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Protocol to fluorescently stain vacuoles in Arabidopsis root cells. 荧光染色拟南芥根细胞液泡的方法。
IF 1.3
STAR Protocols Pub Date : 2024-12-21 DOI: 10.1016/j.xpro.2024.103537
Wylie Kristopher Tiernan, Graciela Veronica Castro, Kurtis Shipman, Cecilia Rodriguez-Furlan
{"title":"Protocol to fluorescently stain vacuoles in Arabidopsis root cells.","authors":"Wylie Kristopher Tiernan, Graciela Veronica Castro, Kurtis Shipman, Cecilia Rodriguez-Furlan","doi":"10.1016/j.xpro.2024.103537","DOIUrl":"10.1016/j.xpro.2024.103537","url":null,"abstract":"<p><p>Plant vacuoles are essential organelles that respond to developmental and environmental signals. Here, we present a protocol for staining the vacuole lumen in Arabidopsis root cells, enabling precise visualization of vacuolar dynamics. We describe steps for preparing plant material and staining with commonly used fluorescent dyes. We then detail procedures for visualizing vacuoles in the blue, green, and red emission spectra, allowing for their combined use with a variety of compatible fluorescent-tagged protein markers. For complete details on the use and execution of this protocol, please refer to Dubrovsky et al.,<sup>1</sup> Fricker,<sup>2</sup> Bassil et al.,<sup>3</sup> Grzam et al.,<sup>4</sup> and Stefano et al.<sup>5</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103537"},"PeriodicalIF":1.3,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11733036/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142878122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to monitor live-cell, real-time, mitochondrial respiration in mouse muscle cells using the Resipher platform. 使用 Resipher 平台实时监测小鼠肌肉细胞线粒体呼吸的活细胞方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-20 DOI: 10.1016/j.xpro.2024.103330
Matthew Triolo, Mireille Khacho
{"title":"Protocol to monitor live-cell, real-time, mitochondrial respiration in mouse muscle cells using the Resipher platform.","authors":"Matthew Triolo, Mireille Khacho","doi":"10.1016/j.xpro.2024.103330","DOIUrl":"10.1016/j.xpro.2024.103330","url":null,"abstract":"<p><p>Mitochondrial function is typically assessed by measuring oxygen consumption at a given time point. However, this approach cannot monitor respiratory changes that occur over time. Here, we present a protocol to measure mitochondrial respiration in freshly isolated muscle stem cells, primary skeletal muscle, and immortalized C2C12 myoblasts in real time using the Resipher platform. We describe steps for preparing and plating cells, performing media changes, setting up the software and device, and analyzing data. This method can be adapted to other cell types. For complete details on the use and execution of this protocol, please refer to Triolo et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103330"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11459069/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142297147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for generating a 3D culture of epiblast stem cells. 生成上胚层干细胞三维培养液的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-27 DOI: 10.1016/j.xpro.2024.103347
Viviane S Rosa, Nanami Sato, Marta N Shahbazi
{"title":"Protocol for generating a 3D culture of epiblast stem cells.","authors":"Viviane S Rosa, Nanami Sato, Marta N Shahbazi","doi":"10.1016/j.xpro.2024.103347","DOIUrl":"10.1016/j.xpro.2024.103347","url":null,"abstract":"<p><p>Mouse gastrulation entails concomitant changes in cell fate, tissue shape, and embryo size. The use of a reproducible in vitro system is crucial for dissecting the mechanisms that coordinate these events. Here, we present a protocol for generating a 3D culture of epiblast stem cells (3D EpiSCs), which grow as epithelial spheroids mimicking key features of the gastrulating mouse embryonic epiblast. We describe steps for spheroid formation, growth, and passaging, followed by imaging or further downstream analyses. For complete details on the use and execution of this protocol, please refer to Sato et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103347"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11465143/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142355529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the acquisition and maturation of oligodendrocytes from neonatal rodent brains. 从新生啮齿动物大脑中获取少突胶质细胞并使其成熟的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-28 DOI: 10.1016/j.xpro.2024.103327
Hanki Kim, Bum Jun Kim, Seungyon Koh, Hyo Jin Cho, Xuelian Jin, Byung Gon Kim, Jun Young Choi
