Methods in cell biology最新文献

{"title":"Methods for analysis of tertiary lymphoid structures and immune activity by multiplex immunofluorescence histology.","authors":"Leon Zheng, Priya Katyal, Ileana Soto Mauldin","doi":"10.1016/bs.mcb.2025.03.008","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.03.008","url":null,"abstract":"<p><p>Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that are correlated with improved patient outcomes in several solid cancers, including melanoma. Multiplex immunofluorescent histology (mIFH) has been used in numerous studies to identify and characterize TLS. However, detailed studies evaluating immune cell subsets and markers of immune activity at TLS sites have been limited. Here, we introduce multiplex immunofluorescence histology methods to identify TLS, their associated immune cell components, and markers of immune activity. We outline two mIFH panels for evaluating and quantifying TLS, and markers of immune activity, offering methodologies that can be used to gain a more nuanced understanding of the role and immunological activity of TLS in cancer prognosis and therapy.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"199 ","pages":"37-49"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145313370","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}

{"title":"Quantitative analysis of the B cell immune synapse using imaging techniques.","authors":"Oreste Corrales Vázquez, Teemly Contreras, Martina Alamo Rollandi, Felipe Del Valle Batalla, Maria-Isabel Yuseff","doi":"10.1016/bs.mcb.2023.08.002","DOIUrl":"10.1016/bs.mcb.2023.08.002","url":null,"abstract":"<p><p>This chapter presents a series of quantitative analyses that can be used to study the formation of the immune synapse (IS) in B cells. The methods described are automated, consistent, and compatible with open-source platforms. The IS is a crucial structure involved in B cell activation and function, and the spatiotemporal organization of this structure is analyzed to provide a better understanding of its mechanisms. The analyses presented here can be applied to other immune cells and are accessible to researchers of diverse fields. In addition, the raw data derived from the results can be further explored to perform quantitative measurements of protein recruitment and tracking of intracellular vesicles. These techniques have the potential to enhance not only our understanding of the IS in B cells but also in other cell models.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"229-252"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370865","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}

{"title":"Assessment of adhering and invading properties of Escherichia coli strains.","authors":"Valerio Iebba","doi":"10.1016/bs.mcb.2024.08.011","DOIUrl":"10.1016/bs.mcb.2024.08.011","url":null,"abstract":"<p><p>Gastrointestinal infections, caused by Enterobacteriaceae, pose a major global health challenge, resulting in significant morbidity and mortality. Enhanced adherence and invasion properties are widespread among enteric pathogenic species, particularly those linked to invasive infections such as some pathovars of Escherichia coli or pathogens like Shigella and Salmonella. Pathogenic E. coli strains are categorized into various pathotypes, including diarrheagenic E. coli (DEC) and extraintestinal pathogenic E. coli (ExPEC). Notably, Enteroinvasive E. coli (EIEC) and Adherent-invasive E. coli (AIEC) demonstrate significant invasive properties. EIEC, similar to Shigella, invades intestinal epithelial cells causing dysentery-like illness, while AIEC persists in the gut epithelium, potentially contributing to chronic inflammatory bowel diseases (IBD). Techniques like cell culture assays are vital for assessing E. coli's adherence and invasion capabilities, with specific virulence factors such as fimbriae and type III secretion systems (T3SS) playing crucial roles. Comparatively, Shigella and Salmonella also utilize T3SS for epithelial cell invasion, but with distinct effector proteins and mechanisms. Understanding these differences is crucial for diagnosis and treatment, as advanced molecular diagnostics improve the identification of invasive E. coli strains. Potential therapeutic interventions targeting fimbrial adherence, T3SS and effector proteins offer promising avenues for developing antivirulence drugs. Here are provided protocols for studying the adherence and invasion properties of E. coli and other Enterobacteriaceae to enhance diagnostic methods, ultimately improving the management of enteric infections.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"169-190"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586224","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}

{"title":"Gauging antigen recognition by human primary T-cells featuring orthotopically exchanged TCRs of choice.","authors":"Vanessa Mühlgrabner, Angelika Plach, Johannes Holler, Judith Leitner, Peter Steinberger, Loïc Dupré, Janett Göhring, Johannes B Huppa","doi":"10.1016/bs.mcb.2024.03.003","DOIUrl":"10.1016/bs.mcb.2024.03.003","url":null,"abstract":"<p><p>Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"127-154"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370853","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}

