Methods in cell biology最新文献_第8页

Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors. 基于流式细胞术的实体瘤患者外周血髓源性抑制细胞监测。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-11-13 DOI: 10.1016/bs.mcb.2024.10.009

Anna-Jasmina Donaubauer, Ilka Scheer, Rainer Fietkau, Udo S Gaipl, Benjamin Frey

{"title":"Flow cytometry-based monitoring of myeloid-derived suppressor cells in the peripheral blood of patients with solid tumors.","authors":"Anna-Jasmina Donaubauer, Ilka Scheer, Rainer Fietkau, Udo S Gaipl, Benjamin Frey","doi":"10.1016/bs.mcb.2024.10.009","DOIUrl":"10.1016/bs.mcb.2024.10.009","url":null,"abstract":"Myeloid-derived suppressor cells (MDSCs) ameliorate inflammation by inhibiting T cell responses. In pathological conditions, such as autoimmunity, chronic infections or cancer they accumulate in the periphery. In cancer, MDSCs can also be part of the tumor microenvironment and are associated with a worse prognosis and limited response to immunotherapy. Nowadays attempts are made to specifically target MDSCs in cancer therapy. Still, the role of MDSCs in standard cancer treatment modalities, such as radiotherapy remains mostly elusive. Here, we describe a flow cytometry-based method to determine and monitor monocytic and granulocytic-derived MDSCs directly from whole blood in an easy, fast and reliable assay. As specific surface markers for MDSCs are lacking, the assay follows a gating strategy that excludes successively the main immune cells types and analyzes the remaining events for a set of molecules that are expressed on MDSCs. This assay is especially appropriate for longitudinal analyses and clinical trials and is suitable for being integrated into more complex immunophenotyping panels to generate a comprehensive immune status.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"191 ","pages":"135-150"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008125","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

Metallic nanoparticles biodistribution for the study of lymphoma in animal models. 金属纳米粒子在淋巴瘤动物模型研究中的生物分布。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mcb.2024.07.004

Barbara Chalhoub, Víctor Franco Puntes, Laura Mondragón

{"title":"Metallic nanoparticles biodistribution for the study of lymphoma in animal models.","authors":"Barbara Chalhoub, Víctor Franco Puntes, Laura Mondragón","doi":"10.1016/bs.mcb.2024.07.004","DOIUrl":"10.1016/bs.mcb.2024.07.004","url":null,"abstract":"T cell lymphoma constitutes a complex group of diseases, characterized by heterogeneous molecular features and clinical symptoms, and a dismal outcome no matter the therapeutic strategy chosen. In an attempt to improve patients' survival chances, treatment combinations (chemotherapy, radiotherapy, immunotherapy, gene therapy and thermotherapy) have been tested for their synergistic effects that may dramatically improve outcomes and reduce the side effects of each single modality treatment when therapeutic effects add up while side effects are distributed. In this context, nanoscale drug delivery agents have been developed and exploited to enhance the release of drugs in the treatment of several diseases, showing potential benefits in terms of pharmaceutical flexibility, selectivity, dose reduction and minimization of adverse effects. Inorganic materials (i.e., metal nanoparticles) can be used as imaging and radiotherapy agents demonstrating that nanoparticle-based therapies can combine and act as \"precision medicine\" for targeting tumors while leaving healthy tissue intact. Therefore, nanoparticles (NPs) appear as ideal platforms for multimodal therapy constituting a more than promising strategy in the search of effective combined treatments for T cell lymphoma. In our laboratory, we aim at validating these therapeutic strategies making use of metal NPs able to provide a diagnostic and therapeutic effect at the same time. Validation of the synthesized NPs will be possible thanks to the availability of an in vivo T cell lymphoma animal model also developed in the lab. Here, we describe basic protocols for the administration and biodistribution studies in solid tumors which could be of significant help for future therapies development and follow-up.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"192 ","pages":"159-180"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143039859","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

