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Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors. 使用 ONE-GO 生物传感器检测原代细胞培养物中内源性 GPCR 活性的规程。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-01 DOI: 10.1016/j.xpro.2024.103355
Remi Janicot, Mikel Garcia-Marcos
{"title":"Protocol for detecting endogenous GPCR activity in primary cell cultures using ONE-GO biosensors.","authors":"Remi Janicot, Mikel Garcia-Marcos","doi":"10.1016/j.xpro.2024.103355","DOIUrl":"10.1016/j.xpro.2024.103355","url":null,"abstract":"<p><p>ONE vector G protein Optical (ONE-GO) biosensors can measure the activity of endogenously expressed G protein-coupled receptors (GPCRs) in primary cells. By detecting G proteins that belong to all four families (G<sub>s</sub>, G<sub>i/o</sub>, G<sub>q/11</sub>, G<sub>12/13</sub>) across cell types, these biosensors provide high experimental versatility. We first describe steps to express ONE-GO biosensors in primary cells using lentiviral transduction. We then detail how to carry out measurements and subsequent analysis to quantify changes in bioluminescence resonance energy transfer (BRET) reporting on endogenous GPCR activity. For complete details on the use and execution of this protocol, please refer to Janicot et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103355"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480228/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142366778","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 simultaneous isolation of DNA, RNA, and miRNA from a single archived Kaposi sarcoma biopsy. 从单个存档卡波西肉瘤活检组织中同时分离 DNA、RNA 和 miRNA 的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-04 DOI: 10.1016/j.xpro.2024.103365
Larissa L S Scholte, Justin Browne, David J Nolan, Peyton St John, Katherine Tracy, Rafaela S Thur, Ghangzhao Li, Susanna L Lamers, Paige Bracci, Michael S McGrath, Jeffrey M Bethony
{"title":"Protocol for the simultaneous isolation of DNA, RNA, and miRNA from a single archived Kaposi sarcoma biopsy.","authors":"Larissa L S Scholte, Justin Browne, David J Nolan, Peyton St John, Katherine Tracy, Rafaela S Thur, Ghangzhao Li, Susanna L Lamers, Paige Bracci, Michael S McGrath, Jeffrey M Bethony","doi":"10.1016/j.xpro.2024.103365","DOIUrl":"10.1016/j.xpro.2024.103365","url":null,"abstract":"<p><p>Kaposi sarcoma (KS) punch biopsies present unique challenges for extracting nucleic acids, which can be exacerbated by their long-term stabilization in RNAlater. Here, we present a protocol for simultaneously isolating DNA, RNA, and miRNA from a single KS punch biopsy. We detail the steps for preparing reagents and supplies, disrupting KS tissue using manual and mechanical methods, isolating DNA and total RNA, evaluating nucleic acid quality, and storing nucleic acids long-term.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103365"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11490689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378398","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 establish turkey oviductal organoids as an in vitro model. 建立火鸡输卵管有机体体外模型的程序。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-09 DOI: 10.1016/j.xpro.2024.103384
Pitchaya Santativongchai, Caitlin C Klaeui, Sunantha Kosonsiriluk, Milena Saqui-Salces, Kent M Reed, Ben W Wileman, Marissa M Studniski, Kahina S Boukherroub
{"title":"Protocol to establish turkey oviductal organoids as an in vitro model.","authors":"Pitchaya Santativongchai, Caitlin C Klaeui, Sunantha Kosonsiriluk, Milena Saqui-Salces, Kent M Reed, Ben W Wileman, Marissa M Studniski, Kahina S Boukherroub","doi":"10.1016/j.xpro.2024.103384","DOIUrl":"10.1016/j.xpro.2024.103384","url":null,"abstract":"<p><p>The study of reproductive function in turkey hens has been difficult due to the lack of a reliable, representative in vitro model for investigating profound physiological aspects. This article presents a protocol to establish turkey oviductal organoids, including steps for isolating turkey oviduct epithelial cells followed by seeding and maintaining 3D organoid cultures. We also detail procedures for organoid fixation for histological analysis. This organoid model could serve as a valuable in vitro tool for understanding the intricacies of turkey reproductive physiology.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103384"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142401509","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
Spatial analysis of transcript and protein levels in skeletal muscle. 骨骼肌转录本和蛋白质水平的空间分析。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-09 DOI: 10.1016/j.xpro.2024.103378
Paola Pisterzi, Clara Martinez Mir, Ouafa Dahri, Isabel de Poorter, Sandra Batlles Parera, Milica Dostanić, Massimo Mastrangeli, Christine Mummery, Niels Geijsen, Fanny Sage
