Methods in cell biology最新文献

{"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":"https://doi.org/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":"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":"https://doi.org/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":"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":"https://doi.org/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":"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":"https://doi.org/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":"https://doi.org/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":"Optimizing protocols for human regulatory T isolation, expansion, and characterization.","authors":"Sánchez-Moreno Inés, Martín-Otal Celia, Juan José Lasarte, Lozano Teresa","doi":"10.1016/bs.mcb.2024.10.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.005","url":null,"abstract":"","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"59-77"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008158","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":"Specific selection of stimulation-responsive γδ T-cells utilizing a short-term activation assay.","authors":"Christian Peters, Jara Simeonov, Daniel Gombert, Dieter Kabelitz","doi":"10.1016/bs.mcb.2024.10.006","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.10.006","url":null,"abstract":"<p><p>T cells expressing the γδ T-cell receptor (TCR) represent a numerically small proportion of total T cells. Unlike αβ T cells they are activated by non-peptide antigens independently of MHC-presentation. γδ T cells have been recognized as a favorable prognostic marker across different tumor entities. Recently, γδ T cells (in particular Vδ2 T cells), have gained attention because of their effective intrinsic anti-tumor reactivity. Moreover, their ability for MHC-independent activation and in vitro expansion to high numbers makes them attractive candidates for tumor immunotherapy by adoptive transfer. In this regard, the ex vitro identification of highly reactive γδ T cells upon stimulation enables us to specifically identify, isolate and expand γδ T cells which potentially represent those with high anti-tumor reactivity. CD137 and CD154 represent suitable markers for identifying specifically activated γδ T cells. In humans, the surface mobilization of CD137 and CD154 reveals antigen-specific activation of regulatory (Treg) and conventional CD4 T cells, respectively. We adapted this method for the analysis of Vδ2 T cells, in which the mobilization of both CD137 and CD154 can be used to investigate their activation, whereby CD137 and CD154 do not discriminate regulatory from conventional cells. Thus, this method provides a new way to rapidly analyze quick changes in Vδ2 T-cell activation and allows for using these markers for cell sorting and subsequent expansion of the specifically reacting Vδ2 T cells.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"79-91"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008169","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":"Induction of sepsis in a rat model by the cecal ligation and puncture technique. Application for the study of experimental acute renal failure.","authors":"María Ángeles González-Nicolás, Alberto Lázaro","doi":"10.1016/bs.mcb.2024.05.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.05.004","url":null,"abstract":"<p><p>Sepsis is a systemic inflammatory response to infection, and its occurrence is associated with a poor prognosis in the context of multiorgan dysfunction syndrome (MODS). Although there are several animal models for the study of its etiology, the cecal ligation and puncture (CLP) model has been considered the \"Gold standard\" because it shows a high degree of similarity to the progression of human sepsis. Currently, it is one of the most frequently chosen options to search for therapeutic alternatives to diminish the progression and organ damage induced by sepsis. Here, we describe in depth the CLP technique in a rat model and its application in the study of acute renal failure (ARF), the most severe complication during sepsis.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"69-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039855","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":"Flow cytometry conjugate formation assay between natural killer cells and their target cells.","authors":"Gilles Iserentant, Carole Seguin-Devaux, Jacques Zimmer","doi":"10.1016/bs.mcb.2024.02.037","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.037","url":null,"abstract":"<p><p>Before being able to kill other cells, natural killer (NK) cells first have to establish contact with those targets. In case of a predominance of activating signals from the target cell over inhibitory ones, the killing process is initiated. It is possible, with a simple two-color flow cytometry method, to evaluate, for any given effector cell-target cell pair, the number of conjugates between both types of cells. The percentage obtained gives an idea of the amplitude of binding of the NK cells to the targets and might be expected to be indicative of the level of cytotoxicity. Nevertheless, there is no absolute correlation, as the percentages of conjugates are sometimes higher with relatively resistant targets than with the highly sensitive cell line K562. Practically, NK cells and target cells are stained with two differently fluorescent dyes and incubated together at the desired effector:target ratio (in our example, 1:1) for various periods of time (0, 10, 30min, etc.) at 37°C. After the incubation time, the cells are carefully introduced into the flow cytometer, where in principle three populations are distinguished: the single positive, unconjugated effector and target cells, respectively, and the double positive subset, which corresponds to the conjugates between both cell types. We describe here in detail the staining and cell culture protocols and procedures, and give several examples. Thus, the very cytotoxic NK leukemia cell line KHYG-1 versus the myeloid leukemia K562 (the \"conventional\" NK cell target) and the Burkitt lymphoma cell line Raji forms a high number of conjugates. In contrast, purified, non-activated, healthy donor-derived peripheral blood NK cells bind less to the targets, in accordance with their low (K562) or absent (Raji) cytotoxic activity.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"213-228"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370852","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":"Image processing approaches for microtubule remodeling quantification at the immunological synapse.","authors":"Daniel Krentzel, Maria Isabella Gariboldi, Marie Juzans, Marta Mastrogiovanni, Florian Mueller, Céline Cuche, Vincenzo Di Bartolo, Andrés Alcover","doi":"10.1016/bs.mcb.2024.02.036","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.036","url":null,"abstract":"<p><p>Immunological synapses result from a T cell polarization process, requiring cytoskeleton remodeling. Actin and microtubules drive synapse architecture and the localization of intracellular organelles, including Golgi and endolysosomal compartments, ensuring the directional localization of synapse components. Microtubule remodeling includes the centrosome polarization and the formation of a radial microtubules network, extending from the centrosome to the synapse periphery. Concomitantly, a ring of filamentous actin forms at the synapse periphery. Microtubule and actin remodeling facilitate vesicle fusion at the synapse, enabling T cell effector functions. Analyzing structural subtleties of cytoskeleton remodeling at the immunological synapse is crucial to understand its role in T cell functions. It may also pinpoint pathological states related with cytoskeletal dysfunctions. Quantifying filamentous protein network properties is challenging due to their complex and heterogeneous architectures and the inherent difficulty of segmenting individual filaments. Here, we describe the development of an image processing approach aimed at quantifying microtubule organization at the immunological synapse without the need for filament segmentation. The method is based on the analysis of the spatial and directional organization of microtubules growing from the centrosome to the synapse periphery. It is applied to investigate the importance of Adenomatous polyposis coli (Apc), a polarity regulator and tumor suppressor, in immunological synapse structure and functions and its potential implication in anti-tumor immune responses. We provide an open-source napari plugin of the outlined methods for analyzing filamentous networks.</p>","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"39-67"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370855","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}