Methods in cell biology最新文献_第7页

Some tips and tricks for a Correlative Light Electron Microscopy workflow using stable expression of fluorescent proteins. 使用荧光蛋白稳定表达的相关光电子显微镜工作流程的一些技巧和窍门。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-02-28 DOI: 10.1016/bs.mcb.2024.02.032

Elina Mäntylä, Paul Verkade

引用次数: 0

Preface. 序言

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Methods in cell biology Pub Date : 2024-01-01 DOI: 10.1016/S0091-679X(24)00127-4

J Paul Robinson, Pratip K Chattopadhyay, James W Jacobberger

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Toward quantitative super-resolution methods for cryo-CLEM. 为低温冷冻电镜开发定量超分辨率方法。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-12 DOI: 10.1016/bs.mcb.2024.02.028

Laura C Zanetti-Domingues, Michael Hirsch, Lin Wang, Tara A Eastwood, Karen Baker, Daniel P Mulvihill, Sheena Radford, Jim Horne, Paul White, Benji Bateman

{"title":"Toward quantitative super-resolution methods for cryo-CLEM.","authors":"Laura C Zanetti-Domingues, Michael Hirsch, Lin Wang, Tara A Eastwood, Karen Baker, Daniel P Mulvihill, Sheena Radford, Jim Horne, Paul White, Benji Bateman","doi":"10.1016/bs.mcb.2024.02.028","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.028","url":null,"abstract":"Cryogenic ultrastructural imaging techniques such as cryo-electron tomography have produced a revolution in how the structure of biological systems is investigated by enabling the determination of structures of protein complexes immersed in a complex biological matrix within vitrified cell and model organisms. However, so far, the portfolio of successes has been mostly limited to highly abundant complexes or to structures that are relatively unambiguous and easy to identify through electron microscopy. In order to realize the full potential of this revolution, researchers would have to be able to pinpoint lower abundance species and obtain functional annotations on the state of objects of interest which would then be correlated to ultrastructural information to build a complete picture of the structure-function relationships underpinning biological processes. Fluorescence imaging at cryogenic conditions has the potential to be able to meet these demands. However, wide-field images acquired at low numeric aperture (NA) using air immersion objective have a low resolving power and cannot provide accurate enough three-dimensional (3D) localization to enable the assignment of functional annotations to individual objects of interest or target sample debulking to ensure the preservation of the structures of interest. It is therefore necessary to develop super-resolved cryo-fluorescence workflows capable of fulfilling this role and enabling new biological discoveries. In this chapter, we present the current state of development of two super-resolution cryogenic fluorescence techniques, superSIL-STORM and astigmatism-based 3D STORM, show their application to a variety of biological systems and discuss their advantages and limitations. We further discuss the future applicability to cryo-CLEM workflows though examples of practical application to the study of membrane protein complexes both in mammalian cells and in Escherichia coli.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140866630","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}

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Flow cytometry detection and quantification of circulating leukocyte subpopulations in mice after brain irradiation. 流式细胞术检测和量化脑部照射后小鼠的循环白细胞亚群。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-07-09 DOI: 10.1016/bs.mcb.2024.06.004

Julie Coupey, Marine M Leblond, Erika S Hue, Samuel Valable

{"title":"Flow cytometry detection and quantification of circulating leukocyte subpopulations in mice after brain irradiation.","authors":"Julie Coupey, Marine M Leblond, Erika S Hue, Samuel Valable","doi":"10.1016/bs.mcb.2024.06.004","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.06.004","url":null,"abstract":"In the context of high-grade gliomas such as glioblastoma (GBM), the immune part of the tumor microenvironment (TME) is involved in tumor growth and tumor recurrence. It is mostly represented by high amount of macrophages and low amount of lymphocytes. GBM in itself as well as x-ray-based radiotherapy, a standard treatment for brain tumors, are also associated with systemic effects like lymphopenia that correlates with a poor prognosis. This contributes to the immune-suppressive nature of the TME and may explain the lack of the anti-tumor immune response. Radiation-induced lymphopenia (RIL) is generally evaluated on CD4+ and CD8+ count or on a CBC (complete blood count), but the heterogeneity of the subtypes prompts us to explore them in detail to better understand the cellular response to brain irradiation. To facilitate and develop the evaluation of x-ray brain exposure on circulating immune cells, we developed a reproducible and reliable method to quantify the variation of lymphoid and myeloid subtypes using flow cytometry after brain irradiation in the rodent.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406539","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

