Methods in molecular biology最新文献

Tracing Stem and Progenitor Cells Using Sequential Administration of Three Thymidine Analogs In Vivo. 使用三种胸腺嘧啶类似物在体内追踪干细胞和祖细胞。

Methods in molecular biology Pub Date : 2026-10-09 DOI: 10.1007/7651_2026_721

Sierra H Ball, Rina N Helt, Claudia Capdevila, Jonathan Miller, Liang Cheng, Hyeonjeong Lee, Laura A Jenny, Elena Wang, Sarah N Weinshel, Arnold S Han, Kelley S Yan

{"title":"Tracing Stem and Progenitor Cells Using Sequential Administration of Three Thymidine Analogs In Vivo.","authors":"Sierra H Ball, Rina N Helt, Claudia Capdevila, Jonathan Miller, Liang Cheng, Hyeonjeong Lee, Laura A Jenny, Elena Wang, Sarah N Weinshel, Arnold S Han, Kelley S Yan","doi":"10.1007/7651_2026_721","DOIUrl":"https://doi.org/10.1007/7651_2026_721","url":null,"abstract":"Imaging of adult stem cells and their progeny in the gastrointestinal (GI) epithelium is useful for constructing cellular hierarchies, revealing stem cell location, and understanding stem cell dynamics, all of which provide an understanding of how they sustain tissues during homeostasis and injury. Pulse-chase approaches can be used to track stem and progenitor cells and understand tissue kinetics by using nucleotide analogs that irreversibly incorporate into DNA during S-phase. Existing pulse-chase approaches that use multiple analogs require harsh DNA denaturation which obscures nuclear staining and the ability to visualize colocalization of analog signals with DNA. This can prevent the ability to distinguish between diluted thymidine analog signal and auto fluorescent secretory granules in cells abundant in the GI epithelium. Here we build upon a method which combines three thymidine analogs for triple pulse-chase labeling of proliferating cells in mice to understand tissue kinetics and S-phase length while maintaining strong nuclear signals in the colon epithelium. This approach may be applied to other tissues to study cell proliferation kinetics.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149137399","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

An Organoid-Based Assay for Evaluating Oxidative Stress-Reactive Fluorescent Probes Using the CERO 3D Bioreactor in a Mouse Hepatic Oxidative Injury Model. 在小鼠肝氧化损伤模型中使用CERO三维生物反应器评估氧化应激反应荧光探针的类器官检测。

Methods in molecular biology Pub Date : 2026-10-09 DOI: 10.1007/7651_2026_722

Eva-Maria Bryan, Ozlem Dilek

{"title":"An Organoid-Based Assay for Evaluating Oxidative Stress-Reactive Fluorescent Probes Using the CERO 3D Bioreactor in a Mouse Hepatic Oxidative Injury Model.","authors":"Eva-Maria Bryan, Ozlem Dilek","doi":"10.1007/7651_2026_722","DOIUrl":"https://doi.org/10.1007/7651_2026_722","url":null,"abstract":"Oxidative stress is a central driver of acute liver injury, and protein carbonylation and lipid aldehyde formation are among its most direct chemical signatures. Most carbonyl-reactive fluorescent probes have been characterized only in two-dimensional culture, and their behavior in three-dimensional tissue is often unclear. Here we describe a protocol for evaluating carbonyl-reactive fluorescent probes in mouse hepatic organoids grown in suspension in the CERO 3D bioreactor, using acetaminophen (APAP) overdose as a well-established model of acute oxidative liver injury. We demonstrate the assay with P7-Hz, a hydrazine-based probe that reacts with carbonyl groups on oxidatively damaged proteins and lipids to form the fluorescent hydrazone product P7-Hz-Ali. The protocol covers organoid culture and maintenance, APAP dosing to induce injury, probe incubation (labeling), and imaging-based quantification of the fluorescence signal as a readout of carbonylation, together with co-staining viability to distinguish probe-specific signal from cytotoxicity. Although P7-Hz is used as the demonstration probe, the same workflow accommodates other carbonyl-reactive probes whose hydrazone (or analogous) product is spectroscopically distinguishable from the unreacted probe, providing a reproducible three-dimensional platform for probe validation.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149137427","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

Characterization of Stem Cell-Laden Nanocomposite Hydrogels for Injectable Therapy. 用于注射治疗的干细胞负载纳米复合水凝胶的表征。

