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Protocol for immunofluorescence characterization of drusen in induced pluripotent stem cell-derived retinal pigment epithelium cultures. 诱导多能干细胞来源的视网膜色素上皮培养物中drusen的免疫荧光表征方法。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-05-02 DOI: 10.1016/j.xpro.2025.103791
Sonal Dalvi, Ruchira Singh
{"title":"Protocol for immunofluorescence characterization of drusen in induced pluripotent stem cell-derived retinal pigment epithelium cultures.","authors":"Sonal Dalvi, Ruchira Singh","doi":"10.1016/j.xpro.2025.103791","DOIUrl":"10.1016/j.xpro.2025.103791","url":null,"abstract":"<p><p>Drusen, lipoprotein-rich deposits underlying retinal pigment epithelium (RPE), are a pathological hallmark of age-related macular degeneration and related macular dystrophies. Here, we describe a protocol for visualizing drusen deposits beneath induced pluripotent stem cell (iPSC)-RPE (iRPE) monocultures. We describe steps for iRPE cell culture and, subsequently, enzymatic removal of the iRPE monolayer and immunostaining of sub-RPE drusen deposits. We then detail steps for confocal microscopy imaging of immunostained drusen in iRPE culture. For complete details on the use and execution of this protocol, please refer to Dalvi et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103791"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12090231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144053045","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 assess the impact of sperm RNA on mouse preimplantation embryos using microinjection. 使用显微注射评估精子RNA对小鼠着床前胚胎影响的方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-05-09 DOI: 10.1016/j.xpro.2025.103772
Grace S Lee, Natalie A Trigg, Colin C Conine
{"title":"Protocol to assess the impact of sperm RNA on mouse preimplantation embryos using microinjection.","authors":"Grace S Lee, Natalie A Trigg, Colin C Conine","doi":"10.1016/j.xpro.2025.103772","DOIUrl":"10.1016/j.xpro.2025.103772","url":null,"abstract":"<p><p>Zygotic microinjections show that sperm RNAs transmit non-genetically inherited phenotypes to offspring and influence embryonic development. Here, we present a protocol for the micromanipulation of mouse zygotes to introduce physiologically relevant levels of sperm RNA. We describe steps for producing functional mRNAs in vitro; purifying mouse sperm RNAs; and preparing, microinjecting, and culturing zygotes. This protocol facilitates causal analysis between sperm RNA and gene regulation postfertilization. For complete details on the use and execution of this protocol, please refer to Trigg et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103772"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12136814/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144056766","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 cGAMP-induced STING oligomerization in cultured cells. 培养细胞中cgamp诱导的STING寡聚化检测方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-21 DOI: 10.1016/j.xpro.2025.103709
Lizhi Liu, James L Manley
{"title":"Protocol for detection of cGAMP-induced STING oligomerization in cultured cells.","authors":"Lizhi Liu, James L Manley","doi":"10.1016/j.xpro.2025.103709","DOIUrl":"10.1016/j.xpro.2025.103709","url":null,"abstract":"<p><p>STING (stimulator of interferon genes) plays a critical role in the innate immune response, including in viral infection, cancer immunity, and several autoimmune diseases. Here, we present a protocol for detection of STING oligomerization, a critical step in STING activation. We describe steps for expressing STING in HEK293T cells, activating STING by 2'3' cyclic GMP-AMP (cGAMP) treatment, and analyzing STING oligomerization by non-reducing SDS-PAGE and blue native PAGE. This protocol is useful for studies of STING functions, regulation, and dysregulation. For complete details on the use and execution of this protocol, please refer to Liu and Manley.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103709"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11976245/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143693626","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 inducing epidermis-specific gene deletion and establishing a UVB-irradiated skin inflammation model. 诱导表皮特异性基因缺失和建立uvb辐照皮肤炎症模型的方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-16 DOI: 10.1016/j.xpro.2025.103701
Ye Liu, George L Sen
