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Protocol for quantifying muscle fiber size, number, and central nucleation of mouse skeletal muscle cross-sections using Myotally software. 使用myotic软件定量小鼠骨骼肌横截面的肌纤维大小、数量和中央成核的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103555
Pieter Both, Soochi Kim, Jengmin Kang, Marina Arjona, Daniel I Benjamin, Christopher W Nutter, Armon Goshayeshi, Thomas A Rando
{"title":"Protocol for quantifying muscle fiber size, number, and central nucleation of mouse skeletal muscle cross-sections using Myotally software.","authors":"Pieter Both, Soochi Kim, Jengmin Kang, Marina Arjona, Daniel I Benjamin, Christopher W Nutter, Armon Goshayeshi, Thomas A Rando","doi":"10.1016/j.xpro.2024.103555","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103555","url":null,"abstract":"<p><p>Here, we present a protocol for using Myotally, a user-friendly software for fast, automated quantification of muscle fiber size, number, and central nucleation from immunofluorescent stains of mouse skeletal muscle cross-sections. We describe steps for installing the software, preparing compatible images, finding the file path, and selecting key parameters like image quality and size limits. We also detail optional features, such as measuring mean fluorescence. By automating these traditionally labor-intensive processes, Myotally improves research efficiency and data consistency.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103555"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972459","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 study neuronal membrane proteasome function in mouse peripheral sensory neurons. 小鼠外周感觉神经元膜蛋白酶体功能的研究方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103552
Emily R Krueger, Taylor R Church, Anna Brennan, Fulya Türker, Eric Villalón Landeros
{"title":"Protocol to study neuronal membrane proteasome function in mouse peripheral sensory neurons.","authors":"Emily R Krueger, Taylor R Church, Anna Brennan, Fulya Türker, Eric Villalón Landeros","doi":"10.1016/j.xpro.2024.103552","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103552","url":null,"abstract":"<p><p>Neuronal membrane proteasomes (NMPs) are expressed on a subset of somatosensory dorsal root ganglion (DRG) neurons and influence mechanical and pain sensitivity. Here, we present a protocol for studying NMP function in mouse peripheral sensory neurons. We describe steps for procuring and culturing primary DRG neurons. We then detail biochemical and antibody feeding approaches to analyze NMP expression and localization. Finally, we include Ca<sup>2+</sup> imaging techniques for investigating NMP function in DRG neurons. For complete details on the use and execution of this protocol, please refer to Villalón Landeros et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103552"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972466","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
Live-cell metabolic analyzer protocol for measuring glucose and lactate metabolic changes in human cells. 用于测量人体细胞中葡萄糖和乳酸代谢变化的活细胞代谢分析仪协议。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103518
Kenji Miki, Mikako Yagi, Ko Igami, Hiroki Kittaka, Akito Tani, Natsuki Horiuchi, Satoshi Fukumoto, Koji Yoshimoto, Takeshi Uchiumi
{"title":"Live-cell metabolic analyzer protocol for measuring glucose and lactate metabolic changes in human cells.","authors":"Kenji Miki, Mikako Yagi, Ko Igami, Hiroki Kittaka, Akito Tani, Natsuki Horiuchi, Satoshi Fukumoto, Koji Yoshimoto, Takeshi Uchiumi","doi":"10.1016/j.xpro.2024.103518","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103518","url":null,"abstract":"<p><p>Understanding metabolic conditions related to glycolysis dependence is crucial for developing new treatments in cancer and regenerative medicine. This protocol details a method for using the live-cell metabolic analyzer (LiCellMo) to measure continuous changes in glucose consumption and lactate production in cultured human cells. LiCellMo provides real-time data on consecutive metabolic changes, improving measurements of these processes in various contexts, including in cancer and regenerative treatments.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103518"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972425","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 assessing immune-target cell interactions using a single-cell cytotoxicity assay. 使用单细胞细胞毒性试验评估免疫靶细胞相互作用的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103558
Zhihao Wei, Konglan Lin, Min Huang, Shicheng Su, Yiwen Lu
