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Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems. 评估类固醇生成和非类固醇生成系统中线粒体胆固醇转运和蛋白质熔融球状态的方案。
IF 1.3
STAR Protocols Pub Date : 2025-07-25 DOI: 10.1016/j.xpro.2025.103971
Himangshu S Bose, Mahuya Bose, Randy M Whittal
{"title":"Protocol for assessing mitochondrial cholesterol transport and protein molten globule state in steroidogenic and nonsteroidogenic systems.","authors":"Himangshu S Bose, Mahuya Bose, Randy M Whittal","doi":"10.1016/j.xpro.2025.103971","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103971","url":null,"abstract":"<p><p>Steroid hormones are essential for the survival of all mammals for carbohydrate metabolism, stress management, and sexual reproduction. Here, we present a protocol for assessing mitochondrial cholesterol transport and protein molten globule state via measurement of pregnenolone or progesterone synthesis in steroidogenic and nonsteroidogenic cellular systems. We describe steps for cell culture, transfection, and measurement of steroidogenic activity from nonsteroidogenic cells. We then detail procedures for metabolic conversion. For complete details on the use and execution of this protocol, please refer to Bose,<sup>1</sup> Bose et al.,<sup>2</sup> Pawlak et al.,<sup>3</sup> and Prasad et al.<sup>4</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103971"},"PeriodicalIF":1.3,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718792","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 differentiating human pluripotent stem cells into midbrain organoids for targeted microinjection of viruses. 将人多能干细胞分化为中脑类器官用于病毒靶向显微注射的方案。
IF 1.3
STAR Protocols Pub Date : 2025-07-25 DOI: 10.1016/j.xpro.2025.103983
Mengdan Tao, Qi Chen, WeiWei Gao, Shanshan Wu, Yan Liu
{"title":"Protocol for differentiating human pluripotent stem cells into midbrain organoids for targeted microinjection of viruses.","authors":"Mengdan Tao, Qi Chen, WeiWei Gao, Shanshan Wu, Yan Liu","doi":"10.1016/j.xpro.2025.103983","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103983","url":null,"abstract":"<p><p>Midbrain organoids serve as a valuable model for studying brain development and disease. Here, we present a viral infection protocol for midbrain organoid models. We describe steps for human pluripotent stem cell (hPSC) differentiation into midbrain organoids; equipment preparation; and targeted virus microinjection into organoids to reduce viral load, minimize cytotoxicity, and preserve structural integrity. We then detail procedures for visualizing infected cells. By combining the virus with specific promoters, this protocol enables precise targeting of tube-like ventricular zone (VZ) regions. For complete details on the use and execution of this protocol, please refer to Zhu et al.,<sup>1</sup> Wu et al.,<sup>2</sup> Xu et al.,<sup>3</sup> and Lu et al.<sup>4</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103983"},"PeriodicalIF":1.3,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718794","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 processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue. 用于处理和分析多路复用图像的协议提高了人体组织中淋巴细胞的识别和空间结构。
IF 1.3
STAR Protocols Pub Date : 2025-07-25 DOI: 10.1016/j.xpro.2025.103976
Justin A Smith, Drew T Bloss, MacKenzie D Williams, Amanda L Posgai, Todd M Brusko, Mark A Atkinson, Clive H Wasserfall, Maigan A Brusko
{"title":"Protocol for processing and analyzing multiplexed images improves lymphatic cell identification and spatial architecture in human tissue.","authors":"Justin A Smith, Drew T Bloss, MacKenzie D Williams, Amanda L Posgai, Todd M Brusko, Mark A Atkinson, Clive H Wasserfall, Maigan A Brusko","doi":"10.1016/j.xpro.2025.103976","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103976","url":null,"abstract":"<p><p>Multiplexed images of human lymphatic tissue are extensively preprocessed before cell phenotyping and spatial analysis. Here, we present KINTSUGI (knowledge integration with new technologies: simplified user-guided image processing), a protocol designed to interactively engage the user in each processing step to ensure quality control. We describe steps for parameter tuning and batch processing of raw image data including illumination correction, stitching, deconvolution, 3D-2D conversion, registration, and autofluorescence subtraction. We then detail procedures for segmentation, feature extraction, phenotyping, and spatial analysis.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103976"},"PeriodicalIF":1.3,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718796","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 developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants. 原生肺和肺植入物血液性扩散后原发性肺结核小鼠模型的建立。
IF 1.3
STAR Protocols Pub Date : 2025-07-25 DOI: 10.1016/j.xpro.2025.103984