{"title":"Spatial analysis of transcript and protein levels in skeletal muscle.","authors":"Paola Pisterzi, Clara Martinez Mir, Ouafa Dahri, Isabel de Poorter, Sandra Batlles Parera, Milica Dostanić, Massimo Mastrangeli, Christine Mummery, Niels Geijsen, Fanny Sage","doi":"10.1016/j.xpro.2024.103378","DOIUrl":"10.1016/j.xpro.2024.103378","url":null,"abstract":"<p><p>Skeletal muscle spatial analyses have revealed unexpected regionalized gene expression patterns challenging the understanding of muscle as a homogeneous tissue. Here, we present a protocol for the spatial analysis of transcript and protein levels in murine skeletal muscle. We describe steps for tibialis anterior dissection, formaldehyde fixation, tissue chopper cutting, and hybridization chain reaction (HCR) detection and amplification. We then detail procedures for immunostaining, tissue clearing, and imaging. This protocol is easily adaptable to other tissues.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103378"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142401510","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 analyzing invadopodia formation and gelatin degradation. 分析内生单体形成和明胶降解的程序。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-18 DOI: 10.1016/j.xpro.2024.103399
Weijian Meng, Qingwen Huang, Rui Zhang, Jie Shen, Daxing Xie, Cong Xu, Liang Liu
{"title":"Protocol for analyzing invadopodia formation and gelatin degradation.","authors":"Weijian Meng, Qingwen Huang, Rui Zhang, Jie Shen, Daxing Xie, Cong Xu, Liang Liu","doi":"10.1016/j.xpro.2024.103399","DOIUrl":"10.1016/j.xpro.2024.103399","url":null,"abstract":"<p><p>Invadopodia are actin-rich protrusions on the tumor cell membrane that degrade the extracellular matrix and play a crucial role in tumor cell invasion and metastasis. Here, we present a protocol to examine invadopodia's ability to form and degrade the extracellular matrix during tumor invasion and metastasis. We detail the procedure for using immunofluorescence staining to indirectly detect invadopodia formation and assess their extracellular matrix degradation capability via the gelatin degradation assay. For complete details on the use and execution of this protocol, please refer to Huang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103399"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513534/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476469","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 constructing whole-genome libraries from mini-biopsies by using laser capture microdissection. 利用激光捕获显微切割技术从微型活组织中构建全基因组文库的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-15 DOI: 10.1016/j.xpro.2024.103396
Yiyi Xi, Shaosen Zhang
{"title":"Protocol for constructing whole-genome libraries from mini-biopsies by using laser capture microdissection.","authors":"Yiyi Xi, Shaosen Zhang","doi":"10.1016/j.xpro.2024.103396","DOIUrl":"10.1016/j.xpro.2024.103396","url":null,"abstract":"<p><p>Disclosure of the key events in tumor progress will help us deepen the understanding of tumorigenesis. Here, we present a protocol for multi-regional tissue capture of malignant continuum. We describe steps for preparing tissue sections, laser capture microdissection, and whole-genome library preparation, which enable the concurrent analysis of potential driver events in precancer initiation. This protocol overcomes challenges posed by limited DNA quantity and preserves the spatial information of the target regions. For complete details on the use and execution of this protocol, please refer to Li et al.,<sup>1</sup> Chang et al.,<sup>2</sup> and Chen et al.<sup>3</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103396"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11525219/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476472","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 detecting homologous recombination activity in cancer cells by adenovirus-based functional assay. 通过基于腺病毒的功能测试检测癌细胞中同源重组活性的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-19 DOI: 10.1016/j.xpro.2024.103403
Shih-Han Huang, Chih-Ying Lee, Peter Chi
{"title":"Protocol for detecting homologous recombination activity in cancer cells by adenovirus-based functional assay.","authors":"Shih-Han Huang, Chih-Ying Lee, Peter Chi","doi":"10.1016/j.xpro.2024.103403","DOIUrl":"10.1016/j.xpro.2024.103403","url":null,"abstract":"<p><p>Cancer cells with homologous recombination deficiency (HRD) exhibit a distinctive vulnerability to poly(ADP-ribose) polymerase inhibitors (PARPis). To address the limitations of existing methodologies incapable of providing real-time insights into homologous recombination (HR) status, we present an adenovirus-based functional assay designed to quantify cellular HR activity. Here, we delineate the step-by-step procedure for producing the adenovirus harboring an HR reporter, processing primary cells, and assessing HR activity in primary ovarian cancer cells. For complete details on the use and execution of this protocol, please refer to Lee et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103403"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11532989/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476474","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 detection of glial complement expression in relation to amyloid plaques in mouse brain with combined FISH and IHC. 利用 FISH 和 IHC 联合方法检测小鼠大脑中与淀粉样蛋白斑块有关的胶质补体表达的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-16 DOI: 10.1016/j.xpro.2024.103388
