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Protocol for investigating light/dark locomotion in larval stage zebrafish using a standardized behavioral assay. 使用标准化行为测定法研究斑马鱼幼虫期光/暗运动的规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-10 DOI: 10.1016/j.xpro.2024.103346
Courtney Hillman, James Kearn, Matthew O Parker
{"title":"Protocol for investigating light/dark locomotion in larval stage zebrafish using a standardized behavioral assay.","authors":"Courtney Hillman, James Kearn, Matthew O Parker","doi":"10.1016/j.xpro.2024.103346","DOIUrl":"10.1016/j.xpro.2024.103346","url":null,"abstract":"<p><p>Zebrafish (Danio rerio) larvae offer a unique avenue for high-throughput in vivo investigation. The light/dark locomotor assay is widely used but lacks experimental consistency. Here, we present a protocol for a standardized light/dark assay by describing the steps for plating, acclimatizing larvae, performing the assay, and preparing drug exposure solutions. We also detail procedures for substance exposure and data analysis. For complete details on the use and execution of this protocol, please refer to Hillman et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103346"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406974","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
Protocols for identifying endogenous interactors of RNA-binding proteins in mammalian cells using the peroxidase APEX2 biotin-labeling method. 使用过氧化物酶 APEX2 生物素标记法鉴定哺乳动物细胞中 RNA 结合蛋白的内源相互作用体的方法。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-10 DOI: 10.1016/j.xpro.2024.103368
Ryota Uozumi, Kohji Mori, Shoshin Akamine, Manabu Ikeda
{"title":"Protocols for identifying endogenous interactors of RNA-binding proteins in mammalian cells using the peroxidase APEX2 biotin-labeling method.","authors":"Ryota Uozumi, Kohji Mori, Shoshin Akamine, Manabu Ikeda","doi":"10.1016/j.xpro.2024.103368","DOIUrl":"10.1016/j.xpro.2024.103368","url":null,"abstract":"<p><p>Engineered ascorbate peroxidase, APEX2, is widely applied for the identification of intracellular molecule-molecule interaction analyses. Here, we present a protocol for identifying interactors of RNA-binding proteins (RBPs) in living HeLa cells using the APEX2 fusion construct. We describe steps for generation of RBP-APEX2, proximity biotin labeling, and preparation of labeled molecules for mass spectrometry analysis. This protocol may be applicable to other cell cultures and RBPs of interest. For complete details on the use and execution of this protocol, please refer to Uozumi et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103368"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142406976","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 live-cell imaging of immune synapse formation and activation of CAR T cells against cancer cells. 对免疫突触的形成和 CAR T 细胞对癌细胞的激活进行活细胞成像的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-01 DOI: 10.1016/j.xpro.2024.103422
Jorge Ibanez, Nikhil Hebbar, Unmesha Thanekar, Zhongzhen Yi, Haley Houke, Meghan Ward, Chris Nevitt, Liqing Tian, Stephen C Mack, Heather Sheppard, Jason Chiang, M Paulina Velasquez, Giedre Krenciute
{"title":"Protocol for live-cell imaging of immune synapse formation and activation of CAR T cells against cancer cells.","authors":"Jorge Ibanez, Nikhil Hebbar, Unmesha Thanekar, Zhongzhen Yi, Haley Houke, Meghan Ward, Chris Nevitt, Liqing Tian, Stephen C Mack, Heather Sheppard, Jason Chiang, M Paulina Velasquez, Giedre Krenciute","doi":"10.1016/j.xpro.2024.103422","DOIUrl":"10.1016/j.xpro.2024.103422","url":null,"abstract":"<p><p>Immune synapse (IS) formation determines T cell antitumor activity. Here, we present a protocol for characterizing the IS formation between chimeric antigen receptor (CAR) T cells and tumor cells by measuring the IS size and calcium flux by live-cell imaging. We describe steps for CAR T cell manufacturing, sample preparation, image acquisition, and data analysis. For complete details on the use and execution of this protocol, please refer to Chockley et al.,<sup>1</sup> Ibanez et al.,<sup>2</sup> and Zoine et al.<sup>3</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103422"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11566859/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568093","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 studying parasitoid-induced long-term effects in Drosophila. 研究寄生虫对果蝇长期影响的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-11-04 DOI: 10.1016/j.xpro.2024.103438