Shivraj M Yabaji, Suruchi Lata, Igor Gavrish, Ming Lo, Aoife K O'Connell, Hans P Gertje, Colleen E Thurman, Nicholas A Crossland, Lester Kobzik, Igor Kramnik
{"title":"Protocol for developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants.","authors":"Shivraj M Yabaji, Suruchi Lata, Igor Gavrish, Ming Lo, Aoife K O'Connell, Hans P Gertje, Colleen E Thurman, Nicholas A Crossland, Lester Kobzik, Igor Kramnik","doi":"10.1016/j.xpro.2025.103984","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103984","url":null,"abstract":"<p><p>Here, we present a protocol for a mouse model for studying mechanisms of post-primary pulmonary tuberculosis (PTB) caused by virulent Mycobacterium tuberculosis (Mtb) using subcutaneous hock infection and lung tissue implantation. We describe steps for collagen instillation of lungs, lung and spleen implantation, preparation of Mtb for infection, and hock infection of mice. We then detail procedures for the perfusion of the lung and collection of organs, tissue processing, and histopathologic interpretation. For complete details on the use and execution of this protocol, please refer to Yabaji et al.<sup>1</sup><sup>,</sup><sup>2</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103984"},"PeriodicalIF":1.3,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718793","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 visualization of ultrafine nuclear structures in cultured cells using protein retention expansion microscopy. 用蛋白保留扩增显微镜观察培养细胞超微核结构的方法。
IF 1.3
STAR Protocols Pub Date : 2025-07-24 DOI: 10.1016/j.xpro.2025.103985
Takaaki Okamoto, Masahide Fukada, Akio Masuda
{"title":"Protocol for visualization of ultrafine nuclear structures in cultured cells using protein retention expansion microscopy.","authors":"Takaaki Okamoto, Masahide Fukada, Akio Masuda","doi":"10.1016/j.xpro.2025.103985","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103985","url":null,"abstract":"<p><p>In the nucleus, multivalent interactions between DNA, RNA, and proteins form functional networks that drive nuclear metabolism, such as transcription and RNA processing. Here, we present a protocol to visualize the subnuclear distribution of nuclear proteins in cultured cells using protein retention expansion microscopy (proExM). We describe steps for immunostaining, sample expansion, sample bonding on glass coverslips, and imaging with confocal microscopy. This protocol is a reproducible and cost-effective procedure for studying ultrafine nuclear organizations. For complete details on the use and execution of this protocol, please refer to Masuda et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103985"},"PeriodicalIF":1.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144715199","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 directed differentiation of human induced pluripotent stem cells into thyroid follicular epithelial cells. 人诱导多能干细胞定向分化为甲状腺滤泡上皮细胞的方法。
IF 1.3
STAR Protocols Pub Date : 2025-07-24 DOI: 10.1016/j.xpro.2025.103979
Hendrik J Undeutsch, Alberto Posabella, Darrell N Kotton, Anthony N Hollenberg
{"title":"Protocol for directed differentiation of human induced pluripotent stem cells into thyroid follicular epithelial cells.","authors":"Hendrik J Undeutsch, Alberto Posabella, Darrell N Kotton, Anthony N Hollenberg","doi":"10.1016/j.xpro.2025.103979","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103979","url":null,"abstract":"<p><p>We provide a directed differentiation protocol deriving thyroid follicular cells (TFCs) from human induced pluripotent stem cells (iPSCs) or embryonic stem cells without exogenous transcription factors, recapitulating the sequence of developmental milestones of thyroid development. We describe a protocol for seeding iPSCs, inducing definitive and anterior foregut endoderm, followed by thyroid lineage specification via Fibroblast Growth Factor/Bone Morphogenetic Protein (FGF/BMP) signaling and subsequent differentiation and maturation into TFCs. Our protocol is validated using stem cell lines from a variety of human genetic backgrounds. For complete details on the use and execution of this protocol, please refer to Undeutsch et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103979"},"PeriodicalIF":1.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144718795","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 direct reprogramming of canine fibroblasts to induced motor neurons. 犬成纤维细胞直接重编程诱导运动神经元的方案。
IF 1.3
STAR Protocols Pub Date : 2025-07-24 DOI: 10.1016/j.xpro.2025.103975
Ryan S Anderson, Amelia C Corona, Masatoshi Suzuki, Susannah J Sample
