Methods in cell biology最新文献_第9页

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":"184 ","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":"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":"185 ","pages":"19-33"},"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":"182 ","pages":"221-236"},"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":"187 ","pages":"1-41"},"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

Assessment of DNA fibers to track replication dynamics. 评估 DNA 纤维以跟踪复制动态。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-03-30 DOI: 10.1016/bs.mcb.2023.02.007

L G Bennett, C J Staples

{"title":"Assessment of DNA fibers to track replication dynamics.","authors":"L G Bennett, C J Staples","doi":"10.1016/bs.mcb.2023.02.007","DOIUrl":"10.1016/bs.mcb.2023.02.007","url":null,"abstract":"DNA replication is a complex and tightly regulated process that must proceed accurately and completely if the cell is to faithfully transmit genetic material to its progeny. Organisms have thus evolved complex mechanisms to deal with the myriad exogenous and endogenous sources of replication impediments to which the cell is subject. These mechanisms are of particular relevance to cancer biology, given that such \"replication stress\" frequently foreshadows genome instability during cancer pathogenesis, and that many traditional chemotherapies and a number of precision medicines function by interfering with the progress of DNA replication. Visualization of the progress and dynamics of DNA replication in living cells was historically a major challenge, neatly surmounted by the development of DNA fiber assays that utilize the fluorescent detection of halogenated nucleotides to track replication forks at single-molecule resolution. This methodology has been widely applied to study the dynamics of unperturbed DNA replication, as well as the cellular responses to various replication stress scenarios. In recent years, subtle modifications to DNA fiber assays have facilitated assessment of the stability of nascent DNA at stalled replication forks, as well as the detection of single-stranded DNA gaps and their subsequent filling by error-prone polymerases. Here, we present and discuss several iterations of the fiber assay and suggest methodologies for the analysis of the data obtained.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"182 ","pages":"285-298"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139741338","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 cell cycle progression and mitotic slippage by videomicroscopy. 通过视频显微镜评估细胞周期进展和有丝分裂滑动。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-08-21 DOI: 10.1016/bs.mcb.2023.03.004

Luca Mattiello, Sara Soliman Abdel Rehim, Gwenola Manic, Ilio Vitale

引用次数: 0

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors. 流式细胞仪辅助量化 CDK4/6 抑制剂治疗的癌细胞的细胞周期停滞。

4区生物学

Methods in cell biology Pub Date : 2024-01-01 Epub Date: 2023-08-22 DOI: 10.1016/bs.mcb.2023.02.018

Vanessa Klapp, Norma Bloy, Carlos Jiménez-Cortegana, Aitziber Buqué, Giulia Petroni

{"title":"Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.","authors":"Vanessa Klapp, Norma Bloy, Carlos Jiménez-Cortegana, Aitziber Buqué, Giulia Petroni","doi":"10.1016/bs.mcb.2023.02.018","DOIUrl":"10.1016/bs.mcb.2023.02.018","url":null,"abstract":"Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"181 ","pages":"197-212"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139672171","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