Methods in cell biology最新文献_第6页

Implementation of the FENIX assay to assess the anti-ferroptotic properties of test compounds. 实施FENIX检测，以评估测试化合物的抗铁腐性。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-19 DOI: 10.1016/bs.mcb.2026.01.013

Juliana A Hüther, Melodie Mallais, Markus Rehm, Derek A Pratt

{"title":"Implementation of the FENIX assay to assess the anti-ferroptotic properties of test compounds.","authors":"Juliana A Hüther, Melodie Mallais, Markus Rehm, Derek A Pratt","doi":"10.1016/bs.mcb.2026.01.013","DOIUrl":"10.1016/bs.mcb.2026.01.013","url":null,"abstract":"Ferroptosis is an iron-dependent form of regulated cell death characterized by the uncontrolled accumulation of lipid peroxides, primarily within phospholipid membranes. This oxidative process, driven by a radical-mediated chain reaction known as autoxidation, plays a central role in numerous pathological conditions including neurodegeneration, ischemia-reperfusion injury, and cancer. Pharmacological inhibition of lipid peroxidation by radical-trapping antioxidants (RTAs) has thus emerged as a promising strategy for therapeutic intervention. However, conventional 'antioxidant assays' such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) or oxygen radical absorbance capacity (ORAC) are inadequate for assessing reactivity toward lipid peroxyl radicals, limiting their utility in ferroptosis research. To address this gap, the Fluorescence-ENabled Inhibited autoXidation (FENIX) platform was developed as a physiologically relevant, high-throughput assay system for the mechanistic and kinetic characterization of lipid peroxidation inhibitors. FENIX-1 quantifies RTA activity by measuring rate constants for RTA-lipidperoxyl radical reactions initiated by a highly lipid-soluble radical initiator in liposomal systems wherein a fluorescent reporter is co-autoxidized along with the lipids. In FENIX-2 lipid peroxidation is initiated via iron-mediated decomposition of pre-formed phospholipid hydroperoxides, enabling the study of both RTA and non-RTA inhibitors, including iron chelators and peroxidase mimics. Together, these assays are complementary and mechanistically informative platforms for the identification and evaluation of inhibitors of lipid peroxidation and - by extension - ferroptosis. Here, we present a detailed overview and standardized protocols for both FENIX-1 and FENIX-2, with the aim of encouraging their application in lipid peroxidation research, ferroptosis modulation, and redox-targeted drug discovery.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"205 ","pages":"63-83"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147663010","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

In vitro expansion of stem-like memory T cells via D-mannose supplementation. d -甘露糖对干细胞样记忆T细胞体外扩增的影响。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-13 DOI: 10.1016/bs.mcb.2026.02.025

Yajing Qiu, Congcong Zhao, Guideng Li, Bo Huang

引用次数: 0

Immunofluorescent microscopy to quantify and characterize chromosomal instability (CIN). 免疫荧光显微镜定量和表征染色体不稳定性（CIN）。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-01-23 DOI: 10.1016/bs.mcb.2025.10.013

Kathryn M Jones, Yeseo Choi, Radchanon Leelasukseree, Nikki N Messick, Ava Bryan, Sophie Bice, Pippa F Cosper

