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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
Protocol for the isolation of brain microvessels and visualization of RNA fluorescence in mice and humans. 小鼠和人脑微血管分离和RNA荧光可视化的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-07 DOI: 10.1016/j.xpro.2024.103530
Olivia M Osborne, Oandy Naranjo, Silvia Torices, Sarah Schmidlin, Destiny Tiburcio, Minseon Park, Michal Toborek
{"title":"Protocol for the isolation of brain microvessels and visualization of RNA fluorescence in mice and humans.","authors":"Olivia M Osborne, Oandy Naranjo, Silvia Torices, Sarah Schmidlin, Destiny Tiburcio, Minseon Park, Michal Toborek","doi":"10.1016/j.xpro.2024.103530","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103530","url":null,"abstract":"<p><p>Here, we present a protocol for isolating microvessels from fresh or snap-frozen brain tissue from mice and humans, followed by visualization of RNA utilizing RNAscope hybridization for quantification of mRNA. We describe the steps for sample preparation and isolation, fixation, and hybridization. This protocol was specifically designed to integrate with RNAscope in situ hybridization. Although the protocol was developed in a mouse model, it can be optimized for use in other organisms, including human brain samples.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103530"},"PeriodicalIF":1.3,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956076","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 a pancreatic cancer organoid associated with heterogeneous tumor microenvironment. 生成与异质肿瘤微环境相关的胰腺癌类器官的方案。
IF 1.3
STAR Protocols Pub Date : 2025-01-07 DOI: 10.1016/j.xpro.2024.103539
Kenta Takeuchi, Shunsuke Tabe, Yuya Yamamoto, Kenta Takahashi, Megumi Matsuo, Yasuharu Ueno, Masayuki Ohtsuka, Soichiro Morinaga, Yohei Miyagi, Tomoyuki Yamaguchi, Naoki Tanimizu, Hideki Taniguchi
{"title":"Protocol for generating a pancreatic cancer organoid associated with heterogeneous tumor microenvironment.","authors":"Kenta Takeuchi, Shunsuke Tabe, Yuya Yamamoto, Kenta Takahashi, Megumi Matsuo, Yasuharu Ueno, Masayuki Ohtsuka, Soichiro Morinaga, Yohei Miyagi, Tomoyuki Yamaguchi, Naoki Tanimizu, Hideki Taniguchi","doi":"10.1016/j.xpro.2024.103539","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103539","url":null,"abstract":"<p><p>Pancreatic ductal adenocarcinoma (PDAC) organoids that simulate the tumor microenvironment (TME) are an effective tool to identify how TME affects PDAC malignancy. We present a protocol for generating a fused pancreatic cancer organoid (FPCO) that partly reproduces the TME, including heterogeneous cancer-associated fibroblasts (CAFs), using patient-derived PDAC cells and human-induced pluripotent cell-derived endothelial and mesenchymal cells. We also describe the procedure for analyzing FPCO characteristics. FPCO can provide a platform for establishing a reliable drug screening system. For complete details on the use and execution of this protocol, please refer to Takeuchi et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103539"},"PeriodicalIF":1.3,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956073","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 fecal microbiota transplantation: A microaerophilic approach for mice housed in a specific pathogen-free facility. 粪便微生物群移植方案：一种在特定无病原体设施中饲养的小鼠的嗜微气方法。
IF 1.3
STAR Protocols Pub Date : 2025-01-07 DOI: 10.1016/j.xpro.2024.103517
Shari Wouters, Hugo Moors, Mieke Verslegers, Natalie Leys, Surbhi Malhotra-Kumar, Samir Kumar-Singh, Mohamed Mysara
{"title":"Protocol for fecal microbiota transplantation: A microaerophilic approach for mice housed in a specific pathogen-free facility.","authors":"Shari Wouters, Hugo Moors, Mieke Verslegers, Natalie Leys, Surbhi Malhotra-Kumar, Samir Kumar-Singh, Mohamed Mysara","doi":"10.1016/j.xpro.2024.103517","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103517","url":null,"abstract":"<p><p>Recently, studies have emerged exploring the potential application of fecal microbiota transplantation (FMT) in pre-clinical settings. Here, we present a protocol for FMT for mice housed in a specific pathogen-free (SPF) facility. We describe steps for sample collection, microaerophilic processing of freshly collected fecal pellets, and administration through oral gavage. We then detail procedures for the engraftment of the bacterial community. This protocol focuses on age- and gender-matched, healthy donor mice using a mobile and cost-effective alternative to an anoxic cabinet.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103517"},"PeriodicalIF":1.3,"publicationDate":"2025-01-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956072","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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