Methods in cell biology最新文献_第3页

A comprehensive guide to study the immunological synapse using imaging flow cytometry. 利用成像流式细胞术研究免疫突触的综合指南。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-04-03 DOI: 10.1016/bs.mcb.2024.03.001

Andrea Michela Biolato, Liza Filali, Max Krecke, Clément Thomas, Céline Hoffmann

{"title":"A comprehensive guide to study the immunological synapse using imaging flow cytometry.","authors":"Andrea Michela Biolato, Liza Filali, Max Krecke, Clément Thomas, Céline Hoffmann","doi":"10.1016/bs.mcb.2024.03.001","DOIUrl":"10.1016/bs.mcb.2024.03.001","url":null,"abstract":"Cytotoxic lymphocytes, such as cytotoxic T cells and natural killer (NK) cells, are instrumental in the recognition and eradication of pathogenic cells, notably those undergoing malignant transformation. Cytotoxic lymphocytes establish direct contact with cancer cells via the formation of a specialized cell-cell junction known as the lytic immunological synapse. This structure serves as a critical platform for lymphocytes to integrate surface signals from potential cancer cells and to direct their cytolytic apparatus toward the confirmed targets. Conversely, cancer cells evolve synaptic defense strategies to evade lymphocyte cytotoxicity. This chapter delineates protocols using imaging flow cytometry to examine and quantify important subcellular processes occurring within cytotoxic lymphocytes and cancer cells engaged into an immunological synapse. These processes encompass the spatial redistribution of cytoskeletal components, vesicles, organelles and cell surface molecules. We specifically describe methods to generate and select conjugates between MDA-MB-231 breast cancer cells or K-562 leukemic cells and either the NK-92MI cell line or primary human NK cells. In addition, we detail procedures to evaluate the synaptic polarization of the actin cytoskeleton, CD63-positive vesicular compartments, MHC class I molecules, as well as the microtubule-organizing center in effector cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"69-97"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370849","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

Matching model with mechanism: Appropriate rodent models for studying various aspects of diabetes pathophysiology. 与机制匹配的模型：适合研究糖尿病病理生理各方面的啮齿动物模型。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-06-19 DOI: 10.1016/bs.mcb.2024.05.003

Lydia F Daniels Gatward, Aileen J F King

{"title":"Matching model with mechanism: Appropriate rodent models for studying various aspects of diabetes pathophysiology.","authors":"Lydia F Daniels Gatward, Aileen J F King","doi":"10.1016/bs.mcb.2024.05.003","DOIUrl":"10.1016/bs.mcb.2024.05.003","url":null,"abstract":"Many rodent models are available for preclinical diabetes research making it a challenge for researchers to choose the most appropriate one for their experimental question. To aid in this, models have classically been categorized according to which type of diabetes they represent, and further into whether the model is induced, spontaneous or the result of genetic manipulation. This fails to capture the complexity of pathogenesis seen in diabetes in humans. This includes pathogenesis specifically involving the beta cell, which is no longer considered to be innocuous in the development and progression of diabetes. In this chapter we explore rodent models that incorporate the initiating factors believed to be involved in type 1 diabetes (autoimmunity) and type 2 diabetes (insulin resistance), before further discussing rodents that can be used to model specific mechanisms involved in a failure of functional beta cell mass (impaired beta cell function and beta cell apoptosis). We segregate models of beta cell pathogenesis based on the beta cell stressor predominantly associated with phenotype, but it is important to consider that most rodent models will exhibit more than one beta cell stressor. Similarly, many models exhibit more than one pathogenic mechanism, for example the same model may show insulin resistance, impaired beta cell function as well as beta cell loss. This can complicate interpretation of results and should be considered, and the model thoroughly researched, during the experimental planning stage.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"39-68"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039857","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

Simplified acid extraction and quantification of histones in human tumor cells. 人肿瘤细胞组蛋白的简化酸提取和定量。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-18 DOI: 10.1016/bs.mcb.2024.10.002

Lourdes Hontecillas-Prieto, Daniel J García-Domínguez, Rocío Flores-Campos, Juan Antonio Flores, Antonio Pérez-Pérez, Luis de la Cruz-Merino, Víctor Sánchez-Margalet, Nabil Hajji

{"title":"Simplified acid extraction and quantification of histones in human tumor cells.","authors":"Lourdes Hontecillas-Prieto, Daniel J García-Domínguez, Rocío Flores-Campos, Juan Antonio Flores, Antonio Pérez-Pérez, Luis de la Cruz-Merino, Víctor Sánchez-Margalet, Nabil Hajji","doi":"10.1016/bs.mcb.2024.10.002","DOIUrl":"10.1016/bs.mcb.2024.10.002","url":null,"abstract":"Histones are essential nuclear proteins that package eukaryotic DNA into chromosomes, play a vital role in gene regulation, DNA replication, DNA repair and chromosome condensation. Understanding histone modifications is crucial for grasping biological and disease-related processes. Specific alterations in histone modifications serve as sensitive and selective biomarkers for conditions like cancer, impacting both tumor and immune cells and affecting their interactions. Indeed, the interest in histone modifications is growing in the field of tumor immunology and immunotherapy. Different techniques have been developed to characterize histone proteins and their modifications. Here, we present a simple acid extraction protocol to identify and quantify histones. The workflow described here can be used to detect and measure histone proteins or specific residues of histone, even capturing changes resulting from treatment with epigenetic drugs (Epi-drugs) or other drugs in in different human cancer cell line models.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"1-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008163","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

