Methods in cell biology最新文献_第3页

Evaluation of lymphocyte infiltration into cancer spheroids by immunofluorescent staining and 3D imaging. 免疫荧光染色和三维成像评价淋巴细胞浸润癌球体。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-14 DOI: 10.1016/bs.mcb.2024.10.015

Mireia Cruz De Los Santos, Andreas Lundqvist

引用次数: 0

Stereotactic injection of murine brain tumor cells for neuro-oncology studies. 立体定向注射小鼠脑肿瘤细胞用于神经肿瘤学研究。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-12 DOI: 10.1016/bs.mcb.2024.07.005

Camille Daviaud, María Cecilia Lira, Claire Vanpouille-Box, Mara De Martino

引用次数: 0

Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers. 利用活体显微镜和层流剪切室测量T淋巴细胞与靶细胞之间的相互作用力。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-08 DOI: 10.1016/bs.mcb.2024.09.001

Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose

{"title":"Measuring interaction force between T lymphocytes and their target cells using live microscopy and laminar shear flow chambers.","authors":"Sophie Goyard, Amandine Schneider, Jerko Ljubetic, Nicolas Inacio, Marie Juzans, Céline Cuche, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover, Thierry Rose","doi":"10.1016/bs.mcb.2024.09.001","DOIUrl":"10.1016/bs.mcb.2024.09.001","url":null,"abstract":"Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported for measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10pN to 20nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"193 ","pages":"175-200"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143370861","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 chromosomal abnormalities in leukemias by imaging flow cytometry. 利用成像流式细胞术评估白血病染色体异常。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-05-18 DOI: 10.1016/bs.mcb.2023.04.001

Stephanie J Lam, Henry Y L Hui, Kathy A Fuller, Wendy N Erber

{"title":"Assessing chromosomal abnormalities in leukemias by imaging flow cytometry.","authors":"Stephanie J Lam, Henry Y L Hui, Kathy A Fuller, Wendy N Erber","doi":"10.1016/bs.mcb.2023.04.001","DOIUrl":"10.1016/bs.mcb.2023.04.001","url":null,"abstract":"Chromosome analysis assists in the diagnostic classification and prognostication of leukemias. It is typically performed by karyotyping or fluorescent in situ hybridization (FISH) on glass slides. Flow cytometry offers an alternative high throughput automated methodology to analyze chromosomal content. With the advent of imaging flow cytometers, specific chromosomes and regions of interest can be identified and enumerated within specific cell types. The inclusion of immunophenotyping increases the specificity of this technique to ensure only the leukemic cell is analyzed. With many thousands of cells acquired, and neoplastic cells of interest identified by antigen expression, this technology has expanded the role of flow cytometry for cytogenomics in oncology. Applications to date have focused on hematological malignancies to detect aneuploidy (chromosome gains and losses) and structural defects (e.g., deletions; translocations) of diagnostic or prognostic significance at the time of diagnosis. With limits of detection of 1 cytogenetically abnormal cell in 100,000, also makes this new flow cytometry protocol eminently suitable for monitoring low level disease, detecting clonal evolution after therapy and identifying circulating tumor cells. The technique is equally applicable to solid tumors, many of which have chromosomal aberrations, with selection of appropriate immunophenotypic markers and FISH probes.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"71-100"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780451","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

Expansion and activation of NK cells supported by accessory cells. Phenotypic and functional characterization. 辅助细胞支持NK细胞的扩增和活化。表型和功能特征。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-03-04 DOI: 10.1016/bs.mcb.2025.02.003

Cecilia Pesini, Pilar M Lanuza, Julián Pardo, Diego Sánchez-Martínez, Ariel Ramírez-Labrada

{"title":"Expansion and activation of NK cells supported by accessory cells. Phenotypic and functional characterization.","authors":"Cecilia Pesini, Pilar M Lanuza, Julián Pardo, Diego Sánchez-Martínez, Ariel Ramírez-Labrada","doi":"10.1016/bs.mcb.2025.02.003","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.02.003","url":null,"abstract":"Natural Killer cells (NK) are cytotoxic lymphocytes from the innate immune system that recognize and eliminate virally infected and tumor cells. Accordingly, manipulation of NK cells has been the focus of several immunotherapy protocols aimed at eradicating cancer cells. Allogeneic NK cell therapy was initially described over two decades ago, emphasizing KIR-mismatch's importance in preventing NK cell inhibition and promoting cytotoxicity and tumor elimination without inducing graft-versus-host disease (GvHD). While unstimulated NK cells have shown limited antitumoral activity in adoptive cell therapy, various activation and expansion protocols have been proposed to enhance their cytotoxic potential. Activated and expanded allogeneic NK cells, especially with the rise of chimeric antigen receptor (CAR) therapies, have attracted significant attention from academic and commercial sectors. Protocols typically involve using cytokines and stimulatory cells, such as Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell lines (LCLs) or K562 leukemic cells, before or after NK cell enrichment. Here we present two different standardized protocols for NK cell activation and expansion, offering insights into NK cell-based immunotherapies for cancer treatment. We also present a comprehensive methodology for assessing NK cell-mediated cytotoxicity against Neuroblastoma cell lines in both 2D and 3D cultures. The comprehensive methodology presented here lays the foundation for further research in the field, driving advancements in NK cell-based therapies against malignancies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"237-250"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667968","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

