Methods in cell biology最新文献_第5页

Cell-type-specific labeling of endogenous proteins using the split GFP system in Drosophila. 利用果蝇分裂GFP系统对内源性蛋白进行细胞类型特异性标记。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2024-09-27 DOI: 10.1016/bs.mcb.2024.08.012

Melissa Ana Inal, Kota Banzai, Rie Kamiyama, Daichi Kamiyama

{"title":"Cell-type-specific labeling of endogenous proteins using the split GFP system in Drosophila.","authors":"Melissa Ana Inal, Kota Banzai, Rie Kamiyama, Daichi Kamiyama","doi":"10.1016/bs.mcb.2024.08.012","DOIUrl":"10.1016/bs.mcb.2024.08.012","url":null,"abstract":"Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP1-10 and GFP11 fragments, creating a fluorescence signal. We insert the GFP11 fragment into a specific genomic locus while expressing its counterpart, GFP1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP1-10 nor GFP11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"200 ","pages":"137-150"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145588056","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

Integrative analysis of lung multiparametric flow cytometry data to study immune cell dynamics in tumors. 肺多参数流式细胞术数据综合分析研究肿瘤免疫细胞动力学。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-07-21 DOI: 10.1016/bs.mcb.2025.06.002

Romina E Araya, Romina S Goldszmid

引用次数: 0

A syngeneic orthotopic mouse model of metastatic colorectal cancer. 转移性结直肠癌小鼠同基因原位模型。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-06-06 DOI: 10.1016/bs.mcb.2025.05.004

Virginie Feliu, Maha Ayyoub, Christel Devaud

引用次数: 0

In situ imaging of pulmonary invariant Natural Killer T cells in precision cut lung slices. 精确肺切片中肺不变性自然杀伤T细胞的原位成像。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-11-27 DOI: 10.1016/bs.mcb.2025.10.014

Tom Langelaar, Karin E de Visser, Claudia Burrello

{"title":"In situ imaging of pulmonary invariant Natural Killer T cells in precision cut lung slices.","authors":"Tom Langelaar, Karin E de Visser, Claudia Burrello","doi":"10.1016/bs.mcb.2025.10.014","DOIUrl":"10.1016/bs.mcb.2025.10.014","url":null,"abstract":"Invariant Natural Killer T (iNKT) cells are key players in bridging innate and adaptive immunity, recognizing lipid antigens presented by CD1d molecules. Flow cytometry has tremendously contributed to the study of iNKT cell biology through the use of CD1d tetramers. While this technique was pivotal to uncover the anti-tumor potential of iNKT cells, spatial characteristics are difficult to assess as effective tools are lacking. Here, we describe a method for detecting iNKT cells in mouse lung tissue by employing Precision Cut Lung Slices (PCLS) alongside CD1d tetramer staining and fluorescence imaging. This technique maintains lung architecture while facilitating the spatial localization of iNKT cells within the pulmonary milieu. The accuracy of our imaging approach was validated with critical negative controls, including unloaded CD1d tetramer staining and iNKT cell-deficient (Traj18-/-) mice. Co-staining with anti-CD3 confirmed the specificity of the CD1d tetramer signal and validated iNKT cell detection. This method provides a valuable tool to map iNKT cell localization and proximity to cells and histological structures, offering a novel angle to study their biology in lungs.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"202 ","pages":"1-16"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146194885","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

A new inducible mouse model of FH loss. 一种新的FH缺失诱导小鼠模型。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-05 DOI: 10.1016/bs.mcb.2026.01.004

Vincent Zecchini, Chrysanthi Moschandrea, Farina Schneider, Christian Frezza

引用次数: 0

Metastasis mouse model of breast cancer derived from circulating tumor cells (CTCs) after primary orthotopic tumor resection. 原发原位肿瘤切除后循环肿瘤细胞转移的乳腺癌小鼠模型。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-12-29 DOI: 10.1016/bs.mcb.2025.12.008

Ángela Pérez-Cervera, Haritz Moreno, Helena Villanueva, Angelina Zheleva, Beatriz Moreno, Fernando Lecanda, Fernando Pastor