{"title":"Immunofluorescent microscopy to quantify and characterize chromosomal instability (CIN).","authors":"Kathryn M Jones, Yeseo Choi, Radchanon Leelasukseree, Nikki N Messick, Ava Bryan, Sophie Bice, Pippa F Cosper","doi":"10.1016/bs.mcb.2025.10.013","DOIUrl":"10.1016/bs.mcb.2025.10.013","url":null,"abstract":"Chromosomal instability (CIN) is the continuous missegregation of chromosomes or chromosome fragments over multiple cell divisions and is present to some degree in nearly all cancer types. Chromosome missegregation causes aneuploidy, or daughter cells with an abnormal chromosome content, which is a hallmark of cancer. CIN can be caused by altered expression of mitotic checkpoint genes, defects in chromosome congression to the metaphase plate, hyperstable kinetochore-microtubule attachments, defective error correction, replication stress, defects in sister chromatid cohesion, and telomere crisis, among other intrinsic factors. CIN can also be induced by exogenous factors such as DNA tumor viruses, ionizing radiation, and microtubule stabilizing drugs. CIN has been shown to promote or suppress tumors or have no effect depending on the rate of chromosome missegregation. However, increasing CIN above a maximally tolerated threshold of chromosome loss leads to cell death, implying that altering the rates of CIN could be a potential therapeutic strategy. Furthermore, CIN leads to the formation of micronuclei, which can release cytosolic DNA and activate the cGAS-STING pathway, leading to increased innate and adaptive immune responses depending on the chronicity of CIN. Here, we detail a simple method to quantify and characterize CIN and micronuclei in cells by immunofluorescent microscopy. The technique described here can be implemented and optimized for human or murine cell lines or tissues.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"202 ","pages":"17-37"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12983417/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146194869","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

Lipid ROS measurement by spectral flow cytometry based on the ratiometric dye BODIPY™ C11 581/591. 基于比例染料BODIPY™C11 581/591的光谱流式细胞术测量脂质ROS。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-06-18 DOI: 10.1016/bs.mcb.2026.05.017

Daniel Butcher, Naiqi Wang, Sam Nettelfield, Di Yu

{"title":"Lipid ROS measurement by spectral flow cytometry based on the ratiometric dye BODIPY™ C11 581/591.","authors":"Daniel Butcher, Naiqi Wang, Sam Nettelfield, Di Yu","doi":"10.1016/bs.mcb.2026.05.017","DOIUrl":"https://doi.org/10.1016/bs.mcb.2026.05.017","url":null,"abstract":"The detection and measurement of lipid reactive oxygen species (ROS) is a critical method widely applied in the study of ferroptosis, a recently defined form of iron-dependent programmed cell death characterized by excessive lipid peroxidation and subsequent membrane damage. As ferroptosis research expands into various pathophysiological processes and human diseases, including cancer, immunity and inflammation, and tissue injuries, there is an increasing need for robust methods to measure lipid ROS in heterogenous cell samples, such as in deep immune cell profiling. BODIPY™ C11 581/591 is a gold-standard ratiometric fluorescent dye that reports the levels of lipid ROS, and has been used in both mechanistic and applied studies demonstrating ferroptosis regulation across multiple cell types. However, its use has largely been confined to traditional flow cytometry due to its extensive spillover into multiple emission channels and its ratiometric properties, which complicate its application in spectral flow cytometry. Herein, we present solutions to these challenges and a comprehensive workflow for the optimization, unmixing, staining, and analysis required to multiplex lipid ROS detection using BODIPY™ C11 581/591 with high-parameter spectral flow cytometry panels. Our unmixing strategy represents a novel method for separating the fluorescent spectra of two forms of a ratiometric dye, which may also be applicable to other ratiometric dyes in spectral flow cytometry.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"210 ","pages":"61-82"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148887895","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

Time-lapse confocal laser scanning microscopy analysis of FOOD formation. 食品形成的延时共聚焦激光扫描显微镜分析。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-05 DOI: 10.1016/bs.mcb.2026.02.011

Ponsuge T M Cooray, Ivan H K Poon, Stephanie F Rutter

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DiLiCre2.0 mouse model: An advanced genome-editing tool to induce mutagenesis in vivo with high spatio-temporal resolution. DiLiCre2.0小鼠模型：一种先进的基因组编辑工具，可在体内诱导突变，具有高时空分辨率。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-12-17 DOI: 10.1016/bs.mcb.2025.12.003

Miguel Vizoso

引用次数: 0

In vitro protocols for inducing and assessing endotoxin tolerance in human and murine macrophages. 诱导和评估人类和小鼠巨噬细胞内毒素耐受性的体外方案。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-06-10 DOI: 10.1016/bs.mcb.2026.05.013