Madhumala K Sadanandappa, Giovanni Bosco
{"title":"Protocol for studying parasitoid-induced long-term effects in Drosophila.","authors":"Madhumala K Sadanandappa, Giovanni Bosco","doi":"10.1016/j.xpro.2024.103438","DOIUrl":"10.1016/j.xpro.2024.103438","url":null,"abstract":"<p><p>Drosophila and its parasitoids provide an ecologically relevant model for studying host-parasitoid biology, focusing on the behavioral and physiological responses involved in host defensive strategies and parasitoid countermeasures. Here, we outline a protocol for rearing Pachycrepoideus, a pupal parasitoid wasp, and a behavioral assay to assess the long-term impact of parasitoid exposure on adult Drosophila. We detail the steps for preparing and cohabiting Drosophila with the wasps, documenting egg-laying, and analyzing reproductive responses and eclosion in fruit flies. For complete details on the use and execution of this protocol, please refer to Sadanandappa et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103438"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11568782/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142584445","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 extracellular vesicles from alveolar macrophages phagocytosing MRSA in vitro cell culture models.
IF 1.3
STAR Protocols Pub Date : 2024-12-20 DOI: 10.1016/j.xpro.2024.103526
Songjie Bai, Ruomeng Hu, Xuehuan Wen, Wenting Zhang, Bingyu Li, Jie Yang, Qing Yu, Xianchang Zeng, Huiqing Xiu, Zhijian Cai, Gensheng Zhang
{"title":"Protocol for isolating extracellular vesicles from alveolar macrophages phagocytosing MRSA in vitro cell culture models.","authors":"Songjie Bai, Ruomeng Hu, Xuehuan Wen, Wenting Zhang, Bingyu Li, Jie Yang, Qing Yu, Xianchang Zeng, Huiqing Xiu, Zhijian Cai, Gensheng Zhang","doi":"10.1016/j.xpro.2024.103526","DOIUrl":"https://doi.org/10.1016/j.xpro.2024.103526","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) play a crucial role in delivering bioactive cargo in infectious diseases. Here, we present a protocol for isolating EVs from alveolar macrophages (AMs) that phagocytose methicillin-resistant Staphylococcus aureus (MRSA) in vitro cell culture models. We describe steps for bacterial preparation; infection of AMs with MRSA; and isolation, purification, and characterization of EVs. This protocol provides a valuable perspective for studying EVs derived from pathogen-infected immune cells. For complete details on the use and execution of this protocol, please refer to Bai et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103526"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142872902","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 whole-cell patch-clamp assay for HBV-mediated sodium influx in THP-1 macrophages.
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-12-02 DOI: 10.1016/j.xpro.2024.103462
Yingbao Tan, Penghui Chen, Xiaojuan Xin, Jingxue Wang
{"title":"Protocol for whole-cell patch-clamp assay for HBV-mediated sodium influx in THP-1 macrophages.","authors":"Yingbao Tan, Penghui Chen, Xiaojuan Xin, Jingxue Wang","doi":"10.1016/j.xpro.2024.103462","DOIUrl":"10.1016/j.xpro.2024.103462","url":null,"abstract":"<p><p>Sodium influx induced by hepatitis B virus (HBV) enhances activation of the NLRP3 inflammasome, in synergy with potassium efflux signals in macrophages. Here, we present a protocol for conducting patch-clamp assays to measure HBV-mediated sodium influx in THP-1 macrophages. We describe steps for differentiating, resting, and activating THP-1 macrophages and then outline the procedures for whole-cell patch-clamp recordings. 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":"5 4","pages":"103462"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11652772/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142781330","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
Molecular protocol for genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by CIMERA-seq.