Generation of human and murine exhausted CD8⁺ T cells in vitro. 体外培养人和小鼠耗竭CD8+ T细胞。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-13 DOI: 10.1016/bs.mcb.2024.10.007

Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes

{"title":"Generation of human and murine exhausted CD8+ T cells in vitro.","authors":"Rosa Ana Lacalle, Raquel Blanco, Rebeca García-Lucena, Santos Mañes","doi":"10.1016/bs.mcb.2024.10.007","DOIUrl":"10.1016/bs.mcb.2024.10.007","url":null,"abstract":"T cell exhaustion is a state of dysfunction that can occur due to persistent exposure to antigens, such as in the tumor microenvironment. The progressive loss of effector functions in exhausted T cells can lead to resistance to immune checkpoint inhibitors and adoptive cell immunotherapies. Improving our understanding of the exhaustion process is thus crucial for optimizing the clinical outcomes of immunotherapy. A significant hurdle in this area is obtaining an adequate quantity of exhausted T cells. One solution could be the in vitro production of exhausted T cells by mimicking exhaustion-induced conditions. We present a simple, repeatable, flow cytometry-assisted method for generating exhausted CD8+ T cells from both human and mouse sources. This flexible protocol works with various cell sources and activation methods. Our results confirm the production of dysfunctional CD8+ T cells, akin to those in mouse tumor models and patient tumor samples. This methodology could help identify genes involved in the exhaustion process and serve as a platform for finding agents capable of altering, reversing, or accelerating this dysfunctional state. By using both mouse and human models, we increase the adaptability of the method, making it a powerful instrument for assessing potential substances with immunotherapeutic utility.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"93-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008126","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

Characterizing tumor-infiltrating group 1 innate lymphoid cells in PyMT breast tumors. PyMT乳腺肿瘤浸润性1组先天淋巴样细胞的特征。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-04-24 DOI: 10.1016/bs.mcb.2024.03.008

Douglas C Chung, Alisha R Elford, Nicolas Jacquelot

引用次数: 0

Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation. 解读人类血液和肿瘤中中性粒细胞的异质性：分离中性粒细胞和评估其对t细胞增殖影响的方法。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-25 DOI: 10.1016/bs.mcb.2024.10.010

Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch

{"title":"Deciphering neutrophil heterogeneity in human blood and tumors: Methods for isolating neutrophils and assessing their effect on T-cell proliferation.","authors":"Nicolas Delhez, Frank Aboubakar Nana, Camille Houbion, Alexandre Bayard, Annika Bruger, Christophe Vanhaver, Sven Brandau, Pierre van der Bruggen, Thibault Hirsch","doi":"10.1016/bs.mcb.2024.10.010","DOIUrl":"10.1016/bs.mcb.2024.10.010","url":null,"abstract":"Neutrophils were historically considered a homogenous population of cells with functions limited to innate immunity against external threats. However, with the rise of immunotherapy, recent works have shown that neutrophils are also important actors in immuno-oncology. In this context, neutrophils appear as a more heterogenous population of cells. However, many reported neutrophil subpopulations, or neutrophils with various transcriptional states, lack functional characterization to confirm their suspected roles. Thus, we believe that functional assays remain essential to define the role of neutrophils in cancer. In this chapter, we present a T-cell proliferation assay based on the use of allogeneic T-cells to assess the suppressive capabilities of neutrophils isolated from human blood or tumor samples. Allogeneic T-cells are isolated in large quantities from the blood of non-cancerous donors and frozen in aliquots to be used in several experiments. This reduces variability by excluding other cancer-derived factors, which would be present if autologous T-cell were used and allows to isolate the effect of neutrophils on T-cell proliferation. Thawed T-cells have poor proliferative capacities and to initiate proliferation they require co-culture with mature dendritic cells that we generate from monocytes isolated from the same blood sample. Initially developed for lung cancer patients, our method to isolate low-density neutrophils (LDN) and normal-density neutrophils (NDN) can be used with any patient and adapted to other kind of samples (e.g., ascites, urine, …).","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"151-196"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008070","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

Imaging the immune synapse: Three-dimensional analysis of the immune synapse. 免疫突触成像：免疫突触的三维分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-03-02 DOI: 10.1016/bs.mcb.2023.04.003