Aoife P Smith, Mathew J Choaji, Emma M Creagh

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Measuring T-cell/CAR T-cell bystander tumor killing in vitro and in vivo. 体外和体内检测t细胞/CAR - t细胞对旁观者肿瘤的杀伤作用。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-04-12 DOI: 10.1016/bs.mcb.2025.03.021

Matthew J Lin, Daniel Charytonowicz, Joshua Brody

引用次数: 0

Super resolution microscopy-based assessment of organellar redox-active iron transfer and its relevance for ferroptosis. 基于超分辨率显微镜的细胞器氧化还原活性铁转移及其与铁下垂的相关性评估。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-06 DOI: 10.1016/bs.mcb.2026.01.005

Francesca Rizzollo, Charlotte Cresens, Benjamin Pavie, Pablo Hernandez-Varas, Patrizia Agostinis

{"title":"Super resolution microscopy-based assessment of organellar redox-active iron transfer and its relevance for ferroptosis.","authors":"Francesca Rizzollo, Charlotte Cresens, Benjamin Pavie, Pablo Hernandez-Varas, Patrizia Agostinis","doi":"10.1016/bs.mcb.2026.01.005","DOIUrl":"10.1016/bs.mcb.2026.01.005","url":null,"abstract":"Intracellular iron is essential for numerous biological processes, yet its redox activity makes it potentially cytotoxic. Because of this, a tight regulation of its cellular compartmentalization is required. Lysosomes and mitochondria play central roles in iron metabolism. Lysosomes are crucial for iron redistribution after its endocytosis, while mitochondria utilize it for heme and Fe-S cluster synthesis. Disruption of the functional crosstalk between these two organelles can lead to iron dyshomeostasis and ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation. Recent evidence highlights the importance of mitochondria-lysosome contact sites (MLCs) in mediating iron trafficking, particularly under pathological conditions. However, studying these nanoscopic, dynamic structures poses significant technical challenges. Here, we describe a novel live-cell imaging protocol combining super-resolution structured illumination microscopy (SIM) with organelle-specific dyes and a selective mitochondrial Fe(II) probe to visualize MLC formation and track iron transfer in real time. This approach enables the precise investigation of subcellular iron dynamics and their implications for ferroptosis and disease.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"205 ","pages":"85-105"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147662422","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 NK cell cytotoxicity induced by IL-15 based immunotherapy against cancer cells. 基于IL-15的免疫治疗对肿瘤细胞NK细胞毒性的评价。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-04-17 DOI: 10.1016/bs.mcb.2026.04.001

Camille Rolin, Gilles Iserentant, Aubin Pitiot, Carole Seguin-Devaux

{"title":"Assessment of NK cell cytotoxicity induced by IL-15 based immunotherapy against cancer cells.","authors":"Camille Rolin, Gilles Iserentant, Aubin Pitiot, Carole Seguin-Devaux","doi":"10.1016/bs.mcb.2026.04.001","DOIUrl":"10.1016/bs.mcb.2026.04.001","url":null,"abstract":"Natural Killer (NK) cells are at the interface of the innate and adaptive immune system. While they are naturally able to kill pathogen-infected, malignant and compromised cells, they also drive the recruitment and activation of other immune cells through the production of inflammatory cytokines and chemokines. Evaluation of NK cells cytotoxic activity is critical to determine accurately the anti-tumor potency of novel immunotherapies, in particular, when the later aim to overcome NK cells exhaustion driven by an immunosuppressive environment such as the tumor microenvironment. Here, we provide a detailed protocol for the assessment of primary NK cells cytotoxic functions against pancreatic cancer cells stimulated by a directed approach on NK cells based on the cytokine IL-15. This article describes the protocol of a flow cytometry-based assay and a calcein acetoxymethyl ester (AM) cell viability assay to quantify NK cell cytotoxic activity. Both methods are robust, fast and sensitive to be applicable in clinics and can readily be adapted for the assessment of other immunotherapies on NK cells cytotoxicity.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"207 ","pages":"203-218"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005515","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