{"title":"Protocol for direct reprogramming of canine fibroblasts to induced motor neurons.","authors":"Ryan S Anderson, Amelia C Corona, Masatoshi Suzuki, Susannah J Sample","doi":"10.1016/j.xpro.2025.103975","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103975","url":null,"abstract":"<p><p>Direct reprogramming of canine fibroblasts to induced motor neurons in vitro opens the door to molecular characterization, functional analysis, and therapeutic screening of canine age-related neurodegenerative diseases. Here, we present a protocol for generating canine induced motor neurons directly from primary dermal fibroblasts. We provide a step-by-step guide for isolating and maintaining primary dermal fibroblasts from dogs, obtaining a stock of reprogrammable cells, and performing the direct reprogramming process. We then detail procedures for validating the motor neuron identity.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103975"},"PeriodicalIF":1.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144715197","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 minicircle production for gene therapy without subsequent cleanup steps. 无后续清理步骤的基因治疗微环生产方案。
IF 1.3
STAR Protocols Pub Date : 2025-07-24 DOI: 10.1016/j.xpro.2025.103982
Jonathan Do, Gautam Verma, Yasmin Maurice, Muniba Abdumanobova, Zicheng Deng, Tanya V Kalin, Vladimir V Kalinichenko
{"title":"Protocol for minicircle production for gene therapy without subsequent cleanup steps.","authors":"Jonathan Do, Gautam Verma, Yasmin Maurice, Muniba Abdumanobova, Zicheng Deng, Tanya V Kalin, Vladimir V Kalinichenko","doi":"10.1016/j.xpro.2025.103982","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103982","url":null,"abstract":"<p><p>Current techniques for producing minicircles have low yields because of the cleanup steps required. Here, we present a protocol that can increase minicircle yield up to 10-fold by eliminating post-induction cleanup steps. We describe steps for bacterial culture, same-day arabinose induction, and DNA harvesting. This protocol is relevant for all minicircles produced from parental plasmids containing attB, attP, and 32 copies of Isce-I endonuclease sites in the minicircle-producing bacteria, ZYCY10PS3T2.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103982"},"PeriodicalIF":1.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144715198","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 update to: High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids. 方案更新为：用于小鼠脑和胰腺类器官单细胞多组学分析的高通量scNMT方案。
IF 1.3
STAR Protocols Pub Date : 2025-07-24 DOI: 10.1016/j.xpro.2025.103980
Santiago Cerrizuela, Oguzhan Kaya, Lukas P M Kremer, Andrea Sarvari, Tobias Ellinger, Jannes Straub, Jan Brunken, Andrés Sanz-Morejón, Aylin Korkmaz, Ana Martín-Villalba
{"title":"Protocol update to: High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids.","authors":"Santiago Cerrizuela, Oguzhan Kaya, Lukas P M Kremer, Andrea Sarvari, Tobias Ellinger, Jannes Straub, Jan Brunken, Andrés Sanz-Morejón, Aylin Korkmaz, Ana Martín-Villalba","doi":"10.1016/j.xpro.2025.103980","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103980","url":null,"abstract":"<p><p>Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth. For complete details on the use and execution of this protocol, please refer to Kremer et al. and other works.<sup>1</sup><sup>,</sup><sup>2</sup><sup>,</sup><sup>3</sup><sup>,</sup><sup>4</sup><sup>,</sup><sup>5</sup><sup>,</sup><sup>6</sup><sup>,</sup><sup>7</sup> This protocol is an update to Cerrizuela et al.<sup>7</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103980"},"PeriodicalIF":1.3,"publicationDate":"2025-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144715201","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 generation, quantification, and phenotyping of brain metastases in preclinical mouse models. 临床前小鼠脑转移瘤模型的生成、定量和表型分析。
IF 1.3
STAR Protocols Pub Date : 2025-07-23 DOI: 10.1016/j.xpro.2025.103978
Francisco Javier Rodriguez-Baena, Berta Sanchez-Laorden
{"title":"Protocol for generation, quantification, and phenotyping of brain metastases in preclinical mouse models.","authors":"Francisco Javier Rodriguez-Baena, Berta Sanchez-Laorden","doi":"10.1016/j.xpro.2025.103978","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103978","url":null,"abstract":"<p><p>The field of brain metastasis is rapidly expanding, yet no consensus exists on the most reliable quantification approach. We present a protocol for assessing metastatic burden in mice following intracarotid injection of tumor cells. We describe steps for surgical procedures, brain processing, cryo-sectioning, and slide preparation, followed by phenotypic characterization. We detail procedures for quantifying brain metastatic area using automated microscopy and semi-supervised image analysis. For complete details on the use and execution of this protocol, please refer to Rodriguez-Baena et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 3","pages":"103978"},"PeriodicalIF":1.3,"publicationDate":"2025-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144709187","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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