{"title":"Metastasis mouse model of breast cancer derived from circulating tumor cells (CTCs) after primary orthotopic tumor resection.","authors":"Ángela Pérez-Cervera, Haritz Moreno, Helena Villanueva, Angelina Zheleva, Beatriz Moreno, Fernando Lecanda, Fernando Pastor","doi":"10.1016/bs.mcb.2025.12.008","DOIUrl":"10.1016/bs.mcb.2025.12.008","url":null,"abstract":"Breast cancer remains one of the most prevalent and deadly malignancies in women. Metastasis remains an unmet clinical need specially in some subtypes such as triple negative breast cancer. Among non-metastatic patients, more than 40 % fail to achieve a complete pathological response during the course of treatment, facing high rates of local failure post-resection and distant metastases. Recent studies suggest an association between the presence of circulating tumor cells (CTCs) and the aggressiveness of the disease. Herein, we describe an orthotopic syngeneic mouse model in which tumor cells are directly inoculated into the exposed mammary gland. This model allows mechanistic studies on tumor progression and the evaluation of novel therapeutic strategies. Furthermore, the inclusion of surgical resection of established tumor closely mimics current clinical approaches in breast cancer treatment and enables the investigation of mechanisms underlying tumor relapse and metastasis. This chapter outlines the complete protocol, including all necessary steps and materials, for establishing a metastatic breast cancer model in mice. The model is based on CTCs that persist in the bloodstream and subsequently colonize distant organs (primarily the lungs) following surgical resection of the primary tumor.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"203 ","pages":"163-174"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147271528","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

Image analysis of mitochondria during T lymphocyte and chimeric antigen receptor (CAR) effector responses. T淋巴细胞和嵌合抗原受体（CAR）效应反应时线粒体的图像分析。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-02-05 DOI: 10.1016/bs.mcb.2026.01.022

Javier Ruiz-Navarro, Víctor Calvo, Manuel Izquierdo

{"title":"Image analysis of mitochondria during T lymphocyte and chimeric antigen receptor (CAR) effector responses.","authors":"Javier Ruiz-Navarro, Víctor Calvo, Manuel Izquierdo","doi":"10.1016/bs.mcb.2026.01.022","DOIUrl":"10.1016/bs.mcb.2026.01.022","url":null,"abstract":"Mitochondria play a central role in cellular metabolism, ATP production, and redox homeostasis, all of which are essential for sustaining T lymphocyte and CAR-T cell effector functions. Mitochondrial dysfunction has been linked to T cell and CAR-T cell exhaustion, reduced cell expansion capacity, and impaired tumor clearance. To investigate the contribution of mitochondria to T lymphocyte and CAR-T cell functionality, this protocol leverages advanced imaging analysis techniques to assess different parameters related to mitochondrial dynamics, structure and function, which may serve as indicators of both metabolic activity and the exhausted state of T cells. Specifically, the protocol focuses on measuring mitochondrial morphology, energization state, and mitochondrial translocation in the three-dimensional space. The image-based methodologies described in this protocol will contribute to a deeper understanding of mitochondrial regulation during T-cell and CAR-T cell responses. This knowledge will facilitate the identification of key metabolic vulnerabilities and support the refinement of therapeutic strategies to enhance T lymphocyte cytotoxic potential, ultimately improving clinical outcomes in T cell-based adoptive cancer immunotherapy.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"207 ","pages":"19-51"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148005607","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

Quantitative high-content profiling of mitochondrial morphology with automated statistical analysis and integrated data visualization. 定量高含量的线粒体形态分析与自动统计分析和集成数据可视化。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-03-07 DOI: 10.1016/bs.mcb.2026.02.007