Guidelines for the assessment of high endothelial venule functionality and health. 高内皮小静脉功能和健康评估指南。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-03-17 DOI: 10.1016/bs.mcb.2025.02.001

Kathryn A Jacobs, Gabriele Bergers

引用次数: 0

Preparation and analysis of monotypic and organotypic tumor spheroids. 单型和器官型肿瘤球体的制备与分析。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-12-14 DOI: 10.1016/bs.mcb.2024.11.003

Ana Carolina M Domingues, Claire Palin, Yi Sun, Hongyan Xie, Elliot C Woods, Russell W Jenkins, Or-Yam Revach

{"title":"Preparation and analysis of monotypic and organotypic tumor spheroids.","authors":"Ana Carolina M Domingues, Claire Palin, Yi Sun, Hongyan Xie, Elliot C Woods, Russell W Jenkins, Or-Yam Revach","doi":"10.1016/bs.mcb.2024.11.003","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.11.003","url":null,"abstract":"Patient-derived tumor models are emerging as promising tools to explore patient-specific tumor behavior and to address a central gap in tumor immunology and immunotherapy drug development - the need for clinically relevant tumor models that recapitulate the complexity of the human tumor ecosystem, in which cancer cells are interacting with various immune and stromal elements. Patient-derived organotypic tumor spheroids (PDOTS), a biomimetic 3D-patient tumor avatar (3D-PTA), are comprised of cancer cells and autologous tumor-infiltrating immune and stromal cells that are grown within collagen hydrogels embedded in a 3D microfluidic culture device to model physiologic conditions and enable the study of tumor-immune dynamics. PDOTS and their murine counterparts (MDOTS, murine-derived organotypic tumor spheroids) are responsive to immune checkpoint blockade (ICB) and mirror in vivo response dynamics. We have also confirmed the utility of MDOTS/PDOTS in examining novel therapeutic strategies to overcome ICB resistance and testing the efficiency of T cell-based immunotherapies, demonstrating the utility of PDOTS profiling in examining the tumor-immune dynamics of immunotherapy response and resistance. Here, we provide a detailed protocol for processing, ex vivo culture, and analysis of patient-derived tumor organotypic tumor spheroids (PDOTS) in 3D microfluidic culture for immuno-oncology applications. The protocol can be readily adapted for ex vivo profiling of murine-derived organotypic tumor spheroids (MDOTS) and cancer cell line-derived monotypic tumor spheroids (MTS) for robust and iterative testing of immuno-oncology targets.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"139-159"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667976","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

Bacterial predators and BALOs: Growth protocol and relation with mitochondria. 细菌捕食者和BALOs：生长方案和与线粒体的关系。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-24 DOI: 10.1016/bs.mcb.2024.07.003

Valerio Iebba

{"title":"Bacterial predators and BALOs: Growth protocol and relation with mitochondria.","authors":"Valerio Iebba","doi":"10.1016/bs.mcb.2024.07.003","DOIUrl":"10.1016/bs.mcb.2024.07.003","url":null,"abstract":"The microbial world is characterized by mechanisms of competition and predation, akin to the animal world. However, while predation's ecological role is well-established in animals, it's less understood in bacteria due to fewer known predators and unclear phylogenetic affiliations. Nevertheless, microorganisms can prey on bacterial cells, including Bacteriophages, Protists, and Predatory Prokaryotes. These predators inhabit various habitats and may play vital roles in bacterial ecology and ecosystem regulation. Predatory interactions between host and parasite are common in nature. Predatory bacteria, such as Bdellovibrio and like organisms (BALOs), employ various strategies, including epibiotic predation and direct invasion. BALOs, which thrive in the periplasmic space of Gram-negative bacterial cells, modulate bacterial populations and could serve as preventive or therapeutic agents against Gram-negative infections. While primarily active against extracellular prey, BALOs may also target mitochondria, which are crucial for cellular processes. The relationship between intracellular bacteria and host mitochondria, including morphology, function, and apoptosis, warrants further exploration. Protocols for growing, propagating, and detecting predatory activities of BALOs, particularly Bdellovibrio bacteriovorus, are provided to assess their presence and activities against potential prey.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"194 ","pages":"151-167"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143586227","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 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

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