Methods in molecular biology Pub Date : 2026-10-07 DOI: 10.1007/7651_2026_718

Shajahan Amitha Banu, Khan Sharun, Stelios Alexandris, Álvaro Díaz Vera, Jesper de Claville Christiansen, Cristian Pablo Pennisi

{"title":"Characterization of Stem Cell-Laden Nanocomposite Hydrogels for Injectable Therapy.","authors":"Shajahan Amitha Banu, Khan Sharun, Stelios Alexandris, Álvaro Díaz Vera, Jesper de Claville Christiansen, Cristian Pablo Pennisi","doi":"10.1007/7651_2026_718","DOIUrl":"https://doi.org/10.1007/7651_2026_718","url":null,"abstract":"Hydrogels are widely used as injectable biomaterials for minimally invasive stem cell delivery, providing a hydrated three-dimensional matrix that supports cell survival and retention. Nanocomposite hydrogels, formed by combining hydrogels with nanoscale fillers, offer additional opportunities to tailor rheological behavior and mechanical performance for injectable cell-delivery applications. However, their translational potential depends on the ability to rationally characterize both material behavior and cell compatibility under injection-relevant conditions. This chapter presents a structured experimental protocol for the preparation and initial characterization of collagen-Laponite nanocomposite hydrogels containing human adipose-derived mesenchymal stem cells as a representative model for injectable therapeutic applications. The protocol describes hydrogel formulation, nanoparticle dispersion, stem cell encapsulation, rheological characterization, injectability testing, post-injection viability assessment, and short-term metabolic activity analysis. Although a collagen-Laponite formulation and human adipose-derived mesenchymal stem cells are used as the working example, the protocol provides a practical and systematic framework for evaluating injectable stem cell-laden nanocomposite hydrogels, with particular focus on balancing injectability, structural integrity, and preservation of cellular function.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149111908","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

Quantitative Single-Cell Time-Lapse Phase-Contrast Microscopy with DeepSea. 定量单细胞延时相衬显微镜与深海。

Methods in molecular biology Pub Date : 2026-09-30 DOI: 10.1007/7651_2026_717

Alia Azadi, Abolfazl Zargari, Avlyn V Singer, Gerrald A Lodewijk, Carly Guiltinan, S Ali Shariati

引用次数: 0

CRISPR/Cas9 Editing of Human Keratinocytes for Organotypic Modeling of Genetic Epidermal Disorders. CRISPR/Cas9编辑人类角化细胞用于遗传性表皮疾病的器官型建模

Methods in molecular biology Pub Date : 2026-08-30 DOI: 10.1007/7651_2026_714

Anthony Coon, Jessica L Ayers, Christopher Cole, Afua Tiwaa, Arti Parihar, Thomas Vincent, Johann E Gudjonsson, Mrinal K Sarkar, Cory L Simpson

引用次数: 0

Immunofluorescence Detection of Lysyl Oxidase in Formalin-Fixed Paraffin-Embedded Breast Tissue. 福尔马林固定石蜡包埋乳腺组织赖氨酸氧化酶的免疫荧光检测。

Methods in molecular biology Pub Date : 2026-08-25 DOI: 10.1007/7651_2026_716

Eva-Maria Bryan, Virginia Espina, Ozlem Dilek

引用次数: 0

Automated Deep Learning Protocol for Quantifying Cell Viability in Live/Dead Fluorescence Microscopy. 在活/死荧光显微镜中定量细胞活力的自动深度学习协议。