Javier Ruiz-Navarro, Sofía Blázquez-Cucharero, Víctor Calvo, Manuel Izquierdo

{"title":"Imaging the immune synapse: Three-dimensional analysis of the immune synapse.","authors":"Javier Ruiz-Navarro, Sofía Blázquez-Cucharero, Víctor Calvo, Manuel Izquierdo","doi":"10.1016/bs.mcb.2023.04.003","DOIUrl":"10.1016/bs.mcb.2023.04.003","url":null,"abstract":"T cell receptor (TCR) stimulation of T lymphocytes by antigen bound to the major histocompatibility complex (MHC) of an antigen-presenting cell (APC), together with the interaction of accessory molecules, induces the formation of the immunological synapse (IS), the convergence of secretion vesicles toward the centrosome, and the polarization of the centrosome to the IS. Upon IS formation, an initial increase in cortical filamentous actin (F-actin) at the IS takes place, followed by a decrease in F-actin density at the central region of the IS, which contains the secretory domain. These reversible, cortical actin cytoskeleton reorganization processes that characterize a mature IS occur during lytic granule secretion in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells and cytokine-containing vesicle secretion in T-helper (Th) lymphocytes. Besides, IS formation constitutes the basis of a signaling platform that integrates signals and coordinates molecular interactions that are necessary for an appropriate antigen-specific immune response. In this chapter we deal with the three-dimensional (3D) analysis of the synaptic interface architecture, as well as the analysis of the localization of different markers at the IS.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"15-37"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370857","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

Quantification of mitochondrial reactive oxygen species in macrophages during sepsis. 脓毒症时巨噬细胞线粒体活性氧的定量分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-02-24 DOI: 10.1016/bs.mcb.2024.01.005

Kanmani Suganya, Paulraj Kanmani, Guochang Hu

{"title":"Quantification of mitochondrial reactive oxygen species in macrophages during sepsis.","authors":"Kanmani Suganya, Paulraj Kanmani, Guochang Hu","doi":"10.1016/bs.mcb.2024.01.005","DOIUrl":"10.1016/bs.mcb.2024.01.005","url":null,"abstract":"Sepsis is the leading causes of death globally, arising from an imbalanced host response to severe infection. It leads to multi-organ failure and poor outcomes in septic patients due to compromised glucose and lipid oxidation, reduced oxygen consumption, elevated levels of circulating substrates, and impaired mitochondrial function. Mitochondria, essential cellular organelles, play a vital role in regulating various cellular activities and the host immune response to infection. Pathogens, particularly bacteria, often disrupt mitochondrial functions to dysregulate host immunity. Additionally, the mitochondrial function is closely associated with most host immune responses, making mitochondria crucial in maintaining host homeostasis during infection. The intrinsic inflammatory response triggered by pathogens in sepsis impairs mitochondrial function, resulting in excessive production of mitochondrial reactive oxygen species (ROS) and subsequently damage to multiple organs. Here, we present a simple protocol for assessing mitochondrial ROS levels in bone marrow-derived macrophages (BMDMs) isolated from mice. We observed a higher level of ROS generation in lipopolysaccharide (LPS)-treated BMDMs, indicating the effectiveness and efficiency of our designed protocol for assessing mitochondrial ROS generation in vitro.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"59-75"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586249","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

Monitoring the mitochondrial localization of mycobacterial proteins. 监测分枝杆菌蛋白的线粒体定位。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-18 DOI: 10.1016/bs.mcb.2024.10.018

Krishnaveni Mohareer, Jayashankar Medikonda, Sriram Yandrapally, Anushka Agarwal, Sharmistha Banerjee

引用次数: 0

Antimicrobial regime for gut microbiota depletion in experimental mice models. 实验性小鼠模型中肠道菌群耗竭的抗菌方案。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-07-01 DOI: 10.1016/bs.mcb.2024.05.005

Laura Carrillo-Serradell, Alessandra Borgognone, Marc Noguera-Julian, Violeta Planells-Romeo, Lucía Aragón-Serrano, Mariona Parera, Francesc Català-Moll, Sergi Casadó-Llombart, María Velasco-de Andrés, Roger Paredes, Francisco Lozano

{"title":"Antimicrobial regime for gut microbiota depletion in experimental mice models.","authors":"Laura Carrillo-Serradell, Alessandra Borgognone, Marc Noguera-Julian, Violeta Planells-Romeo, Lucía Aragón-Serrano, Mariona Parera, Francesc Català-Moll, Sergi Casadó-Llombart, María Velasco-de Andrés, Roger Paredes, Francisco Lozano","doi":"10.1016/bs.mcb.2024.05.005","DOIUrl":"10.1016/bs.mcb.2024.05.005","url":null,"abstract":"Mice models serve as a valuable tool to study microbiome-immune system interactions. While the use of germ-free mice may represent the gold-standard method, antibiotic-based microbiome depletion provides a more cost-efficient and feasible system. The protocol here in presented provides a mild antimicrobial regime to deplete basal microbiota in 8-week-old C57BL/6 mice, aiming to ensure reproducibility in microbiota studies. The antibiotic regime combines five different antimicrobial agents delivered either ad libitum or via oral gavage, aiming at depleting core gut microbiota in mice. Various administration timings were explored, concluding there were no differences when the antimicrobial treatment was applied for 3, 5 or 7 consecutive days. By offering a detailed antimicrobial preparation and mouse administration, as well as fecal sample processing and 16s rRNA sequencing, this protocol provides an initial framework to develop mice microbiota studies, with perceptible results in fecal microbiota composition.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"101-114"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039808","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