{"title":"Protocol for the acquisition and maturation of oligodendrocytes from neonatal rodent brains.","authors":"Hanki Kim, Bum Jun Kim, Seungyon Koh, Hyo Jin Cho, Xuelian Jin, Byung Gon Kim, Jun Young Choi","doi":"10.1016/j.xpro.2024.103327","DOIUrl":"10.1016/j.xpro.2024.103327","url":null,"abstract":"<p><p>The generation of an oligodendrocyte primary culture model encompassing the diverse stages of the lineage is essential for the in vitro research of oligodendrocyte physiology and pathophysiology. Here, we provide a protocol for generating oligodendrocytes from the neonatal rodent brain. We describe steps for isolating oligodendrocyte progenitor cells (OPCs) through differential centrifugation, their subsequent expansion, passaging, and differentiation. For complete details on the use and execution of this protocol, please refer to Kim et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103327"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11465150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142355544","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to study oxygen dynamics in the in vivo mouse brain using bioluminescence microscopy. 利用生物发光显微镜研究活体小鼠大脑中氧动态的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-26 DOI: 10.1016/j.xpro.2024.103334
Antonios Asiminas, Ryszard S Gomolka, Stefanie Gregoriades, Hajime Hirase, Maiken Nedergaard, Felix R M Beinlich
{"title":"Protocol to study oxygen dynamics in the in vivo mouse brain using bioluminescence microscopy.","authors":"Antonios Asiminas, Ryszard S Gomolka, Stefanie Gregoriades, Hajime Hirase, Maiken Nedergaard, Felix R M Beinlich","doi":"10.1016/j.xpro.2024.103334","DOIUrl":"10.1016/j.xpro.2024.103334","url":null,"abstract":"<p><p>Bioluminescence imaging (BLI) relies on the biochemical reaction between substrate and enzyme that triggers light emission upon convergence. Here, we present a protocol to study molecular oxygen dynamics in the in vivo mouse brain using the oxygen-dependent reaction between luciferase and its substrate. We describe steps for acute craniotomy, viral transfection, substrate administration, imaging, and analysis of hypoxic pockets. This protocol offers superior spatiotemporal properties compared to established approaches like electrodes and phosphorescence. For complete details on the use and execution of this protocol, please refer to Beinlich et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103334"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11460448/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142355553","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol to study monocyte transmigration across primary human liver endothelial cells under physiological shear flow conditions in vitro. 在体外生理剪切流条件下研究单核细胞跨原代人肝内皮细胞迁移的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-05 DOI: 10.1016/j.xpro.2024.103431
Alex L Wilkinson, Megan E Bannister, Ayla O'Keeffe, Chris J Weston, Patricia F Lalor, Shishir Shetty, Daniel A Patten
{"title":"Protocol to study monocyte transmigration across primary human liver endothelial cells under physiological shear flow conditions in vitro.","authors":"Alex L Wilkinson, Megan E Bannister, Ayla O'Keeffe, Chris J Weston, Patricia F Lalor, Shishir Shetty, Daniel A Patten","doi":"10.1016/j.xpro.2024.103431","DOIUrl":"10.1016/j.xpro.2024.103431","url":null,"abstract":"<p><p>Modeling immune cell recruitment by liver endothelial cells in vitro is important to better understand the pathology of chronic inflammatory liver diseases and cancers. Here, we present a protocol for the study of monocyte transmigration across activated primary human liver endothelial cells, under physiological flow conditions. We describe primary endothelial cell isolation from human liver tissues and monocyte isolation from human blood. We then detail the shear flow-based assay and subsequent analysis of the different stages of monocyte transmigration. For complete details on the use and execution of this protocol, please refer to Wilkinson et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103431"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11577181/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591558","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP. 利用 split-GFP 的荧光组装检测 mRNA 翻译抑制剂的体外方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-15 DOI: 10.1016/j.xpro.2024.103448
Ana Quiroz-Huanca, Ana Sanchez-Castro, Pablo Soriano-Castillo, Chiara Poletti, Therese Manuela Nloh Tientcheu, Attilio Fabbretti, Anna Maria Giuliodori, Pohl Milon
{"title":"An in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of the split-GFP.","authors":"Ana Quiroz-Huanca, Ana Sanchez-Castro, Pablo Soriano-Castillo, Chiara Poletti, Therese Manuela Nloh Tientcheu, Attilio Fabbretti, Anna Maria Giuliodori, Pohl Milon","doi":"10.1016/j.xpro.2024.103448","DOIUrl":"10.1016/j.xpro.2024.103448","url":null,"abstract":"<p><p>Here, we present an in vitro protocol to assay mRNA translation inhibitors using the fluorescent assembly of split-GFP for translation test (FAST), based on the small fragment GFP11 binding to GFP1-10<sub>fast</sub>. We detail the expression and purification of the GFP1-10<sub>fast</sub> protein, DNA template amplification, in vitro GFP11-tagged CspA synthesis, FAST detection of the GFP11-tagged protein, and optional recovery of the fluorescent complex. In vitro synthesis of GFP11 maximizes the molar yield of synthesized proteins, providing enhanced sensitivity to test translation inhibitors. For complete details on the use and execution of this protocol, please refer to Pham et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103448"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11609478/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142645072","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for generation of PD modeling induced neurons and detection of α-synuclein forms. 产生帕金森病模型诱导神经元和检测α-突触核蛋白形式的程序。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-15 DOI: 10.1016/j.xpro.2024.103447