Sadhna Rao, Joanna Yung, Melissa Gonzalez Edick, Jesse E Hanson
{"title":"Protocol for detection of glial complement expression in relation to amyloid plaques in mouse brain with combined FISH and IHC.","authors":"Sadhna Rao, Joanna Yung, Melissa Gonzalez Edick, Jesse E Hanson","doi":"10.1016/j.xpro.2024.103388","DOIUrl":"10.1016/j.xpro.2024.103388","url":null,"abstract":"<p><p>Complement proteins contribute to neurodegeneration in Alzheimer's disease (AD) and are secreted by glia surrounding beta-amyloid (Aβ) plaques. We present an optimized protocol for Aβ plaque detection with tyramide-digoxigenin signal amplification. This is combined with a multiplex mRNA fluorescence in situ hybridization (FISH) panel to assay glial-specific complement expression proximal to Aβ plaques in TauPS2APP mice. We describe steps for tissue preparation and mRNA detection. We then detail steps for the detection of Aβ plaques, image acquisition, and analysis.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103388"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11530921/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476475","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 identification of NEMF-mediated C-terminal extensions on mitochondrial nonstop proteins via customized MS/MS spectra database searching. 通过定制的 MS/MS 图谱数据库搜索，确定线粒体不停跳蛋白上由 NEMF 介导的 C 末端延伸。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-11 DOI: 10.1016/j.xpro.2024.103366
Leijie Chen, Jinyou Mo, Yuyong Tan, Liang Lv, Jia Liu
{"title":"Protocol for identification of NEMF-mediated C-terminal extensions on mitochondrial nonstop proteins via customized MS/MS spectra database searching.","authors":"Leijie Chen, Jinyou Mo, Yuyong Tan, Liang Lv, Jia Liu","doi":"10.1016/j.xpro.2024.103366","DOIUrl":"10.1016/j.xpro.2024.103366","url":null,"abstract":"<p><p>The ribosome-associated protein quality control (RQC) core factor nuclear export mediator factor (NEMF) appends C-terminal extended sequences (CESs) to ribosome-stalled nascent chains (NCs). Specific CESs compositions could be directly recognized by enzymes and facilitate NC degradation. Yet, NEMF-mediated CESs remains largely unidentified. Here, we present a protocol for identifying and characterizing NEMF-mediated C-terminal modifications on mitochondrial NCs (mitoNCs) via tandem mass spectrometry (MS/MS) analysis. We describe strategies aimed at constructing a customized MS/MS spectra database for unknown CESs and detail the steps for CES-modified sample preparation. For complete details on the use and execution of this protocol, please refer to Lv et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103366"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476478","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 isolating antigen-specific monoclonal antibodies from immunized mice utilizing the Beacon platform. 利用 Beacon 平台从免疫小鼠体内分离抗原特异性单克隆抗体的方案。
IF 1.3
STAR Protocols Pub Date : 2024-12-20 Epub Date: 2024-10-12 DOI: 10.1016/j.xpro.2024.103389
Meilan Fu, Xiangwei Wang, Shubing Tang, Shimeng Bai, Longfei Ding, Yangyang Hu, Kangli Cao, Tianhan Yang, Chen Zhao, Xiaoyan Zhang, Jianqing Xu
{"title":"Protocol for isolating antigen-specific monoclonal antibodies from immunized mice utilizing the Beacon platform.","authors":"Meilan Fu, Xiangwei Wang, Shubing Tang, Shimeng Bai, Longfei Ding, Yangyang Hu, Kangli Cao, Tianhan Yang, Chen Zhao, Xiaoyan Zhang, Jianqing Xu","doi":"10.1016/j.xpro.2024.103389","DOIUrl":"10.1016/j.xpro.2024.103389","url":null,"abstract":"<p><p>Isolation of antigen-specific plasma B cells had been challenging until the recent arrival of the Beacon platform. Leveraging light-sorting technology, Beacon can perform high-throughput screening of plasma B cells on a chip to sort single cells with the desired antigen specificity. Here, we present a protocol for isolating antigen-specific plasma B cells from immunized mice using Beacon, sequencing the encoded B cell receptors (BCRs), and cloning and expressing the resulting antibodies. This protocol can easily be extended to human samples.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"5 4","pages":"103389"},"PeriodicalIF":1.3,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142476486","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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