{"title":"Measuring interaction kinetics between T cells and their target tumor cells with optical tweezers.","authors":"Edison Gerena, Sophie Goyard, Nicolas Inacio, Jerko Ljubetic, Amandine Schneider, Sinan Haliyo, Thierry Rose","doi":"10.1016/bs.mcb.2024.09.002","DOIUrl":"10.1016/bs.mcb.2024.09.002","url":null,"abstract":"<p><p>T cell adhesion kinetics are a powerful indicator of target cell recognition during the cell-cell exploration process and formation of the immunological synapse facilitating cell communication and activation through specific intercellular molecular interactions. Various techniques have been used to document these binding kinetics, which foreshadow the dynamics of immunological synapse formation. Here, optical tweezers were used for studying at the level of single cells, the adhesion kinetics of human leukemia T lymphocyte cell line (CEM) to mouse mast cell line (P815) used as a tumor cell model. The P815 FcγRII receptors were saturated with the mouse anti-human CD3ɛ immunoglobulin G (OKT3) for initiating the T cell-P815 interaction through the engagement of the T cell CD3 nucleating the TCR complex formation structuring the synapse. Methods were developed to assess the time required to turn a contact between a T cell and a tumor cell into a stable interaction, and thus initiate the synapse formation. Single T cells were manipulated with the optical tweezers while the tumor cells were adhered to the glass surface under culture conditions. Three adhesions scenario were investigated by exerting either repetitive contacts engaging the same area of the two cells, repetitive contacts engaging the same area of the T cell but different areas on the tumor cell surface, or rolling the T cell over the tumor cell surface. With these methods, we observed that the median time of contact of CEM on P815 decreased in the presence of anti-CD3 OKT3 from 46s to 1.3s and the median rolling distance decreased from 50μm to 1.8μm prior the T cell immobilization. T cell adhesion speed assays can be used for measuring their lack of early response, identifying molecules involved in cell adhesion, or screening potential modulators. The techniques and quantitative methods, described here for studying T cell/target cell interaction based on manipulations using optical tweezers, can be generalized to all types of immunological or virological synapses as between T cell/dendritic cell, cytotoxic T cell/target, T cell/macrophage, T cell/B cell, NK cell/target, immune cell/infected cell and others.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"155-174"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370863","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}

{"title":"Isolation and characterization of primary NK cells and the enrichment of the KIR2DL1⁺ population.","authors":"Batel Sabag, Abhishek Puthenveetil, Mira Barda-Saad","doi":"10.1016/bs.mcb.2023.09.001","DOIUrl":"10.1016/bs.mcb.2023.09.001","url":null,"abstract":"<p><p>Natural killer (NK) cells are cytotoxic innate lymphoid cells that play critical roles in the mitigation of viral infections and cancer through the secretion of cytolytic granules and immunomodulatory cytokines. Abnormalities in NK function can lead to viral infections, autoimmunity, and cancer. The current protocol provides an NK isolation technique to study the signaling pathways downstream to the Killer cell immunoglobulin-like receptors (KIR) that serve as key human NK cell function regulators. This procedure enables investigating mechanisms specific to individual KIRs to improve our understanding of NK cell function in health and disease.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"201-211"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370859","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}

{"title":"Efficient discrimination of functional hematopoietic stem cell progenitors for transplantation by combining alkaline phosphatase activity and CD34⁺ immunophenotyping.","authors":"Laura G Rico, Jordi Juncà, Roser Salvia, Michael D Ward, Jolene A Bradford, Jordi Petriz","doi":"10.1016/bs.mcb.2023.08.003","DOIUrl":"10.1016/bs.mcb.2023.08.003","url":null,"abstract":"<p><p>Alkaline phosphatase (ALP) is a membrane-associated hydrolase enzyme with dimeric structure that catalyzes phosphate esters, optimally at alkaline pH. ALP has a focus of interest, since this enzyme is highly expressed in primitive stem cells, such as progenitor cells, non-differentiating cells, and primordial cells. We previously adapted a fluorescent microscopy-based assay for quantifying ALP^high and ALP^low cells by flow cytometry in combination with immunophenotyping. Our method uses a minimal sample perturbation approach, avoiding the use of erythrocyte lysing solutions and washing steps, and offering opportunities to combine live cell response and functional assessment with cell immunophenotyping, while minimizing sample preparation effects on the cell biology. Here we provide a detailed experiment protocol to determine alkaline phosphatase activity in CD34⁺ hematopoietic stem cells from blood and apheresis products obtained from patients involved in a stem cell mobilization process for allo- or auto-transplant. This study may provide the early detection of progenitors at different levels of differentiation and therefore, relate this information to long-term engraftment in hematopoietic stem cell transplants.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"101-113"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780452","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}