My-Anne Hong, Sophie Charrasse, Richard E Frye, Abdel Aouacheria

{"title":"Quantitative high-content profiling of mitochondrial morphology with automated statistical analysis and integrated data visualization.","authors":"My-Anne Hong, Sophie Charrasse, Richard E Frye, Abdel Aouacheria","doi":"10.1016/bs.mcb.2026.02.007","DOIUrl":"10.1016/bs.mcb.2026.02.007","url":null,"abstract":"High-content screening (HCS) is a powerful approach for rapidly and efficiently assessing the harmfulness of numerous compounds across a wide range of cultured cell types. We recently developed a fully automated, miniaturized HCS wet-plus-dry pipeline (MITOMATICS) which leverages mitochondrial morphology as a sensitive and dynamic biomarker of cellular health or damage. Mitochondria are indeed not only vital for energy production and homeostasis, but also serve as critical gatekeepers of apoptotic cell death. MITOMATICS incorporates a proprietary software tool (MitoRadar) designed in-house to perform fast, comprehensive and cost-effective analysis of mitochondrial morphology in live cells. Together, the pipeline and its associated big data analytics software provide a valuable framework for early detection of acute mitotoxic effects of chemicals agents or physical stressors. To illustrate this, we present here a complete protocol for quantifying the impact of the pesticide chlorpyrifos-methyl on mitochondrial morphology of human lung epithelial BEAS-2B cells. Our results show that chlorpyrifos-methyl, even as a single compound, induces profound disruptions in mitochondrial subcellular structure. Beyond this case study, MitoRadar opens up promising avenues for investigating mitotoxicity across diverse cell types and environmental exposures, paving the way for a new generation of cellular diagnostics that could be of interest to the cell death community.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"208 ","pages":"29-53"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226608","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 lysosomal drug sequestration and release using fluorescence microscopy. 利用荧光显微镜评价溶酶体药物的分离和释放。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2025-10-27 DOI: 10.1016/bs.mcb.2025.09.006

Lucille Ferret, Samy Dehissi, Jean Guillon, Karla Alvarez-Valadez, Guido Kroemer, Mojgan Djavaheri-Mergny

{"title":"Assessment of lysosomal drug sequestration and release using fluorescence microscopy.","authors":"Lucille Ferret, Samy Dehissi, Jean Guillon, Karla Alvarez-Valadez, Guido Kroemer, Mojgan Djavaheri-Mergny","doi":"10.1016/bs.mcb.2025.09.006","DOIUrl":"10.1016/bs.mcb.2025.09.006","url":null,"abstract":"Lysosomotropism refers to the ability of certain basic lipophilic compounds to accumulate in lysosomes via pH partitioning. Various drugs including anticancer agents are trapped in lysosomes, and this process can prevent such drugs from reaching their primary target, thereby limiting their effectiveness. Strategies aimed at preventing drug sequestration or inducing drug release from lysosomes have garnered considerable interest. Chloroquine is a widely used anti-malarial drug that triggers lysosome membrane permeabilization (LMP) to liberate sequestered drugs from these organelles. In this study, we first evaluate the lysosomotropism of various fluorescent anticancer agents in silico. Next, we outline a simple, fast and robust method for the visualization and quantification of their lysosomal sequestration and release by fluorescence microscopy. The method is used on live cells and consists of two steps: (i) visualization of the compounds in lysosomes by analyzing their colocalization with a specific fluorescent lysosomal marker, and (ii) assessment of drug release from lysosomes. Furthermore, we present fluorescence microscopy protocols for monitoring LMP by analyzing the subcellular localization of LGALS3 (Galectin-3), which normally distributes diffusely in the cytoplasm but translocates into lysosomes upon LMP. This can be achieved on fixed cells by detecting endogenous LGALS3 with immunostaining or by the visualization of a transgenic LGALS3-mCherry fusion protein on live cells. Altogether, these methods facilitate qualitative and quantitative fluorescence imaging of lysosomal sequestration and liberation of lysosomotropic drugs.","PeriodicalId":18437,"journal":{"name":"Methods in cell biology","volume":"204 ","pages":"171-192"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147581890","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

Methods for the detection of canonical markers of apoptosis, necroptosis and pyroptosis. 细胞凋亡、坏死、焦亡典型标志物的检测方法。

4区生物学

Methods in cell biology Pub Date : 2026-01-01 Epub Date: 2026-01-28 DOI: 10.1016/bs.mcb.2026.01.014

Yuanxin Yang, Daichao Xu

引用次数: 0