Methods in molecular biology Pub Date : 2026-08-25 DOI: 10.1007/7651_2026_715

Álvaro Díaz Vera, Khan Sharun, Shajahan Amitha Banu, Cristian Pablo Pennisi

{"title":"Automated Deep Learning Protocol for Quantifying Cell Viability in Live/Dead Fluorescence Microscopy.","authors":"Álvaro Díaz Vera, Khan Sharun, Shajahan Amitha Banu, Cristian Pablo Pennisi","doi":"10.1007/7651_2026_715","DOIUrl":"https://doi.org/10.1007/7651_2026_715","url":null,"abstract":"The assessment of live/dead cells by means of fluorescence microscopy is a standard technique for assessing cell viability but relies on manual counting, which is laborious and prone to operator bias. Here we present a step-by-step protocol for automated cell viability quantification from two-channel live/dead fluorescence images using a deep learning pipeline built on a U-Net architecture with a ResNet-50 encoder pretrained on ImageNet. The protocol is organized into two tracks: a no-code inference track that applies a pre-trained model to standard live/dead images and an adaptation track for generating custom annotations and retraining the model for new cell types or staining protocols. The pipeline integrates percentile-based normalization, centroid-based (weakly supervised) annotation, patch-based transfer learning, and distance-transform watershed post-processing for instance-level counting. On an independent 25-image test set of human dermal fibroblasts, the model achieved counting R2 values of 0.986 (live) and 0.999 (dead) and a mean absolute viability error of 0.61 percentage points, providing a reproducible framework adaptable to diverse cell types and imaging platforms.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148813358","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

Nanotechnology-Stem Cell Strategies in 3D Glioblastoma Organoid: Targeting Glioma Stem Cells Within a Complex Tumor Microenvironment. 纳米技术-干细胞在三维胶质母细胞瘤类器官中的策略：在复杂的肿瘤微环境中靶向胶质瘤干细胞。

Methods in molecular biology Pub Date : 2026-07-04 DOI: 10.1007/7651_2026_713

Saptaparna Pal, Sejal Mhatre, Puja Sandbhor

{"title":"Nanotechnology-Stem Cell Strategies in 3D Glioblastoma Organoid: Targeting Glioma Stem Cells Within a Complex Tumor Microenvironment.","authors":"Saptaparna Pal, Sejal Mhatre, Puja Sandbhor","doi":"10.1007/7651_2026_713","DOIUrl":"https://doi.org/10.1007/7651_2026_713","url":null,"abstract":"Therapeutic failure in glioblastoma (GBM) is increasingly attributed not only to tumor cell-intrinsic factors but also to the adaptive/supportive tumor microenvironment that nurtures glioma stem cells (GSCs) and drive therapy resistance. GSCs reside within specialized niches shaped by extracellular matrix architecture, stromal interactions, metabolomic gradients, and immune-modulatory cues, enabling their survival, plasticity, and repopulation following conventional therapy. Effective targeting of GBM therefore requires strategies that disrupt both GSC-intrinsic niche and the supportive microenvironment context that limit drug penetration, retention, and therapeutic benefit.Traditional two-dimensional (2D) culture systems fail to capture these spatial and biological complexities, resulting in poor clinically actionable predictive power for successful outcomes. In contrast, three-dimensional (3D) models offer an opportunity to recapitulate relevance. Building on this context, this chapter highlights recent advances that integrate nanotechnology with stem cell-based 3D GBM organoid platforms to enable effective therapeutic delivery and resistance niche-level targeting.We discuss the design and functional evaluation of nanoparticle systems engineered for deep tumor penetration and selective delivery, including polymeric nanoparticles, mesoporous silica nanoparticles, and ultrasmall gold nanostructures. Emphasis is placed on mesenchymal and neural stem cell-mediated nanodelivery, biomimetic hydrogel- and nanofiber-based scaffolds for recreating GSC-associated niche, and advanced analytical readouts including electron microscopy, confocal Z-stack imaging, and ICP-MS. Collectively, this chapter presents a translational framework for leveraging 3D models and stem cell-directed nanotechnologies as preclinical tools to overcome therapy resistance and improve therapeutic outcomes in GBM.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148382320","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

Isolation of Mesenchymal Stem Cell-Derived Extracellular Vesicles. 间充质干细胞来源的细胞外囊泡的分离。

Methods in molecular biology Pub Date : 2026-06-26 DOI: 10.1007/7651_2026_712

Ilgin Kimiz Gebologlu, S Furkan Demirden, Suphi S Oncel

引用次数: 0

Modeling Melanoma Immune Surveillance by CAR-T Cells in Human Skin Organoids. CAR-T细胞在人体皮肤类器官中模拟黑色素瘤免疫监测。

Methods in molecular biology Pub Date : 2026-06-05 DOI: 10.1007/7651_2026_711

Korvell Russell, Anam Adil, Rohan Shivde, Brian McGrath, Hanin Abuzer, Cormac Cosgrove, Madalyne Juarez, I Caroline LePoole, Bethany E Perez White

引用次数: 0