Assessment of the primary cancer cell secretome using amino acid-analog labeling. 用氨基酸类似物标记评估原发性癌细胞分泌组。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2024-12-15 DOI: 10.1016/bs.mcb.2024.11.002

Annemieke C Bouwman, Antoinette van Weverwijk, Onno B Bleijerveld, Liesbeth Hoekman, Bob J Ignacio, Kimberly M Bonger, Karin E de Visser

{"title":"Assessment of the primary cancer cell secretome using amino acid-analog labeling.","authors":"Annemieke C Bouwman, Antoinette van Weverwijk, Onno B Bleijerveld, Liesbeth Hoekman, Bob J Ignacio, Kimberly M Bonger, Karin E de Visser","doi":"10.1016/bs.mcb.2024.11.002","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.11.002","url":null,"abstract":"It is well established that reciprocal communication between cancer cells and other cells in the tumor microenvironment plays a crucial role in cancer progression and therapy response. There are multiple ways by which cells communicate, including direct cell-cell contact and the secretion of soluble mediators. The secretome of cancer cells contains valuable information to disentangle the complex conversation that is happening between cancer cells and neighboring or distant cells such as immune cells, fibroblasts and endothelial cells. Here, we provide a workflow of mapping the cancer cell secretome in an unbiased way using amino acid-analog labeling in combination with mass spectrometry. The generation of single cells from fresh tumors, isolation of primary cancer cells from a complex multi-cellular pool, and the detection of newly synthesized proteins that are secreted into the medium is described in detailed protocols. Using this experimental pipeline the secretome of cancer cells across different tumors can be determined, paving the way to unravel cell-cell communication networks in the tumor microenvironment, which may uncover novel therapeutic targets.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"43-65"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667962","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

Phenotypic assessment of dendritic cell maturation by cost-effective custom ELISA assays. 树突状细胞成熟的表型评估成本效益定制ELISA检测。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2025-01-31 DOI: 10.1016/bs.mcb.2025.01.011

Peng Liu, Yuhong Pan, Misha Mao, Guido Kroemer, Oliver Kepp, Liwei Zhao

{"title":"Phenotypic assessment of dendritic cell maturation by cost-effective custom ELISA assays.","authors":"Peng Liu, Yuhong Pan, Misha Mao, Guido Kroemer, Oliver Kepp, Liwei Zhao","doi":"10.1016/bs.mcb.2025.01.011","DOIUrl":"https://doi.org/10.1016/bs.mcb.2025.01.011","url":null,"abstract":"Dendritic cells (DCs) are professional antigen-presenting cells that are pivotal in operating tumor immunosurveillance and orchestrating anticancer immune responses. Endowed with phagocytic and migratory capacities, DCs can capture and process tumor antigens, travel to lymphoid organs and prime naïve T cells, altogether leading to the clonal expansion of cytotoxic T lymphocytes (CTLs) that can specifically target and lyse cancer cells. Additionally, DCs contribute to the formation of immunological memory, ensuring durable therapeutic effects and long-term surveillance against tumor recurrence. Upon antigen engagement, DCs undergo a maturation process characterized by the production of specific cytokines as well as the increased expression of costimulatory molecules and chemokine receptors on their surface. Here we propose a panel of custom sandwich enzyme linked immunosorbent assays (ELISAs) for assessing DC maturation via the precise quantification of cytokines. This economic approach achieves high precision and reproducibility and can be readily applied in labs equipped with basic molecular cell biology facilities. With appropriate automatization, this protocol can be employed for high-throughput screening campaigns for the discovery of DC maturation modulators.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"196 ","pages":"271-290"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144667975","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

Multiplexed cytometry for single cell chemical biology. 用于单细胞化学生物学的多路细胞术。

4区生物学

Methods in cell biology Pub Date : 2025-01-01 Epub Date: 2023-07-14 DOI: 10.1016/bs.mcb.2023.03.007