{"title":"Protocol for assessing immune-target cell interactions using a single-cell cytotoxicity assay.","authors":"Zhihao Wei, Konglan Lin, Min Huang, Shicheng Su, Yiwen Lu","doi":"10.1016/j.xpro.2024.103558","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103558","url":null,"abstract":"<p><p>Standard flow cytometry-based assays can determine the cytotoxicity of immune effector cells, but it is challenging to monitor the dynamic processes of cytotoxicity. Here, we present a protocol for continuous observation of natural killer (NK) cell-mediated cytotoxicity with microwell arrays using an automated microscope. We describe steps for isolating and labeling primary NK cells, loading cells onto microwell arrays, monitoring target wells, and image analysis. This protocol facilitates observation of the dynamics of immune-target cell interactions at the single-cell level. 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 1","pages":"103558"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972426","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 purification of the plastid-encoded RNA polymerase from transplastomic tobacco plants. 从转质体烟草植株中纯化质体编码RNA聚合酶的方法。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103528
Xiao-Xian Wu, Fan Li, Chuxia Zhu, Shu-Yi Sun, Wen-Hui Mu, Stephanie Ruf, Ralph Bock, Yu Zhang, Fei Zhou
{"title":"Protocol for the purification of the plastid-encoded RNA polymerase from transplastomic tobacco plants.","authors":"Xiao-Xian Wu, Fan Li, Chuxia Zhu, Shu-Yi Sun, Wen-Hui Mu, Stephanie Ruf, Ralph Bock, Yu Zhang, Fei Zhou","doi":"10.1016/j.xpro.2024.103528","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103528","url":null,"abstract":"<p><p>The plastid-encoded RNA polymerase (PEP) plays an essential role in the transcription of the chloroplast genome. Here, we present a strategy to purify the transcriptionally active protein complex from transplastomic tobacco (Nicotiana tabacum) lines in which one of the PEP core subunits is fused to an epitope tag. We describe experimental procedures for designing transformation constructs for PEP purification, selection, and analysis of transplastomic tobacco plants. We then detail the steps for purifying PEP from the transplastomic tobacco leaves. 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 1","pages":"103528"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972460","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 three-dimensional analysis of rodent skeletal muscle. 啮齿动物骨骼肌三维分析规程。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103549
Smrithi Karthikeyan, Yoko Asakura, Mayank Verma, Atsushi Asakura
{"title":"Protocol for the three-dimensional analysis of rodent skeletal muscle.","authors":"Smrithi Karthikeyan, Yoko Asakura, Mayank Verma, Atsushi Asakura","doi":"10.1016/j.xpro.2024.103549","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103549","url":null,"abstract":"<p><p>Confocal imaging is a powerful tool capable of analyzing cellular spatial data within a given tissue. Here, we present a protocol for preparing optically cleared extensor digitorum longus (EDL) skeletal muscle samples suitable for confocal imaging/computational analysis. We describe steps for sample preparation (including perfusion fixation and tissue clearing of muscle samples), image acquisition, and computational analysis, with sample segmentation/3D rendering outlined. This protocol can be applied to characterize various cell types, including muscle satellite cells (muscle stem cells) and capillary endothelial cells within rodent skeletal muscle. For complete details on the use and execution of this protocol, please refer to Verma et al.,<sup>1</sup> Verma et al.,<sup>2</sup> Karthikeyan et al.,<sup>3</sup> and Karthikeyan et al.<sup>4</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103549"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972461","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 study inter-tissue communication between the hypothalamus and white adipose tissue and lifespan using a chemogenetic approach in aged mice. 使用化学发生方法研究老年小鼠下丘脑和白色脂肪组织之间的组织间通讯和寿命的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-10 DOI: 10.1016/j.xpro.2024.103551
Kyohei Tokizane, Shin-Ichiro Imai
{"title":"Protocol to study inter-tissue communication between the hypothalamus and white adipose tissue and lifespan using a chemogenetic approach in aged mice.","authors":"Kyohei Tokizane, Shin-Ichiro Imai","doi":"10.1016/j.xpro.2024.103551","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103551","url":null,"abstract":"<p><p>Here, we present a protocol for assessing the impact of a chemogenetic manipulation in a subpopulation of the hypothalamic neurons on aging and lifespan control using a mouse model developed specifically for this purpose. We describe steps for stereotaxic viral injection and assess inter-tissue communication between protein phosphatase 1 regulatory subunit 17 (Ppp1r17)-expressing neurons in the dorsomedial hypothalamus and white adipose tissue. We then detail procedures for lifespan measurements following chemogenetic manipulation in aged mice. For complete details on the use and execution of this protocol, please refer to Tokizane et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103551"},"PeriodicalIF":1.3,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972465","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 investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients. 利用单细胞RNA-seq技术研究sars - cov -2阳性和康复患者免疫细胞内微生物多样性的方案