Tumor slice culture system for ex vivo immunotherapy studies. 用于体外免疫疗法研究的肿瘤切片培养系统。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-06-15 DOI: 10.1016/bs.mcb.2024.05.007

Leire Arrizabalaga, Joan Salvador Russo-Cabrera, Virginia Belsúe, Pedro Berraondo, Ignacio Melero, Fernando Aranda

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Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells. 从外周血单核细胞中鉴定和分析异体反应 T 淋巴细胞。

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Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-07-16 DOI: 10.1016/bs.mcb.2024.05.011

Alberto Susana, Giovanni Galletti, Gabriele De Simone, Chiara Camisaschi, Enrico Lugli

{"title":"Identification and analysis of alloreactive T lymphocytes from peripheral blood mononuclear cells.","authors":"Alberto Susana, Giovanni Galletti, Gabriele De Simone, Chiara Camisaschi, Enrico Lugli","doi":"10.1016/bs.mcb.2024.05.011","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.05.011","url":null,"abstract":"Alloreactive T-cell responses against mismatched MHC or minor histocompatibility antigens may result in deleterious graft-versus-host disease (GVHD) and increased morbidity and mortality in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, these T-cell responses may be directed against residual tumor cells (the graft-versus-tumor effect, GVT), thus preventing relapse of the disease. Recent findings have shown that CD45RA+ naïve T cells, but not CD45RA- memory T cells are the major contributors to GVHD, thus leading to clinical trials where CD45RA+-depleted, memory-enriched T-cell products are adoptively transferred following allo-HSCT to prevent GVHD and enhance immune reconstitution. However, residual alloreactivity may still be present in the memory T-cell compartment, thus contributing to prevent disease relapse by GVT. Here, we describe a simple cell-based protocol to identify alloreactive naïve and memory T cells by co-culturing T-cell subsets and third-party antigen-presenting cells. The responding cells are identified following dilution of carboxyfluorescein succinimidyl ester (CFSE) and upregulation of the activation marker CD25. These CFSE-diluting cells can be further phenotyped by high-dimensional flow cytometry, or purified with a cell sorter for downstream genomic and functional assays.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406542","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

Cell metabolism: Functional and phenotypic single cell approaches. 细胞代谢：功能和表型单细胞方法。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-25 DOI: 10.1016/bs.mcb.2024.02.024

Sara De Biasi, Julien Paul Gigan, Rebecca Borella, Elena Santacroce, Domenico Lo Tartaro, Anita Neroni, Nikolaos Paschalidis, Katarzyna Piwocka, Rafael José Argüello, Lara Gibellini, Andrea Cossarizza

{"title":"Cell metabolism: Functional and phenotypic single cell approaches.","authors":"Sara De Biasi, Julien Paul Gigan, Rebecca Borella, Elena Santacroce, Domenico Lo Tartaro, Anita Neroni, Nikolaos Paschalidis, Katarzyna Piwocka, Rafael José Argüello, Lara Gibellini, Andrea Cossarizza","doi":"10.1016/bs.mcb.2024.02.024","DOIUrl":"10.1016/bs.mcb.2024.02.024","url":null,"abstract":"Several metabolic pathways are essential for the physiological regulation of immune cells, but their dysregulation can cause immune dysfunction. Hypermetabolic and hypometabolic states represent deviations in the magnitude and flexibility of effector cells in different contexts, for example in autoimmunity, infections or cancer. To study immunometabolism, most methods focus on bulk populations and rely on in vitro activation assays. Nowadays, thanks to the development of single-cell technologies, including multiparameter flow cytometry, mass cytometry, RNA cytometry, among others, the metabolic state of individual immune cells can be measured in a variety of samples obtained in basic, translational and clinical studies. Here, we provide an overview of different single-cell approaches that are employed to investigate both mitochondrial functions and cell dependence from mitochondria metabolism. Moreover, besides the description of the appropriate experimental settings, we discuss the strengths and weaknesses of different approaches with the aim to suggest how to study cell metabolism in the settings of interest.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140851310","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 chronological aging in Saccharomyces cerevisiae. 评估酿酒酵母的时间衰老。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-07-12 DOI: 10.1016/bs.mcb.2022.09.006

