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Protocol for monitoring the stability of transcription factor EB using global protein stability assay. 使用全局蛋白稳定性测定法监测转录因子EB稳定性的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-11 DOI: 10.1016/j.xpro.2025.104141
Boju Wang, Yueling Zhao, Ruiyan Li, Xinying Li, Cheng Dong, Wenyi Mi, Xiaolu Wang
{"title":"Protocol for monitoring the stability of transcription factor EB using global protein stability assay.","authors":"Boju Wang, Yueling Zhao, Ruiyan Li, Xinying Li, Cheng Dong, Wenyi Mi, Xiaolu Wang","doi":"10.1016/j.xpro.2025.104141","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104141","url":null,"abstract":"<p><p>Transcription factor EB (TFEB), the master regulator of autophagy-lysosomal networks, undergoes multiple regulatory mechanisms to maintain proteostasis. Here, we present a protocol for quantifying TFEB stability following loss of function of its regulator (YEATS domain-containing protein 2 [YEATS2] knockdown), utilizing a dual-fluorescent reporter system based on flow cytometry. We describe strategies for vector construction, cell transfection, and fluorescence detection. We then detail procedures for analytical workflows. This protocol is designed to enhance sensitivity and reduce processing duration. For complete details on the use and execution of this protocol, please refer to Wang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104141"},"PeriodicalIF":1.3,"publicationDate":"2025-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145281312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for generating liver organoids containing Kupffer cells using human iPSCs. 利用人多能干细胞生成含库普弗细胞的肝类器官的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104138
Yang Li, Yunzhong Nie, Hideki Taniguchi
{"title":"Protocol for generating liver organoids containing Kupffer cells using human iPSCs.","authors":"Yang Li, Yunzhong Nie, Hideki Taniguchi","doi":"10.1016/j.xpro.2025.104138","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104138","url":null,"abstract":"<p><p>Integration of resident immune cells into in vitro organoid models is important for accurately recapitulating native tissue physiology. Here, we present a protocol for integrating liver-resident macrophages, Kupffer cells, into liver organoid models derived from human induced pluripotent stem cells (iPSCs). We describe procedures for generating Kupffer cell progenitors and hepatic endoderm from iPSCs, followed by detailed steps for establishing liver organoids containing Kupffer cells (KuLOs). This protocol provides a platform for investigating the roles of Kupffer cells in liver development and diseases. For complete details on the use and execution of this protocol, please refer to Li et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104138"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276172","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for AI-assisted quantitative analysis and setup of tumor spheroid invasion into tissue. 人工智能辅助的肿瘤球体侵入组织的定量分析和设置方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104140
Celestine Z Ho, Daniella B Lawther, Christina Bao-Xian Huang, Sriram Bharathkumar, Shi Wei Beh, Ernest W Poon, Phoebe S Lim, Kang Wei Lee, Carla Felisha, Shu Chian Tay, Ivan Yow, Jennifer L Young, Selwin K Wu, Boon Chuan Low
{"title":"Protocol for AI-assisted quantitative analysis and setup of tumor spheroid invasion into tissue.","authors":"Celestine Z Ho, Daniella B Lawther, Christina Bao-Xian Huang, Sriram Bharathkumar, Shi Wei Beh, Ernest W Poon, Phoebe S Lim, Kang Wei Lee, Carla Felisha, Shu Chian Tay, Ivan Yow, Jennifer L Young, Selwin K Wu, Boon Chuan Low","doi":"10.1016/j.xpro.2025.104140","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104140","url":null,"abstract":"<p><p>Recent work has revealed an active mechanical role for the tissue barrier in collective invasion, offering a new perspective on tissue invasion and morphogenesis. Here, we present a protocol for AI-assisted quantitative analysis and setup of tumor spheroid invasion into mesothelial tissue. We describe steps for co-culture setup, time-lapse imaging, AI training, and segmentation. We then detail procedures for cell tracking and quantitative analysis to study the fracturing process of the mesothelium. For complete details on the use and execution of this protocol, please refer to Wu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104140"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the differentiation of HL-60 cells into a neutrophil-like state. HL-60细胞向中性粒细胞样状态分化的方法。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104135
Tamara Hornstein, Klaus Unfried
{"title":"Protocol for the differentiation of HL-60 cells into a neutrophil-like state.","authors":"Tamara Hornstein, Klaus Unfried","doi":"10.1016/j.xpro.2025.104135","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104135","url":null,"abstract":"<p><p>The human myeloid HL-60 cell line serves as a substitute model in neutrophil research. Here, we present a protocol for HL-60 neutrophil differentiation with combined DMSO/all-trans retinoic acid (ATRA) treatment for 5 days. We describe steps for treating and incubating HL-60 cells. We then detail procedures for harvesting and staining of differentiated HL-60 cells for validation of neutrophil maturation. For complete details on the use and execution of this protocol, please refer to Hornstein et al.<sup>1</sup><sup>,</sup><sup>2</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104135"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for identifying DNA damage responders through proximity biotinylation of proliferating cell nuclear antigen interactors. 通过增殖细胞核抗原相互作用物的近距离生物素化鉴定DNA损伤应答者的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104136
Roberta Borosta, István Szepesi-Nagy, István Kató, Gergely Róna
{"title":"Protocol for identifying DNA damage responders through proximity biotinylation of proliferating cell nuclear antigen interactors.","authors":"Roberta Borosta, István Szepesi-Nagy, István Kató, Gergely Róna","doi":"10.1016/j.xpro.2025.104136","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104136","url":null,"abstract":"<p><p>TurboID-based proximity labeling is a powerful approach to capture protein-protein interactions within their native cellular environment. Here, we present a step-by-step protocol for fusing proliferating cell nuclear antigen (PCNA) to TurboID and generating stable cell lines via lentiviral transduction. We describe steps for cell synchronization, DNA damage induction, and proximity labeling, followed by fractionation, affinity purification, and mass spectrometry to identify biotinylated proteins. For complete details on the use and execution of this protocol, please refer to Rona et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104136"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for tendon stem/progenitor cell isolation from human tendon tissue. 从人肌腱组织中分离肌腱干/祖细胞的方法。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104143
Stephanie Michelena-Tupiza, Asawari Parulekar, Sepideh Shemshad, Eleonore Bolle, Tristan Shuker, Kenneth Cutbush, Lisbeth Grøndahl, Justin Cooper-White
{"title":"Protocol for tendon stem/progenitor cell isolation from human tendon tissue.","authors":"Stephanie Michelena-Tupiza, Asawari Parulekar, Sepideh Shemshad, Eleonore Bolle, Tristan Shuker, Kenneth Cutbush, Lisbeth Grøndahl, Justin Cooper-White","doi":"10.1016/j.xpro.2025.104143","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104143","url":null,"abstract":"<p><p>Tendon stem/progenitor cells (TSPCs) are critical for tendon maintenance and repair, yet standardized approaches for their isolation and characterization remain limited. We present a protocol to isolate TSPCs from human tendons and establish them for in vitro culture. We describe steps for confirming phenotype and potency using a comprehensive marker panel through flow cytometry, qPCR, immunofluorescence, and colorimetric staining. This approach consistently yields a homogeneous TSPC population, enabling reproducible in vitro studies of tendon biology and applications in regenerative therapies.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104143"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for isolating and characterizing effector functions of murine bone marrow-derived eosinophils following bacterial challenge. 细菌攻击后小鼠骨髓源性嗜酸性粒细胞效应功能的分离和表征方法。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104104
Katelyn M Parrish, Tyler L Williams, Monica C Gestal
{"title":"Protocol for isolating and characterizing effector functions of murine bone marrow-derived eosinophils following bacterial challenge.","authors":"Katelyn M Parrish, Tyler L Williams, Monica C Gestal","doi":"10.1016/j.xpro.2025.104104","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104104","url":null,"abstract":"<p><p>Understanding eosinophil-associated mechanisms, especially in the context of infection, has been steadily increasing. Here, we present a protocol for isolating and differentiating murine bone marrow-derived eosinophils (bmEos) followed by multi-well plate assays to evaluate eosinophilic responses to bacterial challenge. We describe steps for evaluating eosinophil effector functions including cytotoxicity, bactericidal activity, and cytokine production through multiplex ELISA panels. We use a Bordetella bronchiseptica infection model, but this approach can be modified for an array of microorganisms and different experimental settings. For complete details on the use and execution of this protocol, please refer to First et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104104"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145281289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to sequentially isolate mouse oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons via magnetic cell sorting. 通过磁细胞分选顺序分离小鼠少突胶质细胞、小胶质细胞、内皮细胞、星形胶质细胞和神经元的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-10 DOI: 10.1016/j.xpro.2025.104139
Samah Houmam, Dominika Siodlak, Kevin D Pham, Casandra Salinas-Salinas, Sarah R Ocañas, Willard M Freeman, Heather C Rice
{"title":"Protocol to sequentially isolate mouse oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons via magnetic cell sorting.","authors":"Samah Houmam, Dominika Siodlak, Kevin D Pham, Casandra Salinas-Salinas, Sarah R Ocañas, Willard M Freeman, Heather C Rice","doi":"10.1016/j.xpro.2025.104139","DOIUrl":"10.1016/j.xpro.2025.104139","url":null,"abstract":"<p><p>Isolation of specific brain cell types is essential to study cell-type-specific differences in complex neurological diseases such as Alzheimer's disease. Here, we present a protocol for isolating oligodendrocytes, microglia, endothelial cells, astrocytes, and neurons from a single mouse brain. We describe steps for tissue homogenization, debris removal, and sequential sorting. We then detail procedures for validating cell purity and viability using flow cytometry and quantitative reverse-transcription PCR (RT-qPCR). This protocol is well suited for a range of downstream applications, including genomics, transcriptomics, and proteomics.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104139"},"PeriodicalIF":1.3,"publicationDate":"2025-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145276180","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for measuring cilium length using 3D confocal fluorescence microscopy, CiliaQ software, and a quality control pipeline. 使用三维共聚焦荧光显微镜、CiliaQ软件和质量控制管道测量纤毛长度的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-08 DOI: 10.1016/j.xpro.2025.104128
Daniel Burgdorf, Seniz Yüksel, Katharina Sieckmann, Jan N Hansen, Dagmar Wachten, Nathalie Jurisch-Yaksi
{"title":"Protocol for measuring cilium length using 3D confocal fluorescence microscopy, CiliaQ software, and a quality control pipeline.","authors":"Daniel Burgdorf, Seniz Yüksel, Katharina Sieckmann, Jan N Hansen, Dagmar Wachten, Nathalie Jurisch-Yaksi","doi":"10.1016/j.xpro.2025.104128","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104128","url":null,"abstract":"<p><p>Primary cilia are cellular antennas responding to various biological cues. Cilia undergo substantial changes in length, affecting their ability to sense extracellular signals and trigger downstream effector responses. Here, we present a protocol to image cilia in 2-dimensional cell culture using immunofluorescence staining and confocal microscopy. We describe steps to analyze cilium length using the CiliaQ plugin in Fiji/ImageJ and for using CiliaQ Explorer, a Python-based pipeline that plots CiliaQ-derived data and performs a statistical analysis after quality control. For complete details on the use and execution of this protocol, please refer to Hansen et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104128"},"PeriodicalIF":1.3,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259568","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for measuring cellular energetics through noncanonical amino acid tagging in human peripheral blood and murine tissue immune cells. 通过人外周血和小鼠组织免疫细胞中的非规范氨基酸标记测量细胞能量学的方案。
IF 1.3
STAR Protocols Pub Date : 2025-10-08 DOI: 10.1016/j.xpro.2025.104134
Frank Vrieling, Hendrik J P van der Zande, Manon Dumont, Rinke Stienstra
{"title":"Protocol for measuring cellular energetics through noncanonical amino acid tagging in human peripheral blood and murine tissue immune cells.","authors":"Frank Vrieling, Hendrik J P van der Zande, Manon Dumont, Rinke Stienstra","doi":"10.1016/j.xpro.2025.104134","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.104134","url":null,"abstract":"<p><p>Cellular metabolism dictates immune cell function, yet we lack tools to functionally profile immunometabolism in low-yield, complex samples. We present a flow cytometry-based protocol for measuring cellular energetics through noncanonical amino acid tagging (CENCAT) in human peripheral blood and murine tissue immune cells. We describe steps for sample preparation, metabolic inhibition, protein synthesis analysis using click chemistry, immunophenotyping, and calculation of metabolic dependencies. For complete details on the use and execution of this protocol, please refer to Vrieling et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 4","pages":"104134"},"PeriodicalIF":1.3,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145259551","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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