Henry A M Schares, Madeline J Hayes, Joseph A Balsamo, Hannah L Thirman, Brian O Bachmann, Jonathan M Irish

{"title":"Multiplexed cytometry for single cell chemical biology.","authors":"Henry A M Schares, Madeline J Hayes, Joseph A Balsamo, Hannah L Thirman, Brian O Bachmann, Jonathan M Irish","doi":"10.1016/bs.mcb.2023.03.007","DOIUrl":"10.1016/bs.mcb.2023.03.007","url":null,"abstract":"Flow cytometry has great potential for screening in translational research areas due to its deep quantification of cellular features, ability to collect millions of cells in minutes, and consistently expanding suite of validated antibodies that detect cell identity and functions. However, cytometry remains under-utilized in discovery chemical biology due to the differences in expertise between chemistry groups developing chemical libraries and cell biologists developing single cell assays. This chapter is designed to bridge this gap by providing a detailed protocol aimed at both chemistry and biology audiences with the goal of helping train novice researchers. Assay users select from three elements: a small molecule input, a target cell type, and a module of cytometry readouts. For each, we explore basic and advanced examples of inputs, including screening fractionated microbial extracts and pure compounds, and target cells, including primary human blood cells, mouse cells, and cancer cell lines. One such module of cytometry readouts focuses on cell function and measures DNA damage response (γH2AX), growth (phosphorylated S6), DNA content, apoptosis (cleaved Caspase3), cell cycle M phase (phosphorylated Histone H3), and viability (membrane permeabilization). The protocol can also be adapted to measure different functional readouts, such as cell identity or differentiation and contrasting cell injury mechanisms. The protocol is designed to be used in 96-well plate format with fluorescent cell barcoding and the debarcodeR algorithm. Ultimately, the goal is to encourage the next generation of chemical biologists to use functional cell-based cytometry assays in discovery and translational research.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"195 ","pages":"143-172"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12236058/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs. 用于监测循环 TREGs 的多参数流式细胞检测法的优化和测定内验证。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-07-09 DOI: 10.1016/bs.mcb.2024.06.005

Iole Macchia, Floriana Iacobone, Francesca Urbani

{"title":"Optimization and intra-assay validation of a multiparametric flow cytometric test for monitoring circulating TREGs.","authors":"Iole Macchia, Floriana Iacobone, Francesca Urbani","doi":"10.1016/bs.mcb.2024.06.005","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.06.005","url":null,"abstract":"Multiparametric flow cytometry (MFC) represents an essential tool for immune monitoring, and validation of MFC panels is a fundamental prerequisite in routine laboratory settings as well as for translational and clinical research purposes. Regulatory T cells (TREGs) constitute a subset of CD4+ effector T cells that modulate the immune response in numerous settings, including autoimmune disease, allergy, microbial infection, tumor immunity, transplantation, and more. These cells comprise a small fraction of total CD4+ T cells in human peripheral blood and mouse spleen. In oncology, TREG cells are highly relevant, as they are involved in the suppression of the anti-tumor response in many types of cancer, to the extent that the first immune checkpoint inhibitor approved for clinical use in humans was a monoclonal antibody directed against CTLA-4, a molecule functionally associated with TREGs. Due to all these factors, robust assays are mandatory to accurately determine TREG cell frequency and function. Here, we describe the validation of an 8-color flow-cytometry protocol for TREG detection and analysis in a real-world laboratory scenario. The entire process includes the workflow plan and the standard operating procedure resembling each phase, from the panel design to the staining, acquisition, and analysis steps. Validation is planned to be performed in replicates on fresh whole blood samples derived from multiple healthy subjects. The analytical validity of the TREG cell assay is ensured by testing the intra-assay accuracy. The detailed procedure for the entire process is accompanied by important troubleshooting suggestions and other useful tips.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"189 ","pages":"169-188"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406543","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