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-12-10 DOI: 10.1016/j.xpro.2024.103477
Xinbei Li, Emily A Eiss, William T Mills, Mollie K Meffert
{"title":"Molecular protocol for genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by CIMERA-seq.","authors":"Xinbei Li, Emily A Eiss, William T Mills, Mollie K Meffert","doi":"10.1016/j.xpro.2024.103477","DOIUrl":"10.1016/j.xpro.2024.103477","url":null,"abstract":"<p><p>Small noncoding RNAs (sncRNAs) can regulate gene expression by guiding the RNA-induced silencing complex (RISC) to targeted transcripts for translational repression and/or target destabilization. Here, we present a robust benchtop protocol, termed CIMERA-seq, for the unambiguous profiling of sncRNA:target RNA interactions in a genome-wide and cell-type-selective manner. We describe steps for in vivo crosslinking and harvesting tissue, immunoprecipitation and covalent ligation of sncRNAs to target RNAs within the RISC, and sequencing of the resulting chimeric sncRNA:target RNA interactions. 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":"5 4","pages":"103477"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11697689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142814431","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 establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages. 建立用于铜绿假单胞菌噬菌体高效基因组编辑的 CRISPR-Cas12a 协议。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-12-11 DOI: 10.1016/j.xpro.2024.103488
Bingjie Yan, Yujia Liu, Yumei Cai, Yuqing Liu, Yibao Chen
{"title":"Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.","authors":"Bingjie Yan, Yujia Liu, Yumei Cai, Yuqing Liu, Yibao Chen","doi":"10.1016/j.xpro.2024.103488","DOIUrl":"10.1016/j.xpro.2024.103488","url":null,"abstract":"<p><p>We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103488"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11697554/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818828","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
Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages. 基于点击化学方法检测原代小鼠巨噬细胞中的 4-辛基-伊他康酸烷基化蛋白质。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-19 DOI: 10.1016/j.xpro.2024.103314
Chaofei Su, Tian Cheng, Hanyi Zhang, Hang Yin
{"title":"Click-chemistry-based protocol for detecting 4-octyl-itaconate-alkylated proteins in primary mouse macrophages.","authors":"Chaofei Su, Tian Cheng, Hanyi Zhang, Hang Yin","doi":"10.1016/j.xpro.2024.103314","DOIUrl":"10.1016/j.xpro.2024.103314","url":null,"abstract":"<p><p>4-Octyl itaconate (4-OI), a derivative of itaconate, inhibits inflammation by alkylating its target proteins. Here, we present a click-chemistry-based protocol for detecting 4-OI-alkylated proteins in mouse primary bone-marrow-derived macrophages (BMDMs) by using an itaconate-alkyne (ITalk) probe. We describe steps for culturing and treating BMDMs and details on using click chemistry in the cell lysate. We also detail procedures for detecting alkylated proteins by western blot. For complete details on the use and execution of this protocol, please refer to Su et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103314"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11426128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142297131","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 assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry. 使用流式细胞仪和成像流式细胞仪评估小鼠细胞双层接合及其后续影响的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-09-21 DOI: 10.1016/j.xpro.2024.103152
Yuval Shapir Itai, Ziv Porat, Rony Dahan
{"title":"Protocol for assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry.","authors":"Yuval Shapir Itai, Ziv Porat, Rony Dahan","doi":"10.1016/j.xpro.2024.103152","DOIUrl":"10.1016/j.xpro.2024.103152","url":null,"abstract":"<p><p>Physical interactions between two immune cells or between immune and cancer cells play a major role in shaping the immune response in the tumor microenvironment, making them prime therapeutic targets for bispecific engagers. Here, we present a protocol for assessing murine cell doublet engagement and subsequent effects using flow cytometry and imaging flow cytometry. We describe steps for identifying bispecific cell engager antibodies at the cell-cell interface, doublet quantification, and characterizing cellular protein morphology and processes within the doublet. For complete details on the use and execution of this protocol, please refer to Shapir Itai et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103152"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11459070/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142297132","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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