IF 1.3
STAR Protocols Pub Date : 2025-01-08 DOI: 10.1016/j.xpro.2024.103546
Jyoti Soni, Priyanka Mehta, Sunita Yadav, Partha Chattopadhyay, Rajesh Pandey
{"title":"Protocol for investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients.","authors":"Jyoti Soni, Priyanka Mehta, Sunita Yadav, Partha Chattopadhyay, Rajesh Pandey","doi":"10.1016/j.xpro.2024.103546","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103546","url":null,"abstract":"<p><p>Intracellular microorganisms like viruses and bacteria impact immune cell function. However, detection of these microbes is challenging as the majority exist in a non-culturable state. This protocol presents detailed steps to investigate intracellular microbial diversity using single-cell RNA sequencing (scRNA-seq) in immune-cells of SARS-CoV-2-positive and recovered patients. We present a workflow from sample collection to library preparation, covering peripheral blood mononuclear cell (PBMC) isolation, single-cell labeling, cartridge priming, and cell lysis. We outline the steps for analyzing the scRNA-seq data, from data quality control (QC) to detection of intracellular microbes. For complete details on the use and execution of this protocol, please refer to Yadav et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103546"},"PeriodicalIF":1.3,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956075","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 applying Tumor Treating Fields in mouse models of cancer using the inovivo system. 使用体内系统在小鼠癌症模型中应用肿瘤治疗场的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-08 DOI: 10.1016/j.xpro.2024.103535
Travis J Gates, Sewar Zbidat, Karina Deniz, Sanyukta Padmanabhan, Katherine Ladner, Akshat Sarkari, Xianda Zhao, Shiri Davidi, Roni Blatt, Shay Cahal, Martin Gabay, Mariell Sellevoll, Itai Tzchori, Yaara Porat, Adi Haber, Moshe Giladi, Subbaya Subramanian, Emil Lou
{"title":"Protocol for applying Tumor Treating Fields in mouse models of cancer using the inovivo system.","authors":"Travis J Gates, Sewar Zbidat, Karina Deniz, Sanyukta Padmanabhan, Katherine Ladner, Akshat Sarkari, Xianda Zhao, Shiri Davidi, Roni Blatt, Shay Cahal, Martin Gabay, Mariell Sellevoll, Itai Tzchori, Yaara Porat, Adi Haber, Moshe Giladi, Subbaya Subramanian, Emil Lou","doi":"10.1016/j.xpro.2024.103535","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103535","url":null,"abstract":"<p><p>Tumor Treating Fields (TTFields) are electric fields clinically approved for cancer treatment, delivered via arrays attached to the patient's skin. Here, we present a protocol for applying TTFields to torso orthotopic and subcutaneous mouse tumor models using the inovivo system. We guide users on proper system component connections, study protocol design, mouse fur depilation, array application, and treatment condition adjustment and monitoring. The inovivo system allows for the concurrent application of TTFields with standard cancer therapies. For complete details on the use and execution of this protocol, please refer to Barsheshet et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103535"},"PeriodicalIF":1.3,"publicationDate":"2025-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142962397","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 and characterizing a nasal epithelial model using imaging with application for respiratory viruses. 使用呼吸道病毒成像生成和表征鼻上皮模型的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-07 DOI: 10.1016/j.xpro.2024.103520
Victor H K Lam, Aleena Ghafoor, Yazan Khan, Shirley Constable, Lane B Buchanan, David Zuanazzi, Reeya Parmar, Zeynep G Tepe, Leigh J Sowerby, Cindy M Liu, Ryan M Troyer, Jessica L Prodger
{"title":"Protocol for generating and characterizing a nasal epithelial model using imaging with application for respiratory viruses.","authors":"Victor H K Lam, Aleena Ghafoor, Yazan Khan, Shirley Constable, Lane B Buchanan, David Zuanazzi, Reeya Parmar, Zeynep G Tepe, Leigh J Sowerby, Cindy M Liu, Ryan M Troyer, Jessica L Prodger","doi":"10.1016/j.xpro.2024.103520","DOIUrl":"10.1016/j.xpro.2024.103520","url":null,"abstract":"<p><p>Air-liquid interface (ALI) culture can differentiate airway epithelial cells to recapitulate the respiratory tract in vitro. Here, we present a protocol for isolating and culturing nasal epithelial cells from turbinate tissues for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We describe steps to overcome challenges of imaging fragile cultures, detect the production of mucus, and quantify intracellular virus post-SARS-CoV-2 infection. We present data on the optimal duration of ALI maturation prior to experimentation and describe which steps can be altered to optimize testing of specific hypotheses.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103520"},"PeriodicalIF":1.3,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956074","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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