Adina Schulze, Andreas Zimmermann, Katharina Kainz, Nadine B Egger, Maria A Bauer, Frank Madeo, Didac Carmona-Gutierrez

{"title":"Assessing chronological aging in Saccharomyces cerevisiae.","authors":"Adina Schulze, Andreas Zimmermann, Katharina Kainz, Nadine B Egger, Maria A Bauer, Frank Madeo, Didac Carmona-Gutierrez","doi":"10.1016/bs.mcb.2022.09.006","DOIUrl":"10.1016/bs.mcb.2022.09.006","url":null,"abstract":"Chronological age represents the time that passes between birth and a given date. To understand the complex network of factors contributing to chronological lifespan, a variety of model organisms have been implemented. One of the best studied organisms is the yeast Saccharomyces cerevisiae, which has greatly contributed toward identifying conserved biological mechanisms that act on longevity. Here, we discuss high- und low-throughput protocols to monitor and characterize chronological lifespan and chronological aging-associated cell death in S. cerevisiae. Included are propidium iodide staining with the possibility to quantitatively assess aging-associated cell death via flow cytometry or qualitative assessments via microscopy, cell viability assessment through plating and cell counting and cell death characterization via propidium iodide/AnnexinV staining and subsequent flow cytometric analysis or microscopy. Importantly, all of these methods combined give a clear picture of the chronological lifespan under different conditions or genetic backgrounds and represent a starting point for pharmacological or genetic interventions.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672164","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

Dynamic and scalable assessment of the senescence-associated secretory phenotype (SASP). 对衰老相关分泌表型（SASP）进行动态和可扩展的评估。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-12-05 DOI: 10.1016/bs.mcb.2022.10.005

Nicolas Malaquin, Francis Rodier

{"title":"Dynamic and scalable assessment of the senescence-associated secretory phenotype (SASP).","authors":"Nicolas Malaquin, Francis Rodier","doi":"10.1016/bs.mcb.2022.10.005","DOIUrl":"10.1016/bs.mcb.2022.10.005","url":null,"abstract":"Dual-faced cellular senescence is responsible for beneficial biological processes and for age-related pathologies. Senescent cells under stable proliferation arrest develop numerous senescence-associated phenotypes such as the potent pro-inflammatory secretome called the senescence-associated secretory phenotype (SASP). The SASP shapes the senescent microenvironment and influences the biology of adjacent cells, including the modulation of proliferation and migration/invasion, reinforcement/induction of peripheral senescence, and immune cell activity or recruitment. The SASP is a dynamic process with multiple waves of secreted factors described to interlace over a period of many days. Whether the senescence phenotype reaches a mature stable state remains controversial. Overall, the complexity of the context-dependent and timely SASP compositions and its varied microenvironmental impact demonstrate the importance of properly assessing SASP over time. In this chapter, we focus on scalable and dynamic experimental procedures to prepare SASP conditioned medium over time from cells receiving senescence-inducing stimuli. This SASP-containing conditioned medium can be used to assess the composition of the SASP, study SASP-related signaling pathways or evaluate the paracrine microenvironmental impact of senescent cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672170","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

Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy. 用于癌症免疫疗法的全封闭、自动化原始血液树突状细胞富集。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-09-15 DOI: 10.1016/bs.mcb.2023.05.008

Gerty Schreibelt, Tjitske Duiveman-de Boer, Jeanette M Pots, Tom G M van Oorschot, Annemiek J de Boer, Nicole M Scharenborg, Mandy W M M van de Rakt, Kevin Bos, Anna L de Goede, Katja Petry, Mareke Brüning, Caroline Angerer, Carola Schöggl, Andreas Dzionek, I Jolanda M de Vries

{"title":"Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy.","authors":"Gerty Schreibelt, Tjitske Duiveman-de Boer, Jeanette M Pots, Tom G M van Oorschot, Annemiek J de Boer, Nicole M Scharenborg, Mandy W M M van de Rakt, Kevin Bos, Anna L de Goede, Katja Petry, Mareke Brüning, Caroline Angerer, Carola Schöggl, Andreas Dzionek, I Jolanda M de Vries","doi":"10.1016/bs.mcb.2023.05.008","DOIUrl":"10.1016/bs.mcb.2023.05.008","url":null,"abstract":"Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3+ DC (cDC1) and CD1c/BDCA1+ DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318582","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