{"title":"Protocol for inducing epidermis-specific gene deletion and establishing a UVB-irradiated skin inflammation model.","authors":"Ye Liu, George L Sen","doi":"10.1016/j.xpro.2025.103701","DOIUrl":"10.1016/j.xpro.2025.103701","url":null,"abstract":"<p><p>When skin is exposed to ultraviolet B (UVB) radiation, damaged keratinocytes release double-stranded RNA to trigger an inflammatory response. Here, we present a protocol to induce epidermal-specific Zfp750 gene-deficient mice and establish a UVB-induced skin inflammation model. We detail procedures for detecting gene expressions in the epidermis by quantitative reverse-transcription PCR (RT-qPCR) and analyzing changes in skin immune cell subsets after UVB radiation using flow cytometry. For complete details on the use and execution of this protocol, please refer to Liu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103701"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957568/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143651158","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 mouse carotid artery perfusion for in situ brain tissue fixation and parallel unfixed tissue collection. 小鼠颈动脉灌注原位脑组织固定及平行非固定组织收集方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-14 DOI: 10.1016/j.xpro.2025.103699
Phung N Thai, Zubayer I Ferdous, Pauline Trinh, Valeriy Timofeev, Daphne A Diloretto, Ning Zong, Chao-Yin Chen, Nipavan Chiamvimonvat, Xiao-Dong Zhang
{"title":"Protocol for mouse carotid artery perfusion for in situ brain tissue fixation and parallel unfixed tissue collection.","authors":"Phung N Thai, Zubayer I Ferdous, Pauline Trinh, Valeriy Timofeev, Daphne A Diloretto, Ning Zong, Chao-Yin Chen, Nipavan Chiamvimonvat, Xiao-Dong Zhang","doi":"10.1016/j.xpro.2025.103699","DOIUrl":"10.1016/j.xpro.2025.103699","url":null,"abstract":"<p><p>As the study of central control of multiple organ function becomes more prominent, there is an increasing need for the collection of fixed brain and unfixed organs and tissues from the same experimental animal. Here, we present a protocol for performing carotid artery cannulation, organ and tissue collection, in situ brain perfusion and fixation, and brain dissection in mice. We describe steps for cannulating the carotid artery, harvesting the heart and other organs, and perfusing, fixing, and dissecting the brain.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103699"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11937653/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634831","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 spatial metabolomics and isotope tracing in the mouse brain. 小鼠大脑空间代谢组学和同位素追踪方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-27 DOI: 10.1016/j.xpro.2025.103716
Watit Sontising, Francine Yanchik-Slade, Carlos Rodriguez-Navas, Md Amir Hossen, Manuel Gonzalez-Rodriguez, Jake Vaynshteyn, Shinichi Yamaguchi, Mohamed Nazim Boutaghou, Isaac Marin-Valencia
{"title":"Protocol for spatial metabolomics and isotope tracing in the mouse brain.","authors":"Watit Sontising, Francine Yanchik-Slade, Carlos Rodriguez-Navas, Md Amir Hossen, Manuel Gonzalez-Rodriguez, Jake Vaynshteyn, Shinichi Yamaguchi, Mohamed Nazim Boutaghou, Isaac Marin-Valencia","doi":"10.1016/j.xpro.2025.103716","DOIUrl":"10.1016/j.xpro.2025.103716","url":null,"abstract":"<p><p>Mass spectrometry imaging enables high-resolution spatial chemical mapping, yet its application for dynamic analysis with tracers poses challenges. Here, we present a protocol for spatial metabolomics and isotope tracing in the mouse brain. We describe steps for tracer administration, tissue collection, and cryosectioning. We then detail procedures for matrix application, ion identification, and data analysis. This protocol delivers high-quality spatial metabolomics data and is well suited for region-specific tracing analysis in the brain.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103716"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11995059/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143735892","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 high-throughput screening of SIRT7 inhibitors using fluorescent peptide technology. 利用荧光肽技术高通量筛选 SIRT7 抑制剂的方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-28 DOI: 10.1016/j.xpro.2025.103720
Tian-Shu Kang, Xiaopeng Lu, Jinke Gu, Yuan Tian, Wei-Guo Zhu