Assessment of pDCs functional capacity upon exposure to tumor-derived soluble factors. 评估接触肿瘤衍生可溶性因子后 pDCs 的功能能力。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-08-23 DOI: 10.1016/bs.mcb.2024.07.002

Vladimír Koucký, Linn A Syding, Klára Plačková, Lucie Pavelková, Anna Fialová

{"title":"Assessment of pDCs functional capacity upon exposure to tumor-derived soluble factors.","authors":"Vladimír Koucký, Linn A Syding, Klára Plačková, Lucie Pavelková, Anna Fialová","doi":"10.1016/bs.mcb.2024.07.002","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.07.002","url":null,"abstract":"Plasmacytoid dendritic cells (pDCs) are a minority subset of dendritic cells that despite their tiny quantity play an important role in the immune system, especially in antiviral immunity. They are known mostly as the major producers of type I IFN, which they secrete upon stimulation of endosomal Toll-like receptors 7 and 9 with viral RNA and DNA. However, the functionality of pDCs is more complex, as they were shown to be also involved in autoimmunity, inflammation, and cancer. In the context of the tumor microenvironment, pDCs mostly show substantial functional defects and thus contribute to establishing immunosuppressive micromilieu. Indeed, tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce IFNα and capacity to prime regulatory T cells via the ICOS/ICOS-L pathway. Here we describe in detail a method to assess the functional capacity of pDCs upon exposure to tumor-derived cell culture supernatants. The same technique can be implemented with minimal variations to test any soluble factor's impact on pDC phenotype and function.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"189 ","pages":"85-96"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406522","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

Flow cytometry analysis of myeloid derived suppressor cells using 6 color labeling. 使用 6 色标记对髓源性抑制细胞进行流式细胞术分析。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-09-28 DOI: 10.1016/bs.mcb.2024.08.006

Rocío Flores-Campos, Daniel J García-Domínguez, Lourdes Hontecillas-Prieto, Carlos Jiménez-Cortegana, Luis de la Cruz-Merino, Víctor Sánchez-Margalet

引用次数: 0

An orthotopic metastatic xenograft model of colorectal cancer. 结直肠癌正位转移异种移植模型。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-09-28 DOI: 10.1016/bs.mcb.2024.08.009

Rafael Blanco-Domínguez, Sofia Mensurado, Leandro Barros, Mariana Carreira, Bruno Silva-Santos

{"title":"An orthotopic metastatic xenograft model of colorectal cancer.","authors":"Rafael Blanco-Domínguez, Sofia Mensurado, Leandro Barros, Mariana Carreira, Bruno Silva-Santos","doi":"10.1016/bs.mcb.2024.08.009","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.08.009","url":null,"abstract":"Colorectal cancer (CRC) presents a substantial global health challenge, prompting the necessity for the development and validation of preclinical models to enhance our comprehension and therapeutic interventions. Among the myriad of murine models available for CRC evaluation, orthotopic implantation via intercaecal microinjection stands out as a preferred method for replicating the intricate tumor microenvironment while ensuring uniformity and standardized applicability. In this study, we delineate a methodology addressing the required steps for tumor cell line selection and reporter transduction, animal model preparation, orthotopic tumor implantation, in vivo monitoring of tumor growth and metastasis formation. We comprehensively describe the generation of a xenograft murine model based on the intercaecal implantation of human GFP+/luciferase+ SW620 CRC cells, facilitating the evaluation of responses to pre-clinical human-based therapeutic approaches. The implementation of these standardized protocols promises to augment the reliability and reproducibility of preclinical studies, ultimately advancing our comprehension of CRC pathogenesis and guiding the development of innovative therapeutic strategies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"190 ","pages":"119-132"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142623728","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

Genetic reporters to detect and quantify homologous recombination in yeast. 检测和量化酵母中同源重组的基因报告。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-11-28 DOI: 10.1016/bs.mcb.2022.10.011

Léa Marie, Michael T Kimble, Lorraine S Symington

引用次数: 0