAMPs). Here, we outline methods to induce necroptosis by activating signaling pathways through the tumour necrosis factor receptor 1, Toll-like receptors (TLR3/4), and the interferon receptor. All pathways converge on the core event of RIPK3 activation, which further phosphorylates the effector MLKL to execute cell death. Furthermore, we describe key techniques and optimized protocols for detecting necroptotic activity and validating it with specific inhibitors, providing a comprehensive overview of both induction and evaluation strategies in necroptosis research.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"209 ","pages":"67-89"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148437503","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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