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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
Protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections. 使用固定的冷冻小鼠脑切片进行 Xenium 空间转录组学研究的规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-12 DOI: 10.1016/j.xpro.2024.103420
Xiaokuang Ma, Peng Chen, Jing Wei, John Zhang, Chang Chen, Hanqiu Zhao, Deveroux Ferguson, Aaron W McGee, Zhiyu Dai, Shenfeng Qiu
{"title":"Protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections.","authors":"Xiaokuang Ma, Peng Chen, Jing Wei, John Zhang, Chang Chen, Hanqiu Zhao, Deveroux Ferguson, Aaron W McGee, Zhiyu Dai, Shenfeng Qiu","doi":"10.1016/j.xpro.2024.103420","DOIUrl":"10.1016/j.xpro.2024.103420","url":null,"abstract":"<p><p>Here, we present a protocol for Xenium spatial transcriptomics studies using fixed frozen mouse brain sections. We describe steps for intracardiac perfusion, cryosectioning, and floating section mounting of brain sections, which enable runs on the Xenium analyzer and data delivery. We demonstrate that, in addition to the 10× Genomics-validated formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections, fixed frozen thin brain sections are compatible with the Xenium platform and provide excellent imaging and quantification results for spatially resolved gene expression. For complete details on the use and execution of this protocol, please refer to Ma et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103420"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11605406/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142629653","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
Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates. 利用分裂荧光素酶互补实验分析人体细胞裂解物中蛋白质与蛋白质之间相互作用的方法。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-04 DOI: 10.1016/j.xpro.2024.103328
Zander Claes, Sarah Lemaire, Mathieu Bollen
{"title":"Protocol for analyzing protein-protein interactions by split-luciferase complementation assays in human cell lysates.","authors":"Zander Claes, Sarah Lemaire, Mathieu Bollen","doi":"10.1016/j.xpro.2024.103328","DOIUrl":"10.1016/j.xpro.2024.103328","url":null,"abstract":"<p><p>Here, we present a lysate-based split-luciferase assay for examining protein-protein interactions (PPIs) in HEK293T cell lysates, exemplified by interactions between subunits of protein phosphatase PP1. We describe steps for storing and re-using lysates, sensor design, assay setup/optimization, and high-throughput screening of compound libraries. We then detail procedures for applying the assay as a research tool to characterize the dynamics of PPIs, which we illustrate with specific examples. For complete details on the use and execution of this protocol, please refer to Claes and Bollen.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103328"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11490698/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381800","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 quantification of intercellular connection transmission in ebolavirus infections using ImageJ. 使用 ImageJ 对埃博拉病毒感染中的细胞间连接传播进行量化的程序。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-05 DOI: 10.1016/j.xpro.2024.103363
Rodrigo I Santos, Alexander Bukreyev
{"title":"Protocol for quantification of intercellular connection transmission in ebolavirus infections using ImageJ.","authors":"Rodrigo I Santos, Alexander Bukreyev","doi":"10.1016/j.xpro.2024.103363","DOIUrl":"10.1016/j.xpro.2024.103363","url":null,"abstract":"<p><p>Previous work demonstrates that ebolaviruses can spread to neighboring cells through intercellular connections. Here, we present a protocol to quantify the intercellular spread of ebolaviruses via immunofluorescence. We describe steps for cell plating, Bundibugyo virus infection, and adding a neutralizing antibody. We detail procedures for quantitative microscopy assay using ebolavirus immunodetection. Strong virus accumulation around the plasma membrane leads to high fluorescence signal preventing quantification of viral spread based on signal intensity. This protocol minimizes the impact of this bias. For complete details on the use and execution of this protocol, please refer to Santos et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103363"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11491943/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142381802","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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