{"title":"Multiparametric analysis of tumor infiltrating lymphocytes in solid tumors.","authors":"Rebecca Borella, Annamaria Paolini, Beatrice Aramini, Lara Gibellini, Valentina Masciale, Domenico Lo Tartaro, Massimo Dominici, Sara De Biasi, Andrea Cossarizza","doi":"10.1016/bs.mcb.2023.03.006","DOIUrl":"10.1016/bs.mcb.2023.03.006","url":null,"abstract":"<p><p>The use of single-cell technologies in characterizing the interactions between immune and cancer cells is in continuous expansion. Indeed, the combination of different single-cell approaches enables the definition of novel phenotypic and functional aspects of the immune cells infiltrating the tumor microenvironment (TME). This approach is promoting the discovery of relevant and reliable predictive biomarkers, along with the development of new promising treatments. In this chapter, we describe the main subsets of tumor-infiltrating lymphocytes from a phenotypical and functional point of view and discuss the use of single-cell technologies used to characterize these cell populations within TME.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"39-70"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780453","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}

{"title":"PD-L1 expression in multiple myeloma myeloid derived suppressor cells.","authors":"Laura G Rico, Roser Salvia, Jolene A Bradford, Michael D Ward, Jordi Petriz","doi":"10.1016/bs.mcb.2024.11.006","DOIUrl":"10.1016/bs.mcb.2024.11.006","url":null,"abstract":"<p><p>The Programmed Cell Death Protein 1/Programmed Cell Death Protein Ligand 1 (PD-1/PD-L1) axis stands as one of the most widely acknowledged targets for cancer immunotherapy. This ligand is considered a therapeutic target for this disease as it might play an important role in tumor immune evasion and drug resistance. In multiple myeloma (MM), PD-L1 is overexpressed in abnormal plasma cells and Myeloid-Derived Suppressor Cells (MDSCs). In MDSCs, unlike tumoral cells or derived cell lines, the PD-L1 protein is presented in a conformation not recognized by the monoclonal antibody. In contrast, when stimulating the sample with PMA, the PD-L1 molecule undergoes a conformational change that enables its recognition. Hence, we have developed a flow cytometric screening assay to determine PD-L1 conformational changes in MDSCs based on a minimal manipulation of the sample, to preserve the structure and functionality of the ligand. In this chapter, we provide detailed protocols to assess PD-L1 levels in MDSCs together with the representative results obtained in multiple myeloma patients. The obtained results enable the classification of MM patients based on the different PD-L1 detection after stimulation, which increases compared with unstimulated samples. We also provide protocols to assess kinetic analysis of PD-L1 expression over time and to compare PD-L1 cell surface expression with cytoplasmic expression. Finally, competitive experiments in the presence of durvalumab are also described to study its interaction with PD-L1. This approach can also be used to study the contribution of potential conformational changes in other proteins.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"115-141"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780455","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}

{"title":"Swiss Webster mice as a model for excessive alcohol binge drinking consumption.","authors":"Amine Cherif, Amine Bourzam, El Hafedh El Mouhab, Oumayma Kouki, Sami Zekri, David Vaudry, Mohamed Jemaà, Olfa Masmoudi-Kouki","doi":"10.1016/bs.mcb.2025.02.009","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.02.009","url":null,"abstract":"<p><p>Binge drinking (BD) is a widespread pattern of excessive alcohol consumption among adolescents and young adults with detrimental consequences for brain development. Animal models are essential for investigating the neurobiological mechanisms underlying BD, but selecting an appropriate model is critical to ensure relevance to human behavior. This study aims to validate a murine model of (BD) using Swiss Webster mice. To achieve this, both adolescent and adult mice were exposed to either a single binge (SB) or multiple binge (MB) of BD through intraperitoneal ethanol injections. The findings reveal that the SB protocol produces high blood alcohol concentrations (BACs) (150-400 mg/dL) sustained for several hours, with no significant differences based on age or episode repetition. However, the neurotoxic effects vary, showing that in adolescents, a single episode of BD reduces brain cell survival by 25 %, whereas in adults, multiple episodes are required to observe a 17 % decrease. This murine model of BD in Swiss Webster mice fulfills the main validation criteria identified in the literature. It presents valuable opportunities for studying individual variability and the neurobiological mechanisms of BD in adolescents, in order to identify potential therapeutic targets.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"197 ","pages":"173-188"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145033721","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}