Methods in cell biology最新文献

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

Assessment of myeloid-derived suppressor cell differentiation ex vivo. 髓源性抑制细胞体内外分化评估

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-10-12 DOI: 10.1016/bs.mcb.2023.05.005

Ester Blanco, David Escors, Grazyna Kochan

{"title":"Assessment of myeloid-derived suppressor cell differentiation ex vivo.","authors":"Ester Blanco, David Escors, Grazyna Kochan","doi":"10.1016/bs.mcb.2023.05.005","DOIUrl":"10.1016/bs.mcb.2023.05.005","url":null,"abstract":"Myeloid-derived suppressor cells (MDSCs) are major promoters of progression and metastasis in cancer. MDSCs inhibit the anti-tumor immune response through multiple mechanisms. The main MDSC functions in cancer are related to the inactivation of T cells and the establishment of an immunosuppressive tumor microenvironment (TME) through the production of pro-inflammatory cytokines, among other mechanisms. MDSCs are phenotypically similar to conventional myeloid cells, so their identification is challenging. Moreover, they infiltrate the tumors in limited numbers, and their purification from within the tumors is technically difficult and makes their study a challenge. Therefore, several ex vivo differentiation methods have been established. Our differentiation method leads to MDSCs that closely model tumor-infiltrating counterparts. In this protocol, MDSCs are differentiated from bone marrow precursors by incubation in differentiation medium produced by murine tumor cell lines engineered to constitutively express granulocyte-monocyte colony stimulating factor (GM-CSF). These ex vivo-generated MDSC subsets show high fidelity compared to their natural tumor-infiltrated counterparts. Moreover, the high yields of purification from these ex vivo differentiated MDSC enable their use for validation of new treatments in high-throughput assays. In this chapter we describe the engineering of a stable cell line overexpressing GM-CSF, followed by production and collection of conditioned media supporting MDSC differentiation. Finally, we detail the isolation procedure of bone marrow cells and the specific MDSC differentiation protocol.","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":"140329946","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

Using the model cestode Taenia crassiceps for the study of cysticercosis. 利用模式绦虫Taenia crassiceps研究囊尾蚴病。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-07 DOI: 10.1016/bs.mcb.2024.02.002

María Eugenia Ancarola, Lucía Celia Abril García, Gustavo Mourglia-Ettlin, Marcela Alejandra Cucher

{"title":"Using the model cestode Taenia crassiceps for the study of cysticercosis.","authors":"María Eugenia Ancarola, Lucía Celia Abril García, Gustavo Mourglia-Ettlin, Marcela Alejandra Cucher","doi":"10.1016/bs.mcb.2024.02.002","DOIUrl":"10.1016/bs.mcb.2024.02.002","url":null,"abstract":"Taenia solium is the aetiological agent of taeniasis/cysticercosis, one of the most severe neglected tropical diseases (NTD) according to the World Health Organization (WHO). The life cycle of T. solium alternates between pigs (intermediate host) and humans (definitive host). In addition, humans can act as accidental intermediate hosts if they ingest infective eggs. In this case, the most severe condition of the disease occurs when parasites invade the central nervous system, causing neurocysticercosis (NCC). The complexity of the life cycle of T. solium imposes a barrier to study this pathogen thoroughly. Thus, related species, such as T. crassiceps are commonly used. Due to its capacity to multiply asexually, T. crassiceps can be maintained by serial passage in laboratory mice in standard biosecurity level facilities. In addition, an in vitro system to generate cysticerci in the presence of feeder cells has been recently developed. Despite model species display biological differences with their zoonotic counterparts, they have historically helped to understand the biology of the related pathogenic species and hence, generate improvements in NTD detection and control. In this chapter, we describe the procedures to carry out both in vivo and in vitro systems for T. crassiceps in the laboratory.","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":"140331811","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

Monitoring Chk1 kinase activity dynamics in live single cell imaging assays. 在活体单细胞成像实验中监测 Chk1 激酶的活性动态。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-02-14 DOI: 10.1016/bs.mcb.2022.12.018

Vivianne Lebrec, Olivier Gavet

{"title":"Monitoring Chk1 kinase activity dynamics in live single cell imaging assays.","authors":"Vivianne Lebrec, Olivier Gavet","doi":"10.1016/bs.mcb.2022.12.018","DOIUrl":"10.1016/bs.mcb.2022.12.018","url":null,"abstract":"The ATR/Chk1 pathway is an important regulator of cell cycle progression, notably upon genotoxic stress where it can detect a large variety of DNA alterations and induce a transient cell cycle arrest that promotes DNA repair. In addition to its role in DNA damage response (DDR), Chk1 is also active during a non-perturbed S phase and contributes to prevent a premature entry into mitosis with an incompletely replicated genome, meaning the ATR/Chk1 pathway is an integral part of the cell cycle machinery that preserves genome integrity during cell growth. We recently developed a FRET-based Chk1 kinase activity reporter to directly monitor and quantify the kinetics of Chk1 activation in live single cell imaging assays with unprecedented sensitivity and time resolution. This tool allowed us to monitor Chk1 activity dynamics over time during a normal S phase and following genotoxic stress, and to elucidate the underlying mechanisms leading to its activation. Here, we review available fluorescent tools to study the interplay of cell cycle progression, DNA damage and DDR in individual live cells, and present the full protocol and image analysis pipeline to monitor Chk1 activity in two imaging 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":"139741409","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

How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question. 如何应用光学和电子显微镜的广泛工具箱来解决特定的生物问题。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-03-26 DOI: 10.1016/bs.mcb.2024.02.030

Erin M Tranfield, Gunar Fabig, Thomas Kurth, Thomas Müller-Reichert

{"title":"How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.","authors":"Erin M Tranfield, Gunar Fabig, Thomas Kurth, Thomas Müller-Reichert","doi":"10.1016/bs.mcb.2024.02.030","DOIUrl":"https://doi.org/10.1016/bs.mcb.2024.02.030","url":null,"abstract":"Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The \"toolbox\" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.","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":"140868117","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

Differences in intratumor innate lymphoid cell composition between orthotopic and spontaneous pancreatic mouse models. 正位胰腺小鼠模型与自发胰腺小鼠模型肿瘤内先天性淋巴细胞组成的差异。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2024-04-24 DOI: 10.1016/bs.mcb.2024.04.001

Sara Lamorte, Alisha R Elford, Douglas C Chung, Kiichi Murakami, Tracy L McGaha, Nicolas Jacquelot

引用次数: 0

Assessing polyglutamine tract aggregation in the nematode Caenorhabditis elegans. 评估线虫秀丽隐杆线虫的多谷氨酰胺束聚集。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2022-10-14 DOI: 10.1016/bs.mcb.2022.09.003

Aggeliki Sotiriou, Christina Ploumi, Nikolaos Charmpilas, Nektarios Tavernarakis

引用次数: 0

High-throughput assessment of cellular senescence. 高通量评估细胞衰老。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-05-30 DOI: 10.1016/bs.mcb.2023.02.017

Giulia Cerrato, Allan Sauvat, Félix Peyre, Oliver Kepp, Guido Kroemer

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":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":"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":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":"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