{"title":"Protocol for high-throughput screening of SIRT7 inhibitors using fluorescent peptide technology.","authors":"Tian-Shu Kang, Xiaopeng Lu, Jinke Gu, Yuan Tian, Wei-Guo Zhu","doi":"10.1016/j.xpro.2025.103720","DOIUrl":"10.1016/j.xpro.2025.103720","url":null,"abstract":"<p><p>Fluorescent peptides combine the polypeptides with fluorescent groups organically to facilitate the measurement of enzyme activity. Here, we present a protocol to evaluate Sirtuin 7 (SIRT7) enzymatic activity by analyzing changes in luminescent signals from its substrate polypeptides. We outline the steps for SIRT7 protein purification, enzymatic reaction setup, fluorescence spectrum detection, and data analysis. Additionally, we describe two applications targeting SIRT7: high-throughput screening of SIRT7 inhibitors using microplate and the validation of potential SIRT7 inhibitors. For complete details on the use and execution of this protocol, please refer to Kang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103720"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11995078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143744092","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 optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells. 人类造血干细胞和祖细胞中CRISPR-Cas9基因编辑过程中体外激活培养条件优化方案
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-03-31 DOI: 10.1016/j.xpro.2025.103722
Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco
{"title":"Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.","authors":"Lucrezia Della Volpe, Roberta Vacca, Raffaella Di Micco","doi":"10.1016/j.xpro.2025.103722","DOIUrl":"10.1016/j.xpro.2025.103722","url":null,"abstract":"<p><p>Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103722"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11999203/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143774268","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 determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae. 确定酿酒酵母菌整体内质网膜蛋白拓扑结构的方案。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-04-04 DOI: 10.1016/j.xpro.2025.103727
Ritika Chaudhary, Vineet Choudhary
{"title":"Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.","authors":"Ritika Chaudhary, Vineet Choudhary","doi":"10.1016/j.xpro.2025.103727","DOIUrl":"10.1016/j.xpro.2025.103727","url":null,"abstract":"<p><p>Here, we present a protocol to determine the topology of Fld1 (few lipid droplets), an integral endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae. We describe steps to generate functional N-terminal GFP and C-terminal mCherry fusion with Fld1. We detail the strategy to perform subcellular fractionation to isolate ER-derived microsomes that were subjected to salt detergent extraction analysis. We then provide procedures to determine the topology of Fld1 using proteinase K treatment.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103727"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12002983/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143789272","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 how expectations guide predictive eye movements and information sampling in humans. 研究期望如何引导人类的预测性眼球运动和信息采样的协议。
IF 1.3
STAR Protocols Pub Date : 2025-06-20 Epub Date: 2025-04-08 DOI: 10.1016/j.xpro.2025.103737
Annika Garlichs, Mark Lustig, Matthias Gamer, Helen Blank
{"title":"Protocol to study how expectations guide predictive eye movements and information sampling in humans.","authors":"Annika Garlichs, Mark Lustig, Matthias Gamer, Helen Blank","doi":"10.1016/j.xpro.2025.103737","DOIUrl":"10.1016/j.xpro.2025.103737","url":null,"abstract":"<p><p>Investigating eye movements provides a unique tool to explore how expectations influence information sampling in the visual domain. Here, we present a protocol for measuring predictive eye movements during face anticipation as well as fixations and dwell time during face recognition in humans. We describe steps for setting up two eye-tracking experiments. We then detail procedures for the measurement and analysis of eye-tracking data. For complete details on the use and execution of this protocol, please refer to Garlichs et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103737"},"PeriodicalIF":1.3,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12005905/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804349","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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