Francesco V Nardi, Gulimiheranmu Maisumu, You Zhou, Bo Liang, Abraam M Yakoub
{"title":"Protocol for generation of PD modeling induced neurons and detection of α-synuclein forms.","authors":"Francesco V Nardi, Gulimiheranmu Maisumu, You Zhou, Bo Liang, Abraam M Yakoub","doi":"10.1016/j.xpro.2024.103447","DOIUrl":"10.1016/j.xpro.2024.103447","url":null,"abstract":"<p><p>Alpha-synuclein (α-Syn) is an important molecule in the pathogenesis of Parkinson's disease and Alzheimer's disease-related dementias such as Lewy body dementia, forming multiple pathological species. In vitro disease models, including human neurons and α-Syn-transfected cells, are instrumental to understand synucleinopathies or test new therapies. Here, we provide a detailed protocol to generate human neurons derived from induced pluripotent stem cells (iPSCs), and HEK cells, with α-Syn mutations. We also describe multiple assays to determine the various α-Syn forms.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103447"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11609654/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142645075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for isolating and culturing neonatal murine cardiomyocytes. 分离和培养新生小鼠心肌细胞的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-22 DOI: 10.1016/j.xpro.2024.103461
Chiara Bongiovanni, Carmen Miano, Francesca Sacchi, Silvia Da Pra, Irene Del Bono, Stefano Boriati, Gabriele D'Uva
{"title":"Protocol for isolating and culturing neonatal murine cardiomyocytes.","authors":"Chiara Bongiovanni, Carmen Miano, Francesca Sacchi, Silvia Da Pra, Irene Del Bono, Stefano Boriati, Gabriele D'Uva","doi":"10.1016/j.xpro.2024.103461","DOIUrl":"10.1016/j.xpro.2024.103461","url":null,"abstract":"<p><p>The isolation and culture of neonatal murine cardiac cells are valuable techniques for studying their properties and molecular mechanisms in response to various treatments or conditions. Here, we present a protocol for isolating a high yield of viable neonatal murine cardiac cells, including functional, beating cardiomyocytes. We describe the steps of heart extraction, washing and pre-digestion, digestion, and cell seeding. We detail procedures for mechanical and enzymatic digestions, conducted in a controlled environment within a cell culture incubator. For complete details on the use and execution of this protocol, please refer to Bongiovanni et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103461"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11625229/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for analyzing functional gene module perturbation during the progression of diseases using a single-cell Bayesian biclustering framework. 利用单细胞贝叶斯双聚类框架分析疾病进展过程中功能基因模块扰动的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-30 DOI: 10.1016/j.xpro.2024.103349
Kunyue Wang, Yuqiao Gong, Zixin Yan, Zhiyuan Dang, Junhao Wang, Maoying Wu, Yue Zhang
{"title":"Protocol for analyzing functional gene module perturbation during the progression of diseases using a single-cell Bayesian biclustering framework.","authors":"Kunyue Wang, Yuqiao Gong, Zixin Yan, Zhiyuan Dang, Junhao Wang, Maoying Wu, Yue Zhang","doi":"10.1016/j.xpro.2024.103349","DOIUrl":"10.1016/j.xpro.2024.103349","url":null,"abstract":"<p><p>The pathogenesis of complex diseases involves intricate gene regulation across cell types, necessitating a comprehensive analysis approach. Here, we present a protocol for analyzing functional gene module (FGM) perturbation during the progression of diseases using a single-cell Bayesian biclustering (scBC) framework. We describe steps for setting up the scBC workspace, preparing and exploring input data, training the model, and reconstructing the data matrix. We then detail procedures for Bayesian biclustering, exploring biclustering results, and uncovering pathway perturbations. For complete details on the use and execution of this protocol, please refer to Gong et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